Final exam Flashcards by M M

What are the four classes of macromolecules?

Proteins, lipids, carbohydrates, nucleic acids

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The ___________ structure of proteins consists of the protein’s amino acid sequence.

Primary

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Secondary protein structure consists of alpha helices and beta-pleated sheets that form from ____________ between the backbones of amino acids, not side chain interactions.

Hydrogen bonds

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Super-secondary secondary structures are structures that form from secondary structures grouped in specific ways; they are also called _____________.

Motifs

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A ___________________ is a similar 3D structure conserved among proteins that serve a similar function.

Motif

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Tertiary structures of proteins result from ______________ interactions: (1) hydrophobic interactions, (2) ionic bonds/salt bridges, (3) hydrogen bonds, and (4) _________________ between sulfhydryl groups of cysteine residues.

Side-chain or R-group interactions

Disulfide bridges (covalent interaction)

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___________________ structure occurs when a protein is composed of more than one polypeptide chain.

Quarternary

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What are stable, independelty folding, compact structural units within a protein that have relative independent structure and function distinguishable from other regions and stabilized through the same kind of linkages at the tertiary level?

Domains

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Why do disulfide bridge not exist in the cytosol?

They are highly oxidized, but the cytosol is highly reduced

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How are proteins regulated?

Quantity, activity, location

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What are the ways in which gene expression can be regulated?

Transcriptional control

RNA processing control

RNA localization

Translational control

Post-translational control

mRNA degradation

Protein-activity control

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What primarily mediates transcriptional control of gene expression?

Transcription factors

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Trans-acting factors or ___________________ factors bind cis-regulatory elements and work to speed up or slow down transcription.

Transcription factors

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Transcriptional _______________ bind to cis-regulatory sequences and turn genes off.

Repressors

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Transcriptional ________________ bind to cis-regulatory sequences and turn genes on.

Activators

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_________________ and ______________ bind to activators and repressors, not DNA, and assist in gene regulation.

Coactivators

Corepressors

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What are two mechanisms through which transcriptional activators function?

Chromatin remodeling

RNA polymerase recruitment

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General transcription factors and RNA polymerase cannot displace nucleosomes on their own; therefore transcription activator promote transcription by triggering changes to chromatin structure near promoters, making the DNA more accessible. Transcription activators do so via four processes: (1) covalent histone modification, (2) _________________ remodeling, (3) nucelosome removal, and (4) histone replacement.

Nucleosome

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What are four ways in which activators trigger chromatin remodeling?

Covalent histone modification

Nucleosome remodeling

Nucleosome removal

Histone replacement

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Transcription activators can act on RNA polymerase in four ways. What are they?

Promote binding of additional regulators

Recruit RNA polymerases to promoters

Release RNA polymerase to begin transcription

Release RNA polymerase from pause

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Transcription activators can promot binding of additional regulators, recruit __________________ to promoter region, release _______________ to begin transcription, and release _______________ from pause (all blanks are the same answer).

RNA polymerase

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Genes encoding the most important developmental regulatory proteins are kept tightly repressed or activated?

Repressed

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What are six mechanisms of transcription repression?

Competitive DNA binding

Masking the acitvation surface

Direct interaction with general transcription factors

Recruitment of chromatin remodeling complexes

Recruitment of histone deacetylases

Recrtuitment of histone methyl transferases

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____________________ of DNA usually increases transcription whereas _____________ of DNA usually decreases transcription.

Acetylation

Methylation

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What is combinatorial control?

Multiple activators or multiple copies of a single activator work synergistically to initiate transcription

What is mean by transcriptional synergy?

Transcription factors often exhibit synergy, meaning that they work together to produce a transcription rate much higher than the sum of their transcription rates working alone

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ refers to the maintenance of cell differentiation through subsequent cell generations.

Cell memory

The early *Drosophila* embryo is a \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_, meaning it has multiple nuclei in a shared cytoplasm.

Syncytium

The position of transcriptional factors is crucial for *Drosophilia* development. Bicoid and Hunchback are localized to the ___________ of the cell while Giant and Kruppel are not.

Bicoid and Hunchback are transcription _______________ while Giant and Kruppel are transcription \_\_\_\_\_\_\_\_\_\_\_\_\_.

Activators Repressors

Nuclei in the syncytium begin to express different genes because they are exposed to different __________________ that are localized to specific areas of the cell.

Transcription regulators

To what does *Eve* refer?

Even-skipped gene, which is important in body patterning in *Drosophilia* flies

Bicoid, Hunchback, Kruppel, and Giant are transcription regulators of *Eve* expression in which stripe?

Stripe 2

If Bicoid and Hunchback are inactivated, what happens to stripe 2?

No stripe 2 develops because Bicoid and Hunchback are activators

If Kruppel and Giant are inhibited, what happens to stripe 2?

Stripe 2 is diffused throughout the cell because Kruppel and Giant are repressors

What are three types of RNA processing discussed in class?

5' cap of 7-methylguanosine 3' cleavage and polyadenylation Alternative splicing

Alternative splicing allows for the production of different ______________ from a single gene.

Proteins

Is alternative splicing uniform across cell types and developmental stages?

No; alternative splicing may differ in different cell types, in a single cell type (due to extracellular signals), or in a single cell type during different stages of development

RNA splicing removes __________ sequences from newly transcribed pre-mRNAs.

Introns

A mRNA molecule only becomes designated as such after which two actions occur?

Addition of 5' 7-methylguanosine cap and 3' cleavage and polyadenylation

Although alternative splicing is energy inefficient, it is proposed that over time, RNA splicing increases ___________________ in organisms and for this reason it has continued.

Genetic variability

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ sequences signal where splicing occurs.

Nucleotide

RNA splicing is performed by the \_\_\_\_\_\_\_\_\_\_\_\_.

Splicesome

ATP is required for assembly and rearrangement of the \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_.

Splicesome

Each __________ codes for one domain.

Exon

A domain is the product of a single \_\_\_\_\_\_\_\_\_\_\_\_\_.

Exon

A protein _____________ is a substructure produced by any contiguous part of a polypeptide chain that can fold independently of the rest of the protein into a compact, stable substructure.

Domain

The _______________ protein is a tyrosine-kinase with three domains: \_\_\_\_\_\_\_\_\_\_\_\_, \_\_\_\_\_\_\_\_\_\_\_, and \_\_\_\_\_\_\_\_\_\_\_\_.

Src SH1 SH2 SH3

SH1 is the _______________ site; SH2 is the _________________ binding site; and SH3 binds proline-rich regions of other proteins.

Enzymatic site Phosphotyrosine-binding site

Domains are highly \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_.

Conserved

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ _______________________ during evolution has resulted in domain replication and shuffling.

Genetic recombination

Some domains are found in many different proteins. These serine proteases share the same catalytic domain. Differential __________ is conferred by other domains.

Regulation

Only about \_\_\_\_\_% of all synthesized RNA ever leaves the nucleus.

What degrades improperly processed RNA molecules in the nucleus?

The nuclear exosome

As RNA is processed, it loses certain proteins; only when specific proteins are present including _______________________________ (hnRNPs) that a mRNA molecule is exported from the nucleus.

Heterogeneous nuclear ribonuclear proteins

Successfully produced mRNAs are guided through _________ \_\_\_\_\_\_\_\_\_\_\_\_ \_\_\_\_\_\_\_\_\_\_\_\_\_, aqueous channels in the nuclear membrane that directly connect the nucleoplasm with the cytosol. The cell uses _______ to move mRNAs through these complexes. This transport occurs via _________ \_\_\_\_\_\_\_\_\_ \_\_\_\_\_\_\_\_\_, which are attached to mRNA and dissociate once mRNA has left the nucleus.

Nuclear pore complexes ATP Nuclear transport receptors

What are three ways in which mRNA can be localized in the cell?

Directed transport on the cytoskeleton Random diffusion and trapping Generalized degradation in combination with local transport by trapping

Where are signals for mRNA localization located?

The 3' untranslated region (UTR)

Three distinct mechanisms account for the asymmetric distribution of mRNAs within cells: (1) localized _____________ from degradation, (2) diffusion-coupled local \_\_\_\_\_\_\_\_\_\_\_\_\_\_, and (3) directed transport along a ________________ cytoskeleton.

Protection Entrapment Polarized

mRNA is localized in spatially distinct patterns depending upon the needs of the cell. In most cell types, we find our microfilament structures near the cell membrane in a region called the \_\_\_\_\_\_\_\_\_\_\_. In some cell types, notably \_\_\_\_\_\_\_\_\_\_\_\_, there is an even high concentration of actin, specifically beta actin, and its mRNA near the cortex. Fibroblasts are cells in connective tissue that produce collage and other fibers, and in these cells, beta actin mRNA localizes to the \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_, where translation is required for cytoskeleton-mediated motility.

Cortex Fibroblasts Lamellipodia

Fibroblasts must be able to move to replair connective tissue, which is why the "foot layer" or ________________________ is important.

Lamellipodia

What experiment did this photo demonstrate?

The experiment demonstrated that the chimeric mRNA was concentrated in the leading edge of the fibroblast, supporting the idea that mRNA is localized for translation. Experiment: Actin gene was cloned into an expression vector, which produced actin-beta-galactosidase chimeric mRNA and protein. The gene was transfected into chick embryo fibroblasts, and *in situ* hybridization with beta-galactosidase-specific probes confirmed mRNA accumulates in the lamellipodia just like the protein.

What did this experiment demonstrate?

The two short regions of the beta-actin gene, both of which were located in the 3' untranslated region, were critical for mRNA localization. One of the regions - the proximal region - was a stronger driver of localization than the distal region.

What is meant by a beta-actin zipcode?

The zipcode refers to two consensus sequences found by scientists that ultimately led to mRNA localization

What is the zipcode-binding protein?

A protein with a gene sequence that shares two highly conserved hnRNP (heterogenous nuclear ribonuclear protein) and nuclear export sequences (NES)

It is currently believed that _____________________ underlie the mechanism for beta-actin localization.

Microfilaments

In *Drosophilia* development, egg ____________ is determined by localized gene expression in the oocyte and early embryo.

Polarity

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ is a morphogen transcription factor at the anterior end of the *Drosophilia* embryo. ____________ is a morphogen transcription factor at the posterior end.

Bicoid Nanos

A mutation in Bicoid results in flies with \_\_\_\_\_\_\_\_\_\_\_. A mutation in Nanos results in flies with \_\_\_\_\_\_\_\_\_\_\_\_.

Two tails No abdomens

What facilitates Nanos mRNA localization?

Oskar

Bicoid mRNA localization requires binding by multiple binding proteins including _________ \_\_\_\_\_\_\_\_\_\_ and \_\_\_\_\_\_\_\_\_\_\_\_.

Motor proteins Microtubules

Nanos mRNA is initiatially __________ in the oocyte cytoplasm but remains translationally repressed, except in the ____________ end, where translation repression is released and mRNA degradation fails.

Diffuse Posterior

Nanos localization requires a ________________ \_\_\_\_\_\_\_\_\_\_\_\_\_\_ ___________ in the 3' untranslated region.

Translational control element (TCE)

Without the translational control element, ____________ mRNA does not localize and does not \_\_\_\_\_\_\_\_\_\_\_\_, remaining diffuse throughout the cytosol.

Nanos Degrade

What did researchers determine binds to the translational control element of Nanos, inhibits translation, and prevents localization?

Smaug

Assays for poly-A tail length showed that degradation of Nanos requires ______________ binding to the 3' \_\_\_\_\_\_\_\_\_\_\_.

Smaug 3' UTR

Smaug represses translation of Nanos and recruits a \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_.

Deadenylase

Both ___________________ and the _____________________ (TCE) are required for translation repression and degradation of Nanos.

Smaug Translational control element (TCE)

Smaug was found to be the protein that binds to the TCE of Nanos, which represses Nanos ________________ and recruits a \_\_\_\_\_\_\_\_\_\_\_, a ribonuclease, to degrade Nanos mRNA.

Translation Deadenylase

Nanos is selectively translated at the posterior pole by binding to \_\_\_\_\_\_\_\_\_\_\_\_\_.

Oskar

How does Oskar protect Nanos mRNA?

It beings the TCE and prevents Smaug from binding, thereby preventing degradation and releasing translational repression

Oskar mRNA is localized to the ______________ pole by binding of _________________ (RNPs) and kinesin-1-dependent transport along \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_.

Posterior Ribonucleoproteins Microtubules

What does RNPs stand for?

Ribonucleoproteins

Which motor protein is responsible for Oskar localization?

Kinesin-1

What are four ways in which translation can be regulated?

1. The protein product of a mRNA performs regulatory action on its own mRNA 2. A stem-loop structure can prevent movement of the ribosome 3. A small molecule can bind stem-loop structure 4. Antisense RNA

Iron regulates the expression of certain genes. ___________ is an iron-binding protein that binds to and holds on to iron in cells until needed.

Ferritin

Ferritin mRNA can be bound by a protein that inhibits its translation. This protein is ____________ \_\_\_\_\_\_\_\_\_\_\_\_\_ or the ________________ \_\_\_\_\_\_\_\_\_\_\_\_\_\_ _______________ (IRE) binding protein.

Cytosolic aconitase Iron Response Element (IRE) binding protein

The Iron Response Element (IRE) forms a stem-loop structure that is stabilized by \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_.

Cytosolic aconitase or IRE binding protein

When iron levels in the body increase, iron attaches to the \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_, causing a conformational change that ultimately releases the protein from the IRE sequence.

Cytosolic aconitase or IRE binding protein

Iron binding to cytosolic aconitase or IRE binding protein induces a conformational change that releases the protein from the IRE sequence and destabilizes the __________________ structure. This ultimately enables the translation of \_\_\_\_\_\_\_\_\_\_\_\_, which can bind to and store increased iron in the cell.

Stem-loop structure Ferritin

What is transferrin?

A blood protein that transports iron throughout the cell

When iron levels are low, what happens to transferrin receptors?

They are upregulated so that more iron can be brought into the cell

Normally, the IRE binding protein binds the stem-loop structure at the 3' UTR of the transferrin receptor mRNA, preventing mRNA degradation. However, when iron levels are \_\_\_\_\_\_\_\_, there is no longer a need for transferrin receptors, so iron binds the IRE binding protein, releasing it from the 3' UTR. This leaves the _____________ structure open to degradation.

High Stem-loop structure

During iron starvation, the binding of ______________ or the IRE binding protein to the 5' UTR of ___________ mRNA blocks translation initiation; its binding to the 3' UTR of the ________ receptor mRNA blocks an endonuclease cleavage site, thereby stabilizing the mRNA. In this way, _______ is downregulated and _______ receptors are upregulated, bring iron into the cell.

Cytosolic aconitase Ferritin Transferrin Ferritin Transferrin

When iron levels are low, we want to _____________ ferritin levels and increase _________________ receptors.

Decrease Transferrin

In response to high iron concentrations, ______________ synthesis is increased. Iron binds to _________________ or the IRE binding protein in the 5' UTR, which releases from the mRNA and enables translation. Iron binding also prevents ______________ or the IRE binding protein from binding to the stem-loop structure in the \_\_\_\_\_\_\_, thereby exposing an ________________ cleavage site and enabling degradation of __________________ mRNA.

Ferritin Cytosolic aconitase Cytosolic aconitase 3' UTRE Endonucleolytic Transferrin receptor

Be able to explain this figure.

Control of mRNA degradation can be regulated by _________ RNAs, like ___________ (miRNAs), ____________________ (siRNAs), and _______________________ (piRNAs).

Noncoding MicroRNAs Small interfering RNAs Piwi-interacting RNAs

microRNAs (miRNAs) regulate mRNA _______ and stability. They are synthesized by RNA polymerase II, undergo capping and polyadenylation, and assemble with ___________ to form the \_\_\_-\_\_\_\_\_\_\_\_\_\_ ____________ \_\_\_\_\_\_\_\_\_\_\_\_ (RISC).

Translation Argonaute RNA-induced silencing complex

\_\_\_\_\_\_\_\_ _________ \_\_\_\_\_\_\_\_\_\_ are short double-stranded RNA molecules that are bound by Argonaute and other RISC components to degrade mRNA.

Short interfering RNAs

\_\_\_\_\_\_\_\_ _________ \_\_\_\_\_ are made in the germ line and block the movement of transposable elements

Piwi interacting RNAs (piRNAs)

What 7 covalent modifications occur in cells that regulate proteins post-translationally?

1. •Proteolysis 2. •Glycosylation 3. •Phosphorylation 4. •Acylation 5. •Acetylation 6. •Methylation 7. •Protein tags

Many proteins are synthesized in larger forms known as "pro." Many enzymes are are also synthesized in this way (i.e., "ogen"). These larger forms are _______ and require modification by _________ to engage biological functioning. A well-known example of this post-translation modification is \_\_\_\_\_\_\_\_\_\_.

Insulin

What is glycosylation?

is the process of adding carbohydrate groups onto proteins

There are ______ types of glycosylation depending upon where and how the carbohydrate is attached: (1) \_\_\_-linked glycosylation occurs when the carbohydrate group is attached to the _______ atom on the R group of asparagine, and (2) \_\_\_-linked glycosylation: carbohydrate group is attached to the oxygen atom on the R group of __________ or \_\_\_\_\_\_\_\_.

Two N-linked Nitrogen O-linked Serine or threonine

Are glycosylated proteinscommonly found in the cytosol?

No, they are most often found in proteins destined for secretion

Proteins that recognize and bind carbohydrate structures are called \_\_\_\_\_\_\_\_\_\_.

Lectins

\_\_\_\_\_\_\_\_\_\_\_\_ are a type of lectin that is important in cell-cell recognition and communication, immune system function, and brain development

Selectins

Glycosylation influences protein \_\_\_\_\_\_\_\_\_\_, inhibits \_\_\_\_\_\_\_\_\_\_, provide binding sites for lectins, and function in cell-cell recognition.

Folding Degradation Lectins

Which three amino acids can be phosphorylated?

Serine Threonine Tyrosine

What enzymes phosphorylate molecules?

Kinases

What enzymes dephosphorylate molecules?

Phosphatases

Most enzymes that can phosphorylate serine can also phosphorylate threonine; these kinases are called _________ \_\_\_\_\_\_\_\_\_\_\_\_\_ kinases.

Serine-threonine kinases

Only ___________ kinases can phosphorylate tyrosine.

Tyrosine kinases

What is the purpose of phosphorylation and dephosphorylation in the cell?

The purpose of phosphorylation and de-phosphorylation is for activation and deactivation; when a protein is phosphorylated or dephosphorylated, conformational changes occur, which will lead to either an activating or deactivating change

Proteins can be acylated by the addition of _________ \_\_\_\_\_\_\_\_\_, like myristate and palmitate, and __________ \_\_\_\_\_\_\_\_, like farnesyl and geranylgeranyl.

Fatty acid chains Isoprenyl chains

Why is the addition of acyl chains important?

They anchor and localize proteins to the plasma membrane

Isoprenyl groups are ____ carbon molecular units that are also called \_\_\_\_\_\_\_\_\_\_. They can be linked together to form longer chains. When two are linked together, a ____ carbon unit is formed and called a \_\_\_\_\_\_\_\_\_. When three are linked together, a ___ carbon unit is formed and called a \_\_\_\_\_\_\_\_. And when four are linked together, a 20 carbon unit is formed and called a geranylgeranyl.

Five Terpenes 10 carbon Geranyl 15 carbon farnesyl

Acetylation and methylation most often occur on histones; _________ groups are always added to __________ residues, and _________ groups are usually, but not always, added to \_\_\_\_\_\_\_\_\_\_\_residues, too

Acetyl groups Lysine Methyl groups Lysine

\_\_\_\_\_\_\_\_ acetyl transferases and deacetylases as well as _______ methyl transferases and demethylases are enzymes recruited by transcription factors that recognize specific DNA sequences

Histone Histone

The amino tails of ____________ can be heavily modified, and modifications usually occur on lysine residues. They may differ throughout the genome and may change during the life of the cell.

Histones

What is the histone code?

a hypothesis that the transcription of genetic information encoded in DNA is in part regulated by chemical modifications to histone proteins, primarily on their unstructured ends

What two protein tags did we discuss in class?

Ubiquitin SUMO (small ubiquitin-related modifier)

\_\_\_\_\_\_\_\_\_\_\_ is a highly conserved 76 amino acid protein that is found in a huge range of organisms and remains nearly identical across them. It is conjugated by its C terminus to an internal _______ residue of the target protein

Ubiquitin Internal lysine (K)

\_\_\_\_\_\_\_\_\_ is a pattern of \_\_\_\_\_ubiquination in which one ubiquitin attaches to an internal ______ of the target protein and additional ubiquitins are added to the 48th lysine residue of the first ubiquitin; ______ is similar, except each additional ubiquitin is added to the 63rd lysine on the previous ubiquitin molecule

Lys48 Polyubiquination Lysine Lys63

Ubiquitination of a protein involves three proteins: \_\_\_\_, \_\_\_\_, and \_\_\_\_\_.

E1 E2 E3

In the process of ubiquination, ______ is the ubiquitin-activating enzyme. E2 is the \_\_\_\_\_\_\_\_\_\_\_; together they complex together, and _____ transfers its ubiquitin to \_\_\_\_\_,

E1 Ubiquitin-conjugating enzyme E1 transfers to E2

\_\_\_\_\_ stores the ubiquitin until E3 or ________ and a target protein arrive; once E3 and the target protein are in the vicinity, _____ facilitates the movement of ubiquitin to the target protein

E2 Ubiquitin ligase E2

What is the role of E3 in ubiquination?

E3 simply serves to recognize the target proteins in need of ubiquitination and is the site of substrate binding

Be familiar with the general process of ubiquination.

\_\_\_\_\_\_ubiquination on histones is part of the histone code. \_\_\_\_\_\_ubiquination, specifically __ \_\_\_, on histones and monoubiquination on many other proteins, are common at sites of DNA repair Multiubiquination via mono, di, or poly (via K63) on a transmembrane protein can trigger ________ and trafficking to the \_\_\_\_\_\_\_\_\_for \_\_\_\_\_\_\_\_\_\_\_. \_\_\_\_\_ubiquination (\_\_ \_\_) marks proteins for proteosomal degradation

Monoubiquination Polyubiquination K63 Endocytosis Lysosome Degradation Polyubiquination K48

K63 polyubiquination is involved in ____ \_\_\_\_\_\_ and _______________ of transmembrane proteins that are trafficked to the lysosome for degradation. K48 polyubiquination is involved in ______ \_\_\_\_\_\_\_\_.

DNA repair Endocytosis of transmembrane proteins Proteosomal degradation

SUMOylation is involved in four things. What are they?

–DNA damage repair –Chromatin organization & transcription –Chromosome segregation & cytokinesis –Nuclear protein import

Covalent modifications are reversible but require a great deal of energy and are therefore reserved for "one-and-done" events. \_\_\_\_\_-\_\_\_\_\_\_\_\_\_\_ modifications do not require such an energy expenditure and can include interactions with other proteins and __________ proteins, like cAMP, calcium ions, and GTP.

Non-covalent Regulatory

Which occurs more frequently in the cell? Covalent or non-covalent modifications?

Non-covalent

Non-covalent binding can result in three things. What are they?

1. Formation of structural complexes 2. Localization of a protein within the cell 3. Regulation of a protein's activity

\_\_\_\_\_\_\_\_\_\_\_\_ binding is mediated by weak intermolecular forces

Reversible

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ interactions are not considered intermolecular forces although they are often included under the umbrella term.

Hydrophobic interactinos

Calcium can directly bind to and regulate other proteins. It can also bind to \_\_\_\_\_\_\_-\_\_\_\_\_\_\_\_\_ \_\_\_\_\_\_\_, which then binds to and regulates other proteins. The most common is \_\_\_\_\_\_\_\_\_, which requires two or more calcium ions to bind for activation.

Calcium-binding proteins (CBPs) Calmodulin

G-proteins are intrinsic \_\_\_\_\_\_\_ases.

GTPases

Proteins that bind to GTP are called GTP-binding proteins, commonly called G-proteins. When bound to GTP, the protein is usually \_\_\_\_\_\_\_. When bound to GDP, the protein is usually \_\_\_\_\_\_\_\_.

Activated Inactivated

There is a great deal of ____ in the cell. High-energy nucleotides are *not* scarce.

GTP

Binding to _____ to a G-protein causes a ______________ change, leading to its activation

GTP Conformational change

How are G-proteins regulated?

GEFs and GAPs

What does GEF stand for?

Guanine nucleotide exchange factor

What does GAP stand for?

GTPase activating protein

What does GEF do?

Assists in releasing GDP from G-proteins; GEF binds to the G-protein, causes a conformational change in the G-protein, which decreases the G-protein’s affinity for GDP, thereby releasing GDP; this conformational change simultaneously increases the G-protein’s affinity for GTP

What does GAP do?

Binding of GAP causes a conformational change in the G-protein that increases the catalytic activity of the G-protein, causing it to hydrolyze its GTP faster

What hydrolyzes the GTP? The G-protein or its GAP?

The G-protein

G-proteins regulate many activites in the cell. For example, ___________ is actually a modified form of G-proteins!

Tubulin

There are two ways to degrade a protein in the cell. What are they?

Degradation via the lysosome Degradation via proteosomes (protein-specific)

\_\_\_\_\_\_\_\_\_\_\_\_\_ contain acid hydrolases because these enzymes only function at a lower pH – lower in comparison to blood and cytosol – usually around pH \_\_\_\_. These hydrolases can target nearly any type of molecule within the cell

Lysosomes pH5

Lysosome membranes have ______ \_\_\_\_\_\_\_\_\_\_\_ ______ to keep the interior acidified

ATPase hydrogen pumps

Why must the lysosome have an acidic enviroment?

Protection; if the lysosome bursts and its contents spill into the cytosol, nothing damaging will occur since the hydrolases are only active at lower pHs; they will immediately denature in the more basic environment of the cell

What is autophagy?

Autophagy is a self eating process cells go through for normal organelle turnover; mitochondria are especially prone to becoming worn out and in need of turnover

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ are cylindrical multi-protein complexes with hollow inner chamber lined with proteases and multi-protein caps on each end ## Footnote •

Proteasomes

Where are proteasomes abudant?

Cytosol and nucleus

How do proteosomes work?

1. Target proteins are ‘tagged’ with polyubiquitin 2. The cap complex contains a ubiquitin receptor, a ubiquitin hydrolase, and unfoldases 3. Proteins are unfolded, threaded through the cylinder, cut into short peptides which are released through the opposite end

\_\_\_\_\_\_\_\_\_\_\_\_ function as sensors and/or molecular switches which regulate a variety of cellular processes

G-proteins

What are the three stages of translation?

Initiation Elongation Termination

What are the 5 steps of translation initiation?

1. eIF2-GTP binds tRNA Met and small ribosomal subunit, forming the 43S pre-initiation complex 2. Multiple IFs bind mRNA 3. 43S complex binds mRNA at 5’ end 4. 43S complex scans in 5’ to 3’ direction 5. The.Kozak sequence (ACCAUGG) is recognized; GTP is hydrolyzed with eIF5 serves as its GAP. eIF2 and most other eIFs detach prior to 60s subunit binding

eIF\_\_ is a G-protein

eIF2

What recognizes the Kozak sequence?

The 43S Pre-Initiation Complex

What triggers hydrolysis of eIF2's GTP?

The ribosome

What actually helps eIF2 hydrolyze its GTP? In other words, what is the GAP for eIF2?

eIF5

Initiation factors are \_\_\_\_\_\_\_\_\_\_\_.

Reused

Once detached from the 43S pre-initiation complex, eIF2 will release its GDP, which requires its GEF: \_\_\_\_\_\_\_\_\_.

eIF2B

What initiation factor plays a global role in translation?

eIF2 plays a really important role as a more global regulator of translation; it is possible to both activate and deactivate eIF2 and thereby control translation

eIF2 can be phosphorylated by ______ kinases, which _________ eIF2 by increasing its affinity for its GEF \_\_\_\_\_\_\_\_\_.

eIF2 kinases Inactivates eIF2B

A fraction of ________________ eIF2 can trap nearly all of the ______ in the cell.

Phosphorylated eIF2B

What activates eIF2 kinases?

Stress

What four cell stressors can activate eIF2 kinases in the cell and thereby shutdown translation?

Nutrient limitation, ER stress, viral infection, and heme deprivation

What eIF2 kinase is activated by nutrient limitation?

GCN2

What specifically activates GCN2?

Lack of amino acids

What is structurally important about GCN2 - an eIF2 kinase?

It shares homology with histidyl-tRNA synthetase (HisRS)

Which amino acid is critical to GCN2 activation?

Histidine (specifically its lack)

3AT, an inhibitor of histidine formation, and SM, an inhibitor of branched-chain amino acids, ultimately _________ GCN2 and thereby inactivated eIF2.

Activated via phosphorylation

Where is the connection between low amino acid concentration and GCN2 kinase activation?

GCN2 (eIF2 kinase) binds directly to uncharged tRNA, meaning that when amino acids are unavailable, GCN2 can bind to tRNA and presumably be activated, thereby enabling GCN2 to phosphorylate eIF2 and inhibit translation initiation

What eIF2 kinase is activated under ER stress?

PEK/PERK

Where is PEK/PERK located?

The ER membrane

What specifically triggers PEK/PERK activation?

The unfolded protein response (UPR)

PEK/PERK is normally bound by __________ \_\_\_\_\_\_\_\_\_, a member of the HSP 70 family. When unfolded proteins are present; however, this protein undergoes a conformational change and releases PEK/PERK. PEK/PERK then dimerze, crossphosphorylate, and activate the catalytic domains responsible for _____ phosphorylation, which inhibits translation initiation.

Binding Protein (BiP) eIF2

What does BiP stand for?

Binding Protein

The association of PEK/PERK and BiP (Binding Protein) was demonstrated using an ________________ experiment with ______ \_\_\_\_\_\_\_ cells.

Immunoprecipitation experiment Pancreatic acinar cells

Researchers demonstrated that under ER stress BiP (Binding Protein) and PEK/PERK dissociate by using ________ (Tg), which inhibits the calcium ATPase in the ER membrane, and ________ (DTT), which is a reducing agent. Tg and DTT *trigger* ER stress.

Thapsigargin Dithiothreitol

What eIF2 kinase is activated under viral infection?

PKR

How does PKR recognize viral infection in cells?

Virus-infected cells have a lot of double stranded RNA; PKR possesses a double stranded RNA binding domain

How is PKR activated under viral infection?

PKR recognizes and binds double-stranded DNA viral fragments, which induces a conformational change in PKR that allows it to dimerize with another PKR, crossphosphorylate, and activate each other, thereby enabling PKR to act as an eIF2 kinase and phosphorylate/deactivate eIF2

Why does the cell shut down translation under viral infection?

Viruses replicate using the same machinery of the cell; therefore stopping all translation inhibits viral replication and the creation of more viral proteins

Hemoglobin synthesis requires \_\_\_\_\_\_\_\_\_.

Heme

What eIF2 kinase is activated under heme deprivation?

Heme-controlled inhibitor (HCI)

What does HCI stand for?

Heme-controlled inhibitor

When is heme-controlled inhibitor activated?

When there is a lack of heme needed to make red blood cells

When heme is present, it binds to the \_\_\_\_-\_\_\_\_\_\_\_\_ _______ and deactivates it, turning off its kinase activity and preventing it from functioning as an eIF2 kinase.

Heme-controlled inhibitor

When the the heme-controlled inhibitor activated?

When there is no heme bound to it

In addition to the 5 steps of initiation previously discussed, there are two more steps. What are they?

7. 60S ribosomal subunit binds along with eIF5B-GTP to form 80s initiation complex 8. GTP is hydrolyzed only after large and small subunits join. A rRNA serves as the GAP likely in small subunit and requires eIF1A. eIF5B detaches before translation begins

What must detach from the A site of the ribosome for translation to begin?

eIF5B

If the G-protein is a sensor for some event and must hydrolyze its GTP before translation can proceed, what is it sensing?

The correct binding of the large and small ribosomal subunits

eIF\_\_-GTP binds tRNA (Methionine) and the samll ribosomal subunit, mediating the formation of the 43S Pre-Initiation Complex. eIF\_\_ serves as the GAP for eIF\_\_. These early initiation factors then dissociate, allowing for the 60S ribosomal subunit and eIF\_\_ \_\_-GTP to for the 80S Initiation Complex. rRNA in the small riibosomal subunit serves as the GAP but requires eIF\_\_ __ to do so. eIF\_\_ __ must detach from the ribosome before translation can begin.

eIF2-GTP eIF5 eIF2 eIF5B-GTP eIF1A eIF5B

eEF\_\_ \_\_-GTP shepherds each tRNA into the A site for elongation. Proper codon:anticodon binding leads to conformational changes in the large ribosomal subunit (rRNA serves as GAP), which triggers GTP hydrolysis by eEF\_\_ \_\_, which then releases the tRNA and detaches before peptide bond formation. eEF\_\_ __ serves as the GEF for eEF\_\_ \_\_.

eEF1alpha eEF1alpha eEFbeta-gamma eEF1alpha

There is __ \_\_\_\_\_\_\_\_ in bringing tRNAs to ribosome; it is just _______ diffusion

No selection Random diffusion

For translocation of the ribosome to occur, what elongation factor is required?

eEF2-GTP

Binding of eEF\_\_-GTP causes a counter-clockwise rotation in the small ribosomal subunit that (1) destabilizes the ribosome-tRNA interaction, (2) widens the channel openings for the mRNA, and (3) triggers GTP hydrolysis followed by eEF\_ detachment. The ribosome possesses the GAP activity, but we do not know where. Once eEF\_\_ detaches, the small subunit rotates clockwise back to its original conformation

eEF2 eEF2 eEF2

What is the mechanism of translocation likened to?

Ratchet

When the ribosome recognizes a stop codon in the A site, release factors bind, catalyzing the addition of a water molecule to cleave the bond between the polypeptide chain and the tRNA molecule. eRF\_\_ interacts with and binds to stop codons. Its binding brings eRF\_\_-GTP closer to the ribsome. eRF\_\_ is the G-protein. Once hydrolyzed, both eRF\_\_ and eRF\_\_ detach. This detachment triggers the recruitment of water to the ribosome.

eRF1 eRF3-GTP eRF3 eRF1 and eRF3

What triggers hydrolysis of the peptide bond and tRNA molecule during translation termination?

The detachment of eRF1 and eRF3 from the stop codon

Experiments using __ \_\_\_ ____________ and ________________ demonstrated that GTP hydrolysis of eRF3 can *only* occur when eRF1 and the ribosome were present.

Thin-layer chromatography Phosphoimaging

The nucleus is studded with hundreds of _________ \_\_\_\_\_\_\_\_\_ \_\_\_\_\_\_\_\_\_\_\_\_\_\_, which are large assemblies of proteins, that form channels through the double phospholipid bilayer that makes up the nuclear membrane.

Nuclear pore complexes

Proteins less than ___ kDa can diffuse freely through the nuclear pore complex, but anything larger than ___ kDa must be "actively" transported.

40 kDa 40 kDa

The interior of the nuclear pore complex is composed of amino acids ____________ and _________ repeats, both of which are hydrophobic and impede the movement of hydrophilic materials through the pore.

Phenylalanine Glycine

Proteins to be imported into or exported out of the nucleus have ___________ \_\_\_\_\_\_\_\_\_\_\_ sequences

Consensus signal sequences

Proteins to reside within the nucleus bear _________ \_\_\_\_\_\_\_\_\_\_ \_\_\_\_\_\_\_\_\_\_\_\_.

Nuclear localization signals

Nuclear localization signals usually have a series of three or more _____ and _____ residues.

Lysine Arginine

What recognizes nuclear localization signals?

Importins

\_\_\_\_\_\_\_\_\_\_ are a family of proteins that bind cargo and shepherd these cargo through the nuclear pore complex, navigating the FG repeats inside the pore

Importins

Proteins and some RNAs to be exported from the nucleus have ________ \_\_\_\_\_\_\_\_ \_\_\_\_\_\_\_\_\_\_\_\_.

Nuclear export signals

Nuclear export signals were first discovered in which disease protein?

HIV protein reverse transcriptase

Nuclear export signals are usually high in which amino acid?

Leucine

Nuclear export signals are recognized and bound by \_\_\_\_\_\_\_\_.

Exportins

Importins bind cargo with nuclear localization signals and these importins diffuse through the nuclear pore complex. Once inside the nucleus, importins interact with the G-protein, \_\_\_\_, which is in its GTP bound state. Binding of the importin to the G-protein causes a conformational change in importin that decreases its affinity for its cargo. Importin and \_\_\_\_-GTP remain complexed together and diffuse ____ of the nucleus. Once in the cytosol, \_\_\_-GTP encounters its GAP, \_\_\_-GAP, and releases importin.

Ran-GTP Ran-GTP Out of Ran-GTP Ran-GAP

Exportins bind cargos with nuclear export signals as they simultaneously bind the G-protein, \_\_\_\_, in its GTP state. This triad diffuses out of the nucleus through the nuclear pore complex. Once in the cytosol, \_\_\_-GTP interacts with its GAP, \_\_\_-GAP, hydrolyzing its GTP to GDP and releasing exportin and its cargo.

Ran Ran-GTP Ran-GAP

Nuclear transport is a form of _________ transport because molecules are being moves _____ their concentration gradients

Active Against

Where does the energy come from to drive nuclear transport?

Only cargo proteins are moved against their concentration gradients; cargo transport is coupled with Ran movement, which moves down its concentration gradients

The directionality of nuclear transport is depended solely on the relative concentration gradients of ________ and ______ across the nuclear membrane

Ran-GTP Ran-GDP

Ran-GTP is higher inside or outside the nucleus?

Inside

Ran-GDP is higher inside or outside the nucleus?

Outside

Why is Ran-GTP higher *inside* the nucleus?

It's GEF, Ran-GEF, is bound to chromatin

Why is Ran-GDP higher *outside* the nucleus?

Because its GAP, Ran-GAP, exists in the cytosol

Ran-\_\_\_ leaves the nucleus by simple diffusion, moving down its concentration gradient. Ran-\_\_ enters the nucleus by simply diffusion, moving down its concentration gradient. As soon as Ran-\_\_\_ enters the nucleus, it encounters Ran-\_\_\_ and becomes Ran-\_\_\_, which then exits the nucleus by following its concentration gradient, where it encounters Ran-GAP and becomes Ran-\_\_\_. The cycle continues.

Ran-GTP leaves nucleus Ran-GDP enters nucleus Ran-GDP encounters Ran-GEF Ran-GTP Ran-GDP

The cell takes advantage of the relative concentrations of Ran by coupling the movement of other molecules against their concentration gradients. In the cytosol, ________ binds to cargo and moves down its own concentration gradient by entering the nucleus. Once in the nucleus, _____ binds Ran-\_\_\_, which triggers cargo release and returns ________ to the cytosol by going down Ran-\_\_\_'s concentration gradient.

Importin Importin Ran-GTP Importin Ran-GTP's

When Ran-GTP is hydrolyzed by its GAP in the cytosol, to what does Ran-GDP bind to return to the nucleus?

Nothing, Ran-GDP binds nothing

\_\_\_\_\_\_\_\_\_ binds Ran-\_\_\_ and then binds to its cargo protein; this three-protein complex diffuse out of the nucleus by moving down Ran-\_\_\_\_'s concentration gradient, enabling both exportin and cargo protein to move against their concentration gradients. Once in the cytosol, Ran-\_\_\_ facilitates the hydrolysis of GTP to GDP and everyone loses affinity for each other; exportin and Ran-GDP then move down their respective concentration gradients *unbound to one another*

Exportin Ran-GTP Ran-GAP

Microfilaments are composed of a single monomeric _____ called G-\_\_\_\_, where “G” stands for globular. These monomers can polymerize and form long filamentous structures called microfilaments

Actin G-actin

In the beginning of polymerization, the shift from G-actin to \_\_-actin is slow – called the “lag phase” or “nucleation” – but once a certain level of polymerization has occurred, a growth phase occurs in which the microfilament exponentially elongates until it eventually reachs an equilibrium phase, where the rate at which actin monomers are removed is the same at the rate at which they are added.

F-actin for "filamentous"

Actin microfilaments are _________ because G-actin is not a symmetrical protein. As the microfilament grows, one end of the filament looks different than the other end. By convention, there is the ____ end and a _____ end

Polarized Plus Minus

Under simple polymerization conditions, koff equals kon on each end, meaning that rate at which G-actin is added to a microfilament is the same as that of G-actin being removed actin. This is also known as keq or the ___________ \_\_\_\_\_\_\_\_\_\_\_\_\_\_.

Critical concentration

In reality, koff and kon is ________ at the plus end than the minus end.

Lower

Kon and koff are higher at which end of the actin microfilament?

The minus end

Polymerization and depolymerization of G-actin occurs more rapidly on the ____ end.

Plus end (lower koff and kon values)

Polymerizing G-actin is bound to \_\_\_\_. Some time after polymerization, ____ is hydrolyzed. Therefore, fast polymerization results in a ____ cap and slow polymerization results in a ___ cap.

ATP ATP Fast = ATP cap Slow = ADP cap

Nucleotides affect G-actin and F-actin binding. Actin-ATP has a __________ affinity for actin than does actin-ADP.

Higher

Because actin has a higher affinity for ATP, koff at the plus end becomes very small, and kon at the minus end becomes very small. As such, the __________ \_\_\_\_\_\_\_\_\_\_\_\_ at the plus end becomes smaller than that of the minus end, meaning that _____ G-actin is required for equilibrium to be established at the _____ end.

Critical concentration Less Plus

When free G-actin is very high, boths ends of the microfilament will \_\_\_\_\_\_\_\_\_\_\_\_\_. When free G-actin is very low, both ends of the microfilament will \_\_\_\_\_\_\_\_\_\_\_\_. When the amount of free G-actin is lower than the critical concentration of the minus end but higher at the positive end, _______________ occurs.

Polymerize Depolymerize Treadmilling

What is treadmilling?

The microfilament grows longer on the + end and becomes shorter on the – end; it looks as if the microfilament is crawling or moving through the cell but maintaining its length the whole time

What binding protein binds to and sequesters actin in the cytosol, thereby prevent microfilament growth?

Thymosin

What protein competes with thymosin for binding with actin, thereby stimulating rapid growth at the plus end of the microfilament?

Profilin

\_\_\_\_\_\_\_\_\_\_\_\_\_\_ inhibits actin polymerization; __________ stimulates actin polymerization

Thymosin Profilin

What protein nucleates G-actin monomers?

Formin

\_\_\_\_\_\_\_\_\_\_\_\_ nucleates G-actin monomers by holding the first couple of monomers together.

Formin

Which proteins are involved in later nucleation and branching of actin microfilaments?

Arp 2 and Arp 3

What does Arp stand for?

Actin-related proteins

\_\_\_\_\_\_\_\_\_\_ and _________ form a heterodimer complex that attaches along the side of microfilaments, triggering nucleation of a new microfilament and branching of microfilaments

Arp 2 and Arp 3

What protein binds to the plus end of the microfilament and stabilizes it, thereby preventing both polymerization and depolymerization at the plus end?

CapZ

\_\_\_\_\_\_\_\_\_\_\_\_\_ is a protein that binds to the plus end of a microfilament and stabilizes it, preventing both polymerization and depolymerization at the plus end

CapZ

What protein is an actin depolymerizing factor?

Cofilin

\_\_\_\_\_\_\_\_\_\_ binds F-actin laterally

Cofilin

Cofilin binds F-actin because it has a higher affinity for actin-\_\_\_\_.

Actin-GDP

How does cofilin prompt depolymerization?

Induces twisting in the actin microfilament that interferes with actin-actin binding

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ are composed closely spaced, parallel bundles of microfilaments and are commonly found in microvilli, or finger-like projections of the plasma membrane

Filipodium

Tight parallel bundles of microfilaments are associated with \_\_\_\_\_\_\_\_\_\_\_\_\_.

Filopodium

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ are formed by branched networking of microfilaments induced by Arp2/3 complexes, which create a “dendritic network” of microfilaments

Lamellipodium

In the ________ \_\_\_\_\_\_\_\_\_\_\_, microfilaments lay across each other with the help of proteins, and in this configuration, microfilaments change the consistency of the cytosol from fluid-like to gel-like

Cell cortex

\_\_\_\_\_\_\_\_\_\_\_\_ _____________ are formed from the anti-parallel bundling of microfilaments with spacing slightly larger than those found in filopodium. These bundles are called “contracile bundles” and are important for contractile movement

Stress fibers

Be familiar with microfilament assemblies

What protein assembles parallel microfilament bundles?

Fimbrin

What helps to space antiparallel actin microfilaments?

Alpha actinin

What is responsible for the cross-linking in the gel-like matrix of actin microfilaments?

Filamin

What causes nucleation sites and leads to branching of actin microfilaments?

Arp2/3 complex

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ cross links and holds parallel microfilament bundles together while _____________ spaces antiparallel bundles apart. ____________ is responsible for the gel-like consistency of microfilaments found in the cell cortex.

Fimbrin Alpha actinin Filamin

Actin-polymerization-dependent ___________ and firm attachment of lamellipodium at the leading edge of the cell move the cell forward and stretch the actin \_\_\_\_\_\_. _________ at the rear of the cell propels the body of the cell forward, thereby relaxing tension. New focal contacts are made at the front, and old ones are disassembled at the back.

Protrusion Cortex Contraction

What are keratocytes?

Cells with high levels of actin microfilaments that can migrate

In lamellipodia, ________ is generated at the leading endge by _________ \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_.

Tension Actin polymerization

What are integrins?

transmembrane proteins that bind to cytoskeletal components inside the cell and proteins in the extracellular matrix in surrounding tissues

What are focal adhesions?

Areas in which the cell membrane links to a substrate, mediated by integrins bound to the extracellular matrix and microfilaments

In the trailing edge of lamellipodium, antiparallel bundles are mediated by alpha-actinin and the motor protein __________ \_\_, which is arranged in a bipolar manner and contracts the actin bundle at the rear lamellipodium

Myosin II

\_\_\_\_\_\_\_\_\_ ___ bipolar filaments bind ___ filaments in the lamellipodial meshwork and cause network \_\_\_\_\_\_\_\_\_\_\_\_, required for retraction of the trailing edge of the moving cell.

Myosin II Actin microfilaments Contraction

Microfilament assembles are regulated by \_\_\_\_\_\_\_\_\_\_\_\_\_

G-proteins

Arp2/3, filamin, and myosin activity (decreased) are regulated by ____ while cofilin, actin bundle growth, myosin activity (increased), stress fiber and focal adhesion formation are regulated by \_\_\_

Rac Rho

What does Rac-GTP regulate?

Arp2/3 Filamin Decreased myosin activity

What does Rho-GTP regulate?

Cofilin Myosin activity (increased) Actin bundle growth Stress fiber formation Integrin clustering and focal adhesion formation

How does Rac-GTP exert its activity?

Via the WASp family of proteins

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ mediate Rac-GTP regulation

WASp (Wiscott-Aldrich Sundrome) proteins

Be familiar with this picture.

Rac dominates ______________ and protrusion while Rho dominates __________________ contraction

Polymerization Actin-myosin contraction

Microtubules are long, hollow cylinders made of the protein \_\_\_\_\_\_\_\_\_\_\_\_.

Tubulin

Tubulin exists in two primary forms: an ____ and a ___ form

Alpha and beta form

Alpha tubulin and beta tubulin form heterodimers that polymerize end-to-end and laterally to each other, thereby forming \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_.

Microtubules

Tubulin monomers can bind nucleotide \_\_\_\_\_. Alpha tubulin binds the nucleotide for stability. Beta tubulin binds the nucleotide for hydrolysis and movement

GTP

Actin binds \_\_\_\_\_\_. Tubulin binds \_\_\_.

ATP GTP

GTP can be hydrolyzed shortly after __________ polymerization. It is held by the \_\_\_\_-tubulin monomer. Hydrolysis weakens the monomer's conformation and the bond in the polymer. GDP bound proteins are ____ stable.

Tubulin Beta-tubulin Less

The basic kinetics observed with actin are conserved with \_\_\_\_\_\_\_\_\_: the presence of plus and minus ends, different rates, nucleotide caps (stabilize in GTP form, destabilize in GDP form)

Tubulin

What is dynamic instability?

The rapid depolymerization of a microtubule when a GTP cap is lost; this can be observed both in vitro and in vivo

Dynamic instability is sometimes called microtubule ______________ but can be _______ if the concentration of the GTP dimer becomes high enough to regrow microtubules

Catastrophe Rescued

Nucleation of microtubules occurs by the \_\_\_\_\_\_-tublin complex, which is composed of 7 copies of tubulin. The 14th tubulin sites atop the 1st tubulin, leading to an assembly of 13 protofilaments.

Gamma

Two copies of gamma-tubulin associate with accessory proteins to form the gamma-tubulin _____ \_\_\_\_\_ or gamma-TuSC. Seven copies of gamma-TuSC associate to form a ________ structure, resulting in 13 protofilaments in a microfilament

Small complex Spiral

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ ___________ \_\_\_\_\_\_\_\_\_\_\_\_ are areas of a high density of g-TuRC, which serves as the nucleation site in most cells. In most animal cells the MTOC is the centrosome

Microtubule organizing center (MTOC)

\_\_\_\_\_\_\_\_\_\_\_\_\_\_ exist in the centrosome surrounded by proteins called _______________ material

Centrioles Pericentriolar

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ are composed of ___ triplets of microtubules. They help to organize pericentriolar material and exist as _________ bodies at the base of cilia and flagella. Right before mitosis, they duplicate and the pairs separate to opposite side of the \_\_\_\_\_\_\_\_\_.

Centrioles 9 Basal bodies Nucleus

What protein forms the coiled-coil dimer of the centroiles?

SAS-6

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ elongate toward the periphery and position the \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Microtubules Centrosome

There are two categories of microtubule-associated proteins (MAPs): _____________ and \_\_-\_\_\_\_\_\_\_\_\_

Motor MAPs Non-motor MAPs

What does MAP stand for?

Microtubule-associated proteins

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ is a long, alpha helical protein that binds free tubulin as a tetramer and sequesters tubulin in the cytosol

Stathmin

What is statmin?

A free tubulin (tetramer) binding protein

\_\_\_\_\_\_\_\_\_\_\_\_ 13 can destabilize microtubules at the plus end and facilitate the separation of tubulin dimers. ____ \_\_\_\_ stabilizes tubulin dimers at the plus ends and prevents depolymerization

Kinesin 13 XMAP215

What protein can destabilize microtubules?

Kinesin13

What protein can stabilize microtubules?

XMAP215

\_\_\_\_\_\_\_\_\_\_ and __________ are bundling proteins

Tau MAP2

Tau bundling protein forms closely spaced ________________ bundles while ______ bundling protein forms more distantly spaced bundles

Microtubules MAP2

Non-motor MAPs include \_\_\_\_\_\_\_\_\_, kinesin13, XMAP215, \_\_\_\_\_\_\_\_\_\_, and MAP2

Stathmin Tau

What are the non-motor MAPs discussed in class?

Stathmin Kinesin13 XMAP215 Tau MAP2

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ are motor MAPs that move toward the plus end of microtubules. _____________ are motor proteins that move toward the minus end

Kinesins Dyneins

There are two types of dyneins: ____________ and _____________ or ciliary

Cytoplasmic dynein Axonemal dynein

The kinesin cycle is a ______ \_\_\_ _____ model of movement. ATP hydrolysis in the ____________ head is accompanied by ADP release/ATP binding by the ____________ head. Conformational change in ____ \_\_\_\_\_\_ causes a pivot, which moves the lagging head in front of the leading head. This is called the _______________ \_\_\_\_\_\_\_\_\_\_\_\_, equivalent to the length of one tubulin dimer

Foot over foot Lagging head Leading head Neck linker Power stroke

The dynein cycle is a \_\_\_\_\_\_\_\_\_\_\_-based model. Dynein consists of a _______ that binds microtubules, a planar ring head that contains the ________ domain, and a tail that binds other microtubules or \_\_\_\_\_\_\_. ATP hydrolysis allows the ________ to attach to microtubules, and the release of ADP and Pi leads to ____________ motion in the head and stalk, the ____________ \_\_\_\_\_\_\_\_\_\_\_\_. Binding of a new ATP molecule causes the __________ to release the microtubule. ATP hydrolysis leads to reversal of the rotational motion and allows stalk to reattach to microtubules

Rotational-based model Stalk Motor domain Cargo Stalk Rotational Power stroke Stalk

How does dynein traffic cargo?

Via connection to the dynactin complex

\_\_\_\_\_\_\_\_\_\_\_\_\_ is a large complex that includes components that bind weakly to microtubules, components that bind to \_\_\_\_\_\_\_\_, and components that form a small, actin-like filament made of the actin-related protein, \_\_\_\_\_\_. It is the differences in dynactin complexes that allow for different types of cargo to be trafficked

Dynactin Dynein Arp1

What allows for dynein to carry different types of cargo despite only one cytoplasmic dynein?

Dynactin complex

There are kinesins specific for each cargo but only _____ dynein

One

Movement from the cell body to the periphery is __________________ transport while movement away from the periphery toward the cell body is ______________ transport

Anterograde Retrograde

Kinesins and dyneins interact with microtubules (or tubulin); __________ interact with microfilaments (or actin)

Myosins

All myosins are plus end directed except for myosin \_\_\_

In the myosin cycle, ATP binds to the ______ domain. When ATP is hydrolyzed, this domain changes conformation, resulting in the ___________ extending and the angle between the _______ and tail increasing. Release of Pi by the _______ domain results in initial weak contact with actin. This initial attachment is immediately followed by a much stronger attachment as the motor domain releases \_\_\_\_\_\_\_\_\_, which induces a conformational change in the motor protein, reduces the angle between the head and tail, and thereby forms the _________ \_\_\_\_\_\_\_\_

Motor domain Head extending Angle between head and tail increases Motor domain ADP Power stroke

Type __ myosin is responsible for organelle and vesicle transport and has a larger step than other myosins

Type V myosin

Type ___ myosin is the major motor protein in skeletal muscle

Type II myosin

The _________________ is a microscope, single contractile unit that when activated contracts and shortens the length of the sarcomere

Sarcomere

The thick filaments of the sarcomere are composed of ______________ \_\_ chains. The thin filaments of the sarcomete are composed of _______ \_\_\_\_\_\_\_\_\_\_\_\_\_

Myosin II chains Actin microfilaments

In the sarcomere thick filaments are bound to an elastic protein called ______ that binds to the ends of thick filaments and to the __ \_\_\_\_\_, helping to hold the thick filaments in between the thin filaments

Titin Z disc

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ acts as a molecular spring with spring like domains that can unfold and refold, keeping the thick filaments poised in the middle of the sarcomere and allowing the muscle fiber to recover after being overstretched

Titin

The actin filament plus ends are anchored in the __ \_\_\_\_, which is built from _____ and alpha-actinin; the __ \_\_\_\_ caps the filaments and prevents depolymerization while holding them together in a regularly spaced bundle

Z disc CapZ Z disc

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ stretches from the Z disc toward the minus end of each thin filament. This protein modulates the length of each thin filament

Nebulin

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ caps the minus ends of the thin filaments; the __ \_\_\_\_ caps the plus ends of the thin filaments

Tropomodulin Z-disc

When activated, myosin motor proteins pull the sarcomere together, shortening the sarcomere, but not the length of the filaments, and pulling the ___ \_\_\_\_\_\_s closer together

Z lines

Skeletal muscle is composed of cylindrical units called \_\_\_\_\_\_\_\_\_\_\_\_, which contain lots of sarcomeres. Dark bands in this figure represent the dark bands of ____ \_\_\_\_\_\_\_\_\_\_\_\_. The lighter regions represent the ________ \_\_\_\_\_\_\_\_\_\_\_\_

Myofibrils Thick filaments Thin filaments

Be familiar with this figure

Every muscle cell has a neuromuscular junction into which it can release \_\_\_\_\_\_\_\_\_\_\_, a neurotransmitter which binds to _______ receptors of adjacent cells. These receptors are ____ channels; once bound by \_\_\_\_\_\_\_\_\_\_\_, these channels open, causing ___ to enter the cell and ___ to leave the cell, which results in \_\_\_\_\_\_\_\_\_\_\_\_\_\_. An action potential forms, spreading across the plasma membrane into the __ \_\_\_\_\_\_, invaginations of the plasma membrane, which surround myofibrils. As the action potential spreads into the __ \_\_\_\_, voltage-gated ___ channels open, allowing ___ to flood into the cytoplasm from the extracellular fluid. These ___ channels are attached to proteins embedded in the ___________ reticulum; when open, they pull at the attached protein, allowing even more ___ to flood into the cytoplasm. The increase in intracellular ____ activates the contraction of each myofibril

Acetylcholine Motor Ca2+ ion Acetylcholine Na+ K+ T-tubules T-tubules Ca2+ Ca2+ Ca2+ ion Sarcoplasmic Ca2+ Ca2+

The majority of calcium required for muscle contraction comes from the _______________ \_\_\_\_\_\_\_\_\_\_\_--

Sarcoplasmic reticulum

What protein blocks myosin binding sites on microfilaments?•

Tropomyosin

Tropomyosin blocks the myosin binding site in a _________ cell

Resting

When stimulated, the skeletal muscle cell releases Ca2+, which binds \_\_\_\_\_\_\_\_\_. This protein is bound to tropomyosin, and when bound to Ca2+, it pulls on tropomyosin, uncovering the myosin binding site on the thin filament.

Troponin

Be familiar with smooth muscle cells

Contraction of the _____________ layer of smooth muscle causes the lumen to shrink

Circular layer

In the _________ muscle cells, the thin actin filaments are poorly organized and interspersed. So too are motor proteins. For contraction to occur, myosin must organize into a thicker filament. To do so, myosin must be activate or ____________ by ____ \_\_\_\_ ____ \_\_\_\_\_\_\_, which stretches the myosin chains, which can intersperse between the thin filaments and pull on them

Smooth Phosphorylated Myosin light chain kinase

Smooth muscle cells crunch up because the actin filaments are dispersed throughout the cell and connect to the plasma membrane; “\_\_\_\_\_\_\_\_\_\_\_\_\_” then stimulates the cell pulling in on itself

Contraction

In skeletal muscle, calcium binds \_\_\_\_\_\_\_\_, activating the sacromere. In smooth muscle, calcium binds ______________ that regulates ____ \_\_\_\_\_\_ _____ \_\_\_\_\_, which leads to "contraction"

Troponin Calmodulin Myosin light chain kinase

In smooth muscle cells, Ca2+ is released upon stimulation. This calcium binds to \_\_\_\_\_\_\_\_\_\_\_\_, which undergoes a conformational change and binds ____ \_\_\_\_\_ _____ \_\_\_\_\_\_. Now activated, it can phosphorylate the ____ \_\_\_\_\_ \_\_\_\_\_, allowing for myosin chains to form thick filaments and "contract" smooth muscle cells

Calmodulin Myosin light chain kinase Myosin light chain

There are two forms of ER protein import: ___ \_\_\_\_\_\_\_\_\_\_ and ____ \_\_\_\_\_\_\_\_\_

Co-translation ER import Post-translation ER import

What is the signal hypothesis?

Proposed by Blobel, the hypothesis proposes that proteins destined for secretion, which involves the movement of the protein across a biological membrane, are originally manufactured with an initial sequence of amino acids that may or may not present in the mature protein

The ER signal sequence is located at the __ terminus and is composed of between 20 and 30 amino acids. It is a basic region (\_\_\_\_\_\_\_\_ or \_\_\_\_\_\_) adjacent to a hydrophobic region

N-terminus Arginine or lysine

The ___________ \_\_\_\_\_\_\_\_\_\_\_ _________ is a cytosolic protein that recognizes and binds to the signal sequence once the polypeptide has started translation, and once bound it interacts with the ribosome to halt translation. It binds with the ____ \_\_\_\_\_\_\_\_\_\_\_ on the ER membrane and directs the \_\_\_-\_\_\_\_\_\_\_ complex to the \_\_\_\_\_\_\_\_\_\_\_

Signal recognition particle (SRP) SRP receptor SRP-ribosome complex Translocator

Be familiar with the mechanism of the SRP receptor

Most polypeptides enter the ER via \_\_\_-translational import

Co-translational

The ____________ is a water-filled channel in the ER membrane through which the polypeptide chain passes. The channel is gated by a short ____ \_\_\_\_ that is thought to function as a pluh. The core of the translocon is called the _______ complex, which is composed of three subunits. When the ribosome binds, a conformational change occurs that causes the plug to move. The signal sequence enters first and is pushed to the side by the plug, keeping the other two alpha helical subunits from reforming the seam. The _____ complex contains a ______ \_\_\_\_\_\_\_\_, which cleaves the signal, and _________ \_\_\_\_\_\_\_\_\_\_, which adds carbohydrate chains to asparagine residues via N-linked glycosylation as the polypeptide is translated

Translocon Alpha helix Sec61 complex Sec61 complex signal peptidase oligosaccharyl transferase

What is a polyribosome?

a complex of an mRNA molecule and two or more ribosomes that act to translate mRNA instructions into polypeptides

A single pass transmembrane protein has an N-terminal signal sequence followed by a stop-transfer sequence, resulting in the N-terminus _________ the ___________ and the C-terminus in the \_\_\_\_\_\_\_\_.

Inside the ER lumen Cytosol

In a single pass transmembrnae protein, the ______________ of the positively charged amino acids determines whether the N-terminus or C-terminus will be in the cytosol

Orientation

The presence of signal anchor will result in the N-terminus in the _________ and the C-terminus in the \_\_\_\_\_\_\_\_\_\_\_\_

Cytosol ER lumen

The presence of a reverse signal anchor will result in the N-terminus in the __________ and the C-terminus in the \_\_\_\_\_\_\_\_\_\_

ER lumen Cytosol

When there is both a signal anchor and a stop anchor, both ends of the polypeptide will reside in the \_\_\_\_\_\_\_\_\_\_

Cytosol

A ______________ can be used to determine which segments of a polypeptide will embed in the membrane

Hydropathy

Some transmembrane proteins are anchored into the membrane at the carboxy terminus and these proteins are inserted post-translationally. The pre-targeting complex binds to the carboxy terminus and captures the protein so that it can facilitate binding with _____ ATPase, which the protein to the ER membrane, where it will interact with the \_\_\_\_-\_\_\_ complex. Hydrolysis of ATP results in the carboxy tail's insertion into the membrane

Get3 ATPase Get1-Get2 complex

What are the 5 functions of the ER discussed?

1. Disulfide bond formation 2. Proteolytic cleavage 3. GPI anchoring 4. N-linked glycosylation 5. Protein folding

What enzyme catalyzes the proper formation of disulfide bond?

Disulfide isomerase

Why can disulfide bonds form in the ER lumen but not the cytosol?

The ER lumen is not as reduced as the cytosol

Protein disulfide isomerases have a sulfide group that facilitates oxidation of sulfhydryl groups on _________ residues

Cysteine

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ (GPI) is a lipid with multiple carbohydrate groups that is used as an anchor for proteins that have a carboxy tail embedded within the ER membrane

Glycosylphosphatidylinositol

Glycosylphosphatidylinositol (GPI) anchors are inserted inside the ER membrane so they will be present on the ________ of the vesicle. They are important in cell-cell reognization and _____ \_\_\_\_\_\_

Outside Lipid rafts

N-linked glycosylation refers to the addition of a carbohydrate group to the nitrogen atom of an asparagine residue. The core oligosaccharide is composed of (1) N-acetylglucosamine (2) _______ and (3) \_\_\_\_\_\_\_\_\_\_\_. N-linked glycosylation is facilitated by \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_, part of the translocon complex

Mannose Glucose oligosaccharyl transferase

Oligosaccharide addition begins in the ________ where sugars are added to a lipid anchor known as \_\_\_\_\_\_\_\_\_\_\_\_. It is to this lipid anchor that the core oligosaccharide is added

Cytosol Dolichol

The oligosaccharide core continues to be processed in the ER as sugars are removed by two glycosidases (\_\_\_ and \_\_\_) that remove glucose residues and one ___________ that removes mannose residue

Glycosidase I Glycosidase II Mannosidase

The ER is full of molecular chaperones that help proteins folder properly. These include the ________ \_\_\_\_\_ \_\_\_\_\_\_\_, including BiP (\_\_\_\_\_\_\_ \_\_\_\_\_\_\_\_). The ER also has many \_\_\_\_\_\_\_\_\_, which recognize and bind carbohydrate groups: __________ is located in the ER transmembrane and _______ is in the ER lumen as it is soluble

Heat shock proteins Binding Protein Lectins Calnexin Calreticulin

The length of time a protein stays in the ER depends largely on its proper folding. If it moves slowly, it becomes a target for \_\_\_\_\_\_\_\_\_\_\_\_; the removal of these carbohydrate monomers makes the protein a target for the ______________ \_\_\_\_\_\_\_\_\_, which binds proteins lacking _________ residues and moves them into the cytosol. Once in the cytosol, ___ \_\_\_\_\_\_\_\_\_ ________ interact with the protein and ______ it; if polyubiquinated via \_\_\_\_\_, it is designated for degradation by the proteasome

Mannosidases Retro-translocation complex Mannose E3 ubiquitin ligase Ubiquinates K48

There are three parallel intracellular pathways in which the cell responds to UPR (unfolded protein response): \_\_\_\_\_\_\_\_\_\_\_, \_\_\_\_\_\_\_, and \_\_\_\_\_\_\_\_.

PERK/PEK IRE1 ATF6 (activating transcription factor 6)

\_\_\_\_\_\_\_\_ is held as a monomer by BiP; the presence of unfolded or misfolded proteins in the ER results in BiP detachment. Once free, ______ dimerizes and crossphosphorylates, activating its ___________ domain, which identifies a pre-mRNA to cleave and removes an intron. This pre-mRNA is put back together, is used to translate a transcription regulator, and then enters the nucleus and upregulates expression of ER \_\_\_\_\_\_\_\_

IRE1 IRE1 Ribonuclease domain Chaperones

\_\_\_\_\_\_\_\_\_ is bound to BiP; in response to unfolded proteins, BiP releases \_\_\_\_\_\_\_\_, allowing for its dimerization and crossphosphorylation. It functions as an eIF\_\_ kinase, phosphorylating eIF\_\_, and shutting down translation. This also stimulates the selective translation of transcription factors that increase expression of ER \_\_\_\_\_\_\_\_

PERK PERK eIF2 kinase eIF2 ER chaperones

\_\_\_\_\_\_\_\_\_\_ is a membrane-anchored protein in the ER that cannot function as a transcription factor in the ER. Normally it is bound on its lumenal side to BiP. When BiP becomes busy, ____ is targeted for consolidation into vesicles destined for the \_\_\_\_\_\_\_. Here _______ encounters a _______ that cleaves \_\_\_\_\_\_\_, activating it and enabling it to diffuse into the nucleus and ________ \_\_\_\_\_\_\_\_ expression

ATF6 (activating factor 6) ATF6 Golgi ATF6 Protease ATF6 Upregulate chaperone expression

How does PERK upregulate translation of molecular chaperones?

Via translation of ATF4 (activating transcription factor 4)

Under conditions of ___ \_\_\_\_\_, eIF2-GTP is plentiful. This allows for upstream opening reading frames 1 and 2 of the ATF4 gene to be processively translated one after the other. uOPR2 overlaps with the ATF4 gene, thereby ____________ its translation. Under conditions of stress, eIF2-GTP is ____ because of \_\_\_\_\_. This causes a temporary delay in the ribosome reaquiring eIF2-GTP, causing it to skip translation of uORF2 and to instead translate ATF4 gene

No stress Inhibiting Low PERK

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ are synthesized on cytosolic face of the smooth ER and redistributed randomly by \_\_\_\_\_\_\_\_\_\_\_\_. The smooth ER is also responsible for synthesis of _____________ and ________ \_\_\_\_\_\_\_\_\_\_\_\_

Phospholipids Scramblases Cholesterol Steroid hormones

\_\_\_\_\_\_\_\_\_\_ cells often have high amounts of ____ ER because their primary job is detoxification. Detoxification in the liver is composed of two phases. Phase I reactions are the \_\_\_\_\_\_\_\_\_\_\_, which are catalyzed by ________ \_\_\_\_\_. This transforms toxic molecules into less toxic ones. Phase II reactions are the \_\_\_\_\_\_\_\_\_, where toxins are added to other molecules that can be excreted from the body. These molecules include glucoronic acid, \_\_\_\_\_\_\_, \_\_\_\_\_\_, and glutathione

Liver Smooth ER Oxidations Cytochrome p450 Conjugations Sulfate Acetyl

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ __________ is accomplished by the presence of different proteins and phospholipids

Membrane identity

Different species of _________________ are produced by specific kinases and phosphatases

Phosphatidylinositol

Phosphoatidyinositol-4-phosphate is enriched in ER ____ sites, the Golgi, and \_\_\_\_-derived vesicles

ER exit sites Golgi-derived vesicles

Phosphatidyinositol-4,5-bisphosphate is enriched at sites of _________ at the plasma membrane and is produced by phosphotidylinositol-4-phosphate \_\_-\_\_\_\_\_\_. Phosphpatidyinositol-4-5-bisphopshate is important in signal transduction, too

Endocytosis PI4P-5-kinase

Phosphatidylinositol-3-phosphate is enriched in _____ \_\_\_\_\_\_ and is propduced by phosphorylation by phosphatidylinositol-3-\_\_\_\_\_\_\_\_

Early endosomes PI3-kinase

Phosphatidylinositol 3,5-bisphosphate is enriched in _____ \_\_\_\_\_\_ and is converted from PI3P by PI3P 5-kinase

late endosomes

Phosphatidylinositol 3,4,5-trisphosphate is important in _______________ and is produced by PI(3,4)P2 3-kinase3

Phagocytosis

PI(4)P is enriched at ER exit sites, the Golgi, and Golgi vesicles PI(4,5)P2 is enriched at sites of \_\_\_\_\_\_\_\_\_\_\_ PI(3)P is enriched in _________ \_\_\_\_\_\_\_\_\_ PI(3,5)P2 is enriched in _______ \_\_\_\_\_\_\_\_\_ PI(3,4,5)P3 is important in \_\_\_\_\_\_\_\_\_\_

Endocytosis Early endosomes Late endosomes Phagocytosis

What three things are required for basic vesicle formation?

1. Cargo and cargo receptors 2. Adaptor proteins 3. Proteins that induce or stabilize membrane curvature ("coat" proteins)

When receptors bind cargo, conformational changes occur that produce binding sites for ___________ \_\_\_\_\_\_\_\_ on the their cytosolic tails

Adaptor proteins

G-proteins interact with adaptor proteins, coat proteins, and other effectors to facilitate coat formation. These G-proteins include Sar1, Arf1, Arfs2-6, and \_\_\_7

Rab7

\_\_\_\_\_\_ facilitates COPII assembly at the ER \_\_\_\_ facilitates COPI and clathrin assembly at the \_\_\_\_ Arfs2-6 facilitate clathrin assembly at most other compartments \_\_\_\_ facilitates retromer coat assembly at \_\_\_\_

Sar1 Arf1 Golgi Rab7 Endosomes

The GEFs for these coat proteins are located in the ________ membranes

Donor

\_\_\_\_\_\_\_\_\_\_ or GDP-G-proteins are \_\_\_\_\_\_\_\_\_\_

Inactive Soluble

When Sar1-GDP ecounters its GEF ____ in the ER membrane, a conformational change occurs that exposes the _____________ \_\_\_\_ _____ that enables Sar-GTP to associate with the ER membrane

Sec12 Amiphathic alpha helix

Arf1-GDP encounters its GEF in the \_\_\_\_\_; the exchange of GDP for GTP exposes its _______ anchor, allowing Arf1-GTP to become associated with the _____ membrane

Golgi Myristate Golgi membrane

Adaptor proteins interact with mutiple binding proteins. The major adaptor protein at the ER is ____ \_\_\_\_\_; the major adaptor proteins at the trans-Golgi network are ____ and GGA1-3; and the major adaptor proteins at the plasma membrane include \_\_2, ARH, beta-arrestin, epsin, and many more

Sec23/24 Adaptor protein 1 (AP1) AP2 (adaptor protein 2)

What is coincidence detection?

The requirement that adaptor proteins bind both the phosphatidyinositol and the receptor

What are the four major coat proteins discussed in class?

COPII COPI ("coatomer") Clathrin Retromer

What do coat proteins do?

Polymerize to form cage-like complexes that surround a vesicle and induce/stabilize membrane curvature

COPII proteins are distributed to the \_\_\_\_; COPI to the \_\_-\_\_\_ \_\_\_\_\_\_; clathrin to the _______ \_\_\_\_\_\_\_ and the trans-Golgi network; and retromer to \_\_\_\_\_\_\_

ER Intra-Golgi network (within the Golgi) Plasma membrane Endosomees

\_\_\_\_\_\_\_\_\_\_\_\_ binds to adapter proteins to form coated patches on the cytosolic face of the membrane

Clathrin

Coated _______ lead to coated pits which lead to coated \_\_\_\_\_\_

Patches Vesicles

Many copies of a single clathrin molecule polymerize to form \_\_\_\_\_\_-\_\_\_\_\_\_\_ structures, which are very common features in nature

Cage-like structures

Regions of curved membrane that can be positive or negative curvature. Positive curvature is \_\_\_\_\_\_\_. Negative curvature is \_\_\_\_\_\_. These terms are used in relation to the cytosol

Convex Concave

What are the 5 steps in generation of membrane curvature?

1. Modification of phospholipid composition 2. Insertion of amphipathic a-helices 3. Scaffolding by peripheral membrane proteins 4. Cytoskeletal changes 5. Insertion of integral membrane proteins

Negative curvature is generated by the ___________ of a head group or the addition of a _____ \_\_\_\_. Positive curvature is generated by enlargement of a ______ \_\_\_\_\_\_\_ or _________ of a fatty acid

Removal of head group Addition of fatty acid Enlargement of head group Removal of fatty acid

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ may transfer phospholipids to generate curvature

Flippases

Phospho\_\_\_\_\_\_\_ alter phospholipids

Phospholipases

Peripheral proteins use ___________ amino acids to directly bind negatively charged phospholipid head groups, thereby inducing curvature. The largest group of these proteins are the \_\_\_-\_\_\_\_\_\_\_ ____________ proteins

Cationic Bar-domain-containing proteins

\_\_\_\_\_ _____ proteins provide a range of degrees of curvature, and some induce tubulation of the membrane

BAR domain proteins

Cytoskeletal changes to the ____ cytoskeleton can push outward on the membrane, help constrict the neck region, and use _____ \_\_ to pull on the membrane - all to assist in membrane curvature

Actin cytoskeleton Myosin I

Some coat proteins, notably ____ \_\_\_\_\_ \_\_\_\_\_, recurit _________ promoting factors for actin

BAR domain proteins Nucleation promoting factors

The best studied fission of vesicles is through \_\_\_\_\_\_\_\_. In \_\_\_\_\_\_\_-coated vesicles, it self-assembles into stacked helical rings around the vesicle neck. It functions as a _______________ enzyme, stimulating GTP hydrolysis that causes a conformational change that ultimately leads to neck constriction

Dynamin Clathrin-coated Mechanochemical enzyme

Fission of COPI and COPII-coated vesicles are believed to be the result of _____ and ____ working like dynamin

Arf1 Sar1

In most cases, vesicles uncoat rapidly after initial formation. In COPI and clathrin vesicles, coat disassembly follows GTP hydrolysis of \_\_\_\_. In COPII vesicles, an adaptor protein ____ is the GAP for \_\_\_\_

Arf1 Sec23 Sar1

Transport of vesicles requires ____ GTPases

Rab

Newly synthesized Rabs are bound by ____ \_\_\_\_ \_\_\_\_\_, which facilitates interaction between Rab and geranylgeranyl transferases that add isoprenyl lipid anchors

Rab escort protein

Geranylgeranyl transferases adds to isoprenyl lipid anchors to \_\_\_\_, and these isoprenylated ____ are bound in the cytosol by _____ \_\_\_\_\_\_\_\_\_\_\_\_ ________ \_\_\_\_\_\_\_, concealing the lipid anchors in the cytosol

Rab Rabs Gunanine nucleotide dissociation inhibitor (GDI)

What does GDI stand for?

Gunanine nucleotide dissociation inhibitor

\_\_\_\_\_\_\_\_ _________ \_\_\_\_\_\_\_ allows Rab-\_\_\_ to release _____ and insert its lipid anchor into the membrane. Rabs manipulate different events of vesicle trafficking by activating specific effector proteins

GDI displacement factor (GDF) Rab-GDP Gunanine nucleotide dissociation inhibitor (GDI)

Transport of vesicles occurs via microfilaments or microtubules. Rabs bind _____ proteins directly or indirectly

Motor

\_\_\_\_\_ directly binds Rabkinesin-6, an effector protein, to facilitate ________ intra-Golgi transport (from cis to medial to trans Golgi)

Rab6 Anterograde

\_\_\_\_\_ indirectly binds a dynein to transport late endosomes to lysosomes via \_\_\_\_-\_\_\_\_\_\_\_\_\_ ______ \_\_\_\_\_

Rab7 Rab-interacing lysosomal protein (RILP)

\_\_\_\_\_ indirectly binds myosin __ \_\_ through Rab11-FIP2 to facilitate recycling of receptors from the recycling endosomes to the plasma membrane

Rab11 Myosin Vb

\_\_\_\_\_ indirectly binds myosin Va via _______ to transport melanosomes to the plasma membrane

Rab27 Melanophilin

\_\_\_\_\_ directly binds motor proteins whereas \_\_\_\_, Rab11, and ____ indirectly bind motor proteins

Rab6 Rab7 Rab27

Rabs regulate vesicle targeting and thethering by binding to ____________ \_\_\_\_\_\_\_\_

Tethering proteins

In addition to Rabs, tethering proteins may also bind coat proteins, phospholipids, \_\_\_\_\_\_\_\_\_\_\_

SNAREs

There are two types of tethering proteins: long _____ \_\_\_ proteins and \_\_\_\_\_-\_\_\_\_\_ ______ \_\_\_\_\_\_, which mostly interact with SNAREs and promote vesicle docking

Coiled-coil proteins Multi-subunit tethering complexes (MTCs)

What does MTC stand for?

Multisubunit tethering complex

\_\_\_\_ a coiled-coil tether tethers __________ vesicles to early \_\_\_\_\_\_\_\_\_\_

EEA1 Endocytosed vesicles Early endosomes

\_\_\_\_\_\_\_\_\_\_\_\_ are the most numerous type of coiled-coil tethers

Golgins

Golgins are found in the \_\_\_\_\_\_\_\_-

Golgi

Different tethering ___________ on Golgins selectively bind vesicles from different donor membranes

Motifs

The best characterized tethering motif is the ______ motif on GMAP-210

ALPS (amphiphathic lipid packing sensor)

What does ALPS mean?

Amphipathic lipid packing sensor

What does the ALPS tethering motif do?

Binds vesicles based on size, lipid composition, and lipid packing

Vesicles may navigate ffrom one Rab bindg site to another down the length of a \_\_\_\_\_\_\_\_\_. Rab binding may induce conformational changes in the \_\_\_\_\_\_, causing the vesicle to move closer to the membrane. Rabs may also allow for multi-subunit tethering complexes to form

Golgin Golgin

Multi subunit tethering complexes in the Golgi include \_\_\_\_-\_\_\_\_\_\_\_ _______ \_\_\_\_\_\_\_ (GARP) and _______ \_\_\_\_\_ _____ (COG), where Lobe A is bound to target membrane and _____ \_ is bound to vesicle

Golgi-associated retrograde protein (GARP) Conserved Oligomeric Golgi (COG) Lobe B

The ‘spidery’ arms of the ______ and ________ complexes are speculated to facilitate the transition from tethering to docking as they promote the pairing of the cognate SNAREs

COG (conserved oligomeric golgin) GARP (Golgi-associate retrograde protein)

The _____________ tethers Golgi-derived vesciles at the plasma membrane

Exocyst

Docking occurs from direct binding between cognate SNAREs in each membrane. Vesicles hold \_\_-SNAREs and target membranes hold \_\_-SNAREs

V-SNAREs T-SNAREs

v-SNARES is a generic term that stands for ____ \_\_\_\_ _____ \_\_\_\_\_\_ (VAMPs)

Vesicle-associated membrane proteins or R-SNAREs

t-SNARES are ______________ and \_\_\_\_\_\_\_\_-\_\_\_

Syntaxins (STX) SNAP-25

v-SNAREs are also known a \_\_-SNAREs and t-SNAREs are also known as \_\_-SNAREs

Q R

SNARE interactions occur by what?

Zippering

SNARE zippering proceeds from _____ terminus to ____ terminus to produce a _____ helical bundle of v-SNARE, \_\_\_\_\_\_, and SNAP-25 (two)

N terminus C terminus Four Syntaxin

The force provided by _____________________ is necessary to push the membranes together

Zippering

SNARE motifs are composed of helices that are hydrophobic except in one location: in the ______ of the helix, there are polar and/or charged amino acids interacting with each other. Q-SNAREs have __________ and R-SNAREs have \_\_\_\_\_\_\_\_\_\_. These is an energetically _______ structure that drives the __________ process

Middle Glutamine Arginine Favorable Fusion

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ mediate membrane fusion

SNAREpins

SNARE pairs dissociate with the help of _____________ \_\_\_\_\_\_\_\_\_ _______ \_\_\_\_\_\_\_ (NSF) and _________ \_\_\_\_\_ ________ \_\_\_\_\_\_\_ (SNAP), an accessorry protein

N-ethylamide sensitive fusion protein (NSF) Soluble NSF-attachment protein (SNAP)

SNARE stands for

SNAP receptor

NSF and SNAP use the power of ____ to dissociate SNARE cognates

N-ethylmaleamide sensitive fusion protein Soluble NSF attachment protein ATP

NSF forms a \_\_\_\_\_\_\_\_\_\_\_, each monomer of which consists of an N-terminal domain that interacts with SNAP and SNAREs and two _______ domains. ATP hydrolysis induces a conformational change that disassembles the SNARE complex

N-ethylmaleamide sensitive fusion protein (NSF) Homohexamer ATPase

There are three current models for SNARE disassembly: distributive, \_\_\_\_\_\_\_\_\_\_, and \_\_\_\_\_\_\_\_\_\_\_

Distributive Processive Global

\_\_\_\_\_\_\_\_\_\_\_\_ coated vesicles form at ER exit sites. ____ is recruited and activated by its GEF ____ in the ER membrane and ultimately binds Sec23 of the \_\_\_\_\_/\_\_\_\_ heterodimer. Cargo receptors bind proteins with a di-acidic region, and ______ of the \_\_\_\_\_/\_\_\_\_ complex binds v-SNARE proteins

COPII Sar1 Sec12 Sec23/Sec24 Sec24 Sec23/Sec24

The __________ heterodimer forms a concave surface with many basic amino acids acting as a scaffolding protein to induce or stabilize membrane curvature

Sec23/Sec24

Sar1 recruits ____________ heterodimer, which contributes further to curvature leading to the formation of a polyhedral cage

Sec13/Sec31

Sar1 activates ________________ \_\_\_ to produce the conical lipid phosphatidic acid (PA), contributing to _________ curvature and engaging in a positive feedback with Sar1

Phospholipase D Negative

Sar1 assembles induce ______________ of membrane

Tubulations

\_\_\_\_\_\_\_ plays a role in both COPII coat formation and vesicle fission

Sar1

\_\_\_\_\_\_\_ (of COPII coat, which Sar1-GTP bound originally) acts as the GAP for Sar1. Its GAP activity is greatly increased in the presence of \_\_\_\_

Sec23 Sec31

Sec23 binds the multi-subunit tethering complex _________ only after Sar1 has released from the vesicle. ________ subunit acts as GEF for \_\_\_\_\_, which activates PI(4)K to produce PI(4)P, which steadily increases in the membrane as the vesicle moves from the Golgi to the trans-Golgi network

TRAPPI A TRAPPI Rab1

What is ERGIC?

ER-Golgi intermediate comparment

How does ER-Golgi intermediate compartment (ERGIC) form?

From vesiculotubular clusters from homotypic fusion of COPII coated vesicles

COPII vesicles tether, uncoat, and fuse to the vesiculotubular clusters (VTCs). Rab1 recruited the coiled-coil tether _______ \_\_\_\_\_. This Golgin and _________ tether COPII vesicles together. Tethering accelerates uncoating and promotes SNARE pairing and fusion

Golgin p115 Golgin p115 and TRAPPI

COPII vesicles enter ERGIC but ______ vesicles leave ERGIC

ER-Golgi intermediate comparment

•COPI vesicles leave ERGIC p23/p24 heterodimer recruits Arf-GDP to the donor membrane * Rab1, PI(4)P, and p115 each provide binding sites for GBF-1 * GBF-1 (Arf-GEF) activates Arf, which detaches from p23/p24 * Arf-GTP, cargo receptors, and p23/p24 all recruit coatamer to form vesicles for both anterograde and retrograde transport

Learn if ya got time

Retrieval signal of membrane proteins is a di\_\_\_\_\_\_ motif or di\_\_\_\_\_\_\_ motif bound directly by COPI while the retrieval signal for soluble proteins is KDEL and bound by the KDEL receptor

Dilysine Diarginine Lysine-Aspartate-Glutamate-Leucine (KDEL)

Retrogrande COPI vesicles directly interacts with the ______ tethering complex at the ER

Dls1

Anterograde COPI vesicles' Golgin p115 binds to Golgin ______ at the cis-Golgi with help of TRAPPII

GM130

What are the functions of the Golgi?

There are two models for intra-Golgi network: the _________ \_\_\_\_\_\_\_\_\_\_\_\_ and the ___________ \_\_\_\_\_\_\_\_\_ models

Cisternal maturation model Vesicle transport model

In the ____________ \_\_\_\_\_\_\_\_\_\_\_\_ \_\_\_\_\_\_\_\_\_\_, cis face forms as vesicles from VTC arrive, cis cisterna matures to medial cisterna and then trans cisterna, enzymes are delivered by retrograde transport, and cargo proteins migrate forward with maturing cisternae

Cisternal maturation model

In the __________ \_\_\_\_\_\_\_\_\_ \_\_\_\_\_\_\_\_\_\_, proteins being modified move forward in vesicles by anterograde transport, and Golgi-resident enzymes are returned to their proper cisterna by retrograde transport

Vesicle transport model

COPI coated intra-Golgi vesicle transport is mediated by \_\_\_\_, Golgins, __________ (anterograde), and COG (retrograde)

Rabs TRAPII COG (conserved oligomeric Golgin)

What are the three main post-Golgi trafficking pathways?

In yeast, ____ defines the membrane through the Golgi and __ defines the membrane through the trans-Golgi and secretory vesicles. ____ defines mature secretory vesicles that bind the ________ tether at the plasma membrane

Ypt1 Ypt32 Sec4

Ypt1 recruits TRAPPII, the GEF for \_\_\_\_\_\_, which recruits Sec7, the GEF for Arf1; Gyp1, the GAP for Ypt1; and Sec2, the GEF for \_\_\_\_, which defines mature secretory vesicles

Ypt32 Sec4

Consistiutive secretion in years occus as secretory vesicles tether to the plasma membrane via an octameric multisubunit complex called the \_\_\_\_\_\_\_\_\_\_\_, which assembles in two subcomplexes, with components on both the secretory vesicles and on the plasma membrane. ______ binds members of the exocyst complex known as \_\_\_\_

Exocyst Sec4 Sec15

Docking and fusion of vesicles occur via Rho family members ____ and ____ on the plasma membrane, which recruit other exocyst members (Exo70 & Sec3). Exocyst subunit _____ interacts with a t-SNARE

Cdc42 RalA Sec6

Secretory vesicle maturation is a _________ feedback loop

Positive

Lysosomal enzymes are defined by the addition of \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Mannose-6-phosphate residues

At the trans-Golgi network, adaptor proteins sort cargo by the "tags." Those with mannose-6-phosphate are sent to the ________ and those with di\_\_\_\_\_\_\_\_ motifs are sent to the plasma membrane

Lysosome Dileucine

At the trans-Golgi network, ___________ assembles and ___________ assembles around the vesicle's neck and promotes fission

Clathrin Dynamin

Trans-Golgi network vesicles are direct by multiple Rabs to _____________ including ____ and Rab14, which recruit __________ for anterograde movement

Endosomes Rab6 Kinesins

At the trans-Golgi network, ______ binds the cytoplasmic tail of mannose-6-phosphate receptors. ___ then binds _____ and facilitates recycling of the receptors back to the Golgi

TIP47 Rab9 TIP47

Be familiar with the coats the are responsible for movement between organelles

ERGIC

Movement of COPII and COPI vesicles

Retrieval of ER proteins

Golgi function

Most Golgi cisternae are involved in oligosaccharide processing, specifically N-linked glycosylation

Addition of mannose-6-phosphate

Trafficking of lysosomal enzymes

There are two pathways for exocytosis. What are they?

Constitutive secretory pathway Regulated secretory pathway

\_\_\_\_\_\_\_\_\_\_\_\_ vesicles were used as an example of regulated secretion

GLUT4

What are GSVs?

GLUT4 sequestering vesicles, a population of vesicles with high levels of GLUT4 receptors

GLUT4 receptors wait in GSVs (GLUT4 sequestering vesicles) until an _______ signal is received. Once received, the GSV anchoring protein _____ is deactivated and activates a kinase that phosphorylates and deactivates AS160, the GAP for Rab10 (adipocytes) and Rab8 (muscle), and RGC2, the GAP for RalA. This shifts the equilibrium to more Rab10-GTP, Rab8-GTP, an RalA-GTP

Insulin TUG

Insulin also activates GEFs for ______ and Rac1, both of which are ___ family members that manipulate the cortical actin cytoskeleton

TC10 Rho

Once stimulated, GSVs are translocated to the plasma membrane. This is facilitated by Rab8 and _______ \_\_ __ in muscle cells and ______ and myosin Va in adipocytes. ____ also recruits the GEF for RalA, Rlf, which interacts with myosin Ic and the _______ complex

Myosin Vb Rab10 Rab10 Exocyst

\_\_\_\_\_\_ also binds the ____ to recruit the exocyst complex to the plasma membrane

TC10 Exo70

Know this damn figure

SM (Sec/Munc) proteins bind t-SNARE proteins and play a regulatory role in ________ and \_\_\_\_\_\_\_\_\_\_. t-SNARE complex consists of syntaxin-4 & SNAP-23, which are bound by two SM proteins: Munc18 and Synip – these proteins interact with syntaxin 4 and prevent SNAP-23 complexing

Docking and fusion

Insulin signaling leads to phosphorylation of ______ and \_\_\_\_\_. _____ releases syntaxin-4 and binds Doc2beta; ____ also releases syntaxin-4 and becomes soluble. This allows GSV v-SNAREs (VAMP-2) to interact with t-SNAREs

Munc18c Synip Munch18C Synip

\_\_\_\_\_\_\_\_\_\_\_ terminals are filled with neurotransmitter-filled vesicles, microtubules and microfilaments, and mitochondria. A small percentage of those vesicles are _______ at distinct sites of the plasma membrane called “\_\_\_\_\_\_\_\_ \_\_\_\_\_\_\_\_”. These docked vesicles are “\_\_\_\_\_\_\_\_\_” and may fuse with the membrane \<1 mSec after an action potential reached the synaptic terminal

Synaptic Docked Active zones Primed

The G-protein _____ is ultimately responsible for the formation of SNAREpins that __________ neurosecretory vesicles. It binds its effector protein RIM1 (Rab-interacting modulator 1) and activates its binding partner Munc13-1

Rab3 Prime

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ binds and stabilizes the primed SNAREpin and prevents premature fusion. When the action potential reactions the axon terminal, voltage-gated _____ ion channels open and _____ Ca2+ levels increases. Ca2+ binds \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_, which undergoes a conformational change and pushes ________ out of the way, thereby enabling primed vesicles to fuse within microseconds

Complexin Ca2+ Cytosolic Synaptotagmin Complexin

RIM (Rab-interacting modulator 1), RIM-BP, & Synaptotagmin all bind voltage-gated ______ channels

Calcium

Neurotransmitter release is a regulated secretory pathway

Clostridium botulinum and Clostridium tetanii produce toxins (botulism and tetanus) that impair vesicle fusion in axon terminals. Botulinum neurotoxins (BoNTs) act on _________ \_\_\_\_\_\_\_\_\_\_\_ \_\_\_\_\_\_\_\_\_\_\_. Tetanus neurotoxins (TeNTs) act on __________ in the __________ nervous system

Peripheral motor neurons Interneurons in the central nervous system

BoNT binds to a glycolipid, \_\_\_\_\_\_\_\_\_\_, on the cell surface and to a synaptic vesicle protein, and becomes internalized by endocytosis

Ganglioside

At lower pH the BoNT undergoes pronounced conformational changes that result in the heavy chains forming a channel through which the light chain protrudes into the cytosol. _________________ \_\_\_\_\_\_\_\_\_\_\_ in the cytosol reduces the disulfide bond to release the light chain as a soluble protein in cytosol

Thioredoxin reductase

BoNT and TeNT are \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_, which are specific for differnt fusion components and cut proteins at unique places

Metalloproteases

There are two endocytotic pathways: ___________ and \_\_\_\_\_\_\_\_\_\_\_

Clathrin-dependent endocytosis Clathrin-independent endocytosis

There are two types of clathrin-independent endocytosis: ____________ and \_\_\_\_\_\_\_\_\_\_

Caveolae-associated endocytosis Clathrin-independent/caveolin-independent cytosis

Macropinocytosis and ____________ are two remaining endocytotic pathways. The primary different between the types of endocytosis involves invaginations of the membrane. Endocytosis invaginates into the cytosol while in macropinocytosis and phagocytosis invaginations protude into the extracellular space

Phagocytosis

Endocytic pathways

Clathrin-dependent endocytosis is utilized for internalization of _____________ like LDL and transferrin and for down--regulation of receptors

Nutrients

In clathrin-dependent endocytosis (CDE), occupied receptors recruit AP-2 \_\_\_\_\_\_\_, which possess a ____ domain with physical curvature. AP-2 recruits Arf-6, and together they activate PI (4)P ________ that produces \_\_\_\_\_, which serves as the binding site for more ____ and \_\_\_\_\_\_\_. Eventually AP2 recruits the GEF for Rab5 \_\_\_\_\_\_, and clathrin is recruited to the area

Endophilin BAR domain AP2 Endophilin PI4(P) 5-kinase PI(4,5)P2 hRME6

In CDE, ____________ is recruited. It binds PI-(4,5)P2 negatively charged phospholipid head groups via a plekstrin homology (PH) domain. It also possesses a _____________ rich region that binds ____ domains on membrane proteins

Dynamin Proline-rich SH3 domains

In CDE, \_\_\_\_\_\_\_\_\_\_\_\_, a PI(4,5)P2 5-phosphatase, increases PI(4)P, which provides binding sites for important proteins like the GAP for Arf6. But most importantly it provides a binding site for \_\_\_\_\_\_\_\_\_, which is absolutely critical for vesicle uncoating. ____________ recruits Hsc70 (heat shock cognate 70) and together they depolymerase \_\_\_\_\_\_\_.

Synaptojanin Auxilin Clathrin

Endocytosed vesicles will begin to fuse with each other or pre-existing early endosomes. This is facilitated by EEA1, which is bounded by \_\_\_\_\_. EEA1 then binds ___________ to mediate docking between the endocytic vesicles and early endosomes

Rab5 Syntaxin6

\_\_\_\_\_\_\_\_\_\_\_\_\_ activates Rab5 on early endosomes. Active Rab5 recruits other proteins including \_\_\_\_\_\_\_, which recruits more \_\_\_\_\_\_, promoting a positive feedback loop

Rabex5 Rabaptin Rabex5

Rab5 recruits two different PI3 kinases to early endosomes and two other PI \_\_\_\_\_\_\_\_\_\_, resulting in increased \_\_\_\_, which is characteristic of early endosomes

PI phosphatases PI(3)P phosphatidylinotisol-3-phosphate

Rab5 ultimately leads to __________ remodeling in early endosomes

Membrane

Cargo is sorted in early endosomes (sorting endosomes). Cargo to be __________ is preferentially trafficked from early endosomes, to late endosomes, and then to lysosomes. Some cargo is sorted into tubulations (tubular projections of endosome) and __________ back to the plasma membrane

Degraded Recycled

Early endosomes mature to late endosomes. Rab5 on early endosomes recruits a MTC the ______ tethering complex. ______ on early endosomes recruits PI 5-kinase (PIKfyve), which produces PI(3,5)P2, characteristic of late endosomes. One of the _____ subunits is the GEF for \_\_\_\_, which recruits the GAP for \_\_\_\_, TBC1D1

HOPS PI(3)P HOPS Rab7 Rab5

Late endosomes mature to become lysosomes as the compartment becomes more \_\_\_\_\_\_\_, and the cell deactivates Rabs that defined the endosomes

Acidic

There are two recycling pathways in the cell: a fast and a slow. The fast pathway is mediated by \_\_\_\_\_-directed vesicles. The slow pathway is mediated by Rab11

Rab4

Decreased __________ signaling leads to increased clathrin-dependent endocytosis of \_\_\_\_\_\_\_\_\_. _______ directs glucose transporters (GLUT4) from endosomes into GLUT4 sequestering vesicles (GSVs) in adipocytes

Insulin GLUT4 Rab14

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ uses recycling endosomes

Transcytosis

What is transcytosis?

Transport of proteins across epithelial membranes

\_\_\_\_\_\_\_\_ on LDL particles bind to the LDL receptor. The cargo is delivered to sorting endosomes where the reduced pH causes ____ to dissociate from its receptor

ApoB100 LDL

People with __________ \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ often die in their early 20s because the LDL receptor is defective, so LDL builds up in the blood. It is an __________ \_\_\_\_\_\_\_ disorder

Familial hypercholesterolemia Autosomal recessive

Other people with high cholesterol usually have __________ hypercholesterolemia

Polygenic

LDL degradation

\_\_\_\_\_\_\_-\_\_\_\_\_ __ in endosomes binds LDL receptors via the NPxY motif, leading to LDL receptor recycling in ____ directed vesicles (rapid recycling)

Sorting-nexin 17 Rab4

There are two LDL receptor degradation mechanisms for targeting LDL receptors to the lysosome: ________ binding (predominate mechanism in the liver) and ___________ by _______ \_\_\_\_\_ _____ (IDOL) (predominant mechanism in macrophages, adrenal glands, and \_\_\_\_\_\_\_\_\_\_\_\_\_)

PCSK9 Ubiquination Inducible Degrader of LDL receptor (IDOL) Intestines

PCSK9 is a member of the _____________ \_\_\_\_\_\_\_\_\_\_ family, which binds LDL receptors extracellularly, leads to endocytosis, and delivers vesicles to sorting endosomes

Proprotein convertase family

Without a ________ conformation, _____________ directs LDL receptors to degradation

Closed PCSK9

\_\_\_\_\_\_\_\_\_\_\_\_\_\_ has become a target of cholesterol medications; binding of antagonists to __________ prevents its binding to the LDL receptor and the receptors' endocytosis, thereby enabling the body to bring more LDL into the cell

PCSK9 PCSK9

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ is upregulated in response to high intracellular cholesterol levels. It monoubiquinates LDL receptors, leading to endocytosis by a clathrin-independent/caveolin-independent pathway

IDOL a ubiquitin ligase

Ubiquinated receptors are engulfed into vesicles within endosome what are called __________________ \_\_\_\_\_\_\_\_ (MVBs)

Multivesicular bodies

Sequential _______ protein complexes recognize ubiquitin tags and ultimately lead to MVB formation

ESCRT (endosomal sorting complex required for transport)

ESCRT-I and ESCRT-II causes membrane _____________ leading to inward buds

Deformation

\_\_\_\_\_\_\_\_\_\_\_-\_\_\_\_ is responsible for fission

ESCRT-III

ESCRT 0 recognizes _______ tags

Ubiquitin

What does ESCRT stand for?

Endosomal sorting complex required for transport

MVBs are delivered to \_\_\_\_\_\_\_\_\_\_-

Lysosomes

MVBs may be secretes as \_\_\_\_\_\_\_\_\_\_\_-

Exosomes

Exosomes may release __________ that may affect gene expression in adjacent or distal cells

miRNAs

What does caveolae mean?

Little caves

What are caveolae?

* Highly stable membrane invaginations enriched in sphingolipids, cholesterol, GPI-anchored proteins, multiple signaling proteins * Coated with caveolins, which are anchored by multiple acyl chains

What functions are cavaeloe implicated in?

Lipid uptake/management, signaling, and endocytosis

Caveolae are membrane invaginations enriched in \_\_\_\_\_\_\_\_\_\_\_\_\_, cholesterol, GPI-anchored proteins, and other signaling proteins

Sphingolipids

Cavaeloe-associated endocytosis requires \_\_\_\_\_-\_\_\_\_\_\_ proteins, \_\_\_\_\_\_\_\_, and actin cytoskeletal rearrangment

BAR-domain bodies Dynamin

Clathrin/caveolin-independent endocytosis internalizes ubiquinated receptors associated with _______ rafts. Arf1 recruits/activates actin remodeling proteins like \_\_\_, \_\_\_, and WASp. There are both dynamin-independent and dynamin-dependent forms

Lipid rafts Rho cdc42

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ is responsible for the bulk uptake of fluids and membranes. It involves \_\_\_\_, Rac, and \_\_\_\_\_. Large __________________ vesicles acidify and fuse with late endosomes. It is also a mechanism by which dendritic cells "taste" their surroundings

Macropinocytosis Arf6 Cdc42 Macropinosome

What is the feeding mechanism for protists?

Phagocytosis

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ is the primary role of macrophages, neutrophils, and dendritic cells of mammalian immune system. The __________________ fuses with the lysosome leading to digestion. The process requires receptors which bind bacterial molecules or antibodies

Phagocytosis Phagosome

\_\_\_\_\_ receptors on phagocytes bind \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_, which bind antigens on bacteria and viruses. _______ \_\_\_\_\_\_\_\_ _________ (PRRs) on phacoytes bind \_\_\_\_\_\_\_\_\_\_\_\_-\_\_\_\_\_\_\_\_\_\_ __________ \_\_\_\_\_\_\_\_\_\_\_(PAMPs) on bacteria and viruses

Fc receptors Immunoglobulins Pattern Recognition Receptors (PRRs) Pathogen-associated molecular patterns (PAMPs)