Final exam Flashcards
What are the four classes of macromolecules?
Proteins, lipids, carbohydrates, nucleic acids
The ___________ structure of proteins consists of the protein’s amino acid sequence.
Primary
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
Super-secondary secondary structures are structures that form from secondary structures grouped in specific ways; they are also called _____________.
Motifs
A ___________________ is a similar 3D structure conserved among proteins that serve a similar function.
Motif
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)
___________________ structure occurs when a protein is composed of more than one polypeptide chain.
Quarternary
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
Why do disulfide bridge not exist in the cytosol?
They are highly oxidized, but the cytosol is highly reduced
How are proteins regulated?
Quantity, activity, location
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
What primarily mediates transcriptional control of gene expression?
Transcription factors
Trans-acting factors or ___________________ factors bind cis-regulatory elements and work to speed up or slow down transcription.
Transcription factors
Transcriptional _______________ bind to cis-regulatory sequences and turn genes off.
Repressors
Transcriptional ________________ bind to cis-regulatory sequences and turn genes on.
Activators
_________________ and ______________ bind to activators and repressors, not DNA, and assist in gene regulation.
Coactivators
Corepressors
What are two mechanisms through which transcriptional activators function?
Chromatin remodeling
RNA polymerase recruitment
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
What are four ways in which activators trigger chromatin remodeling?
Covalent histone modification
Nucleosome remodeling
Nucleosome removal
Histone replacement
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
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
Genes encoding the most important developmental regulatory proteins are kept tightly repressed or activated?
Repressed
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
____________________ of DNA usually increases transcription whereas _____________ of DNA usually decreases transcription.
Acetylation
Methylation
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.
Anterior
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.
5
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?
- The protein product of a mRNA performs regulatory action on its own mRNA
- A stem-loop structure can prevent movement of the ribosome
- A small molecule can bind stem-loop structure
- 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?
- •Proteolysis
- •Glycosylation
- •Phosphorylation
- •Acylation
- •Acetylation
- •Methylation
- •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?
- Formation of structural complexes
- Localization of a protein within the cell
- 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
•
Proteasomes
Where are proteasomes abudant?
Cytosol and nucleus
How do proteosomes work?
- Target proteins are ‘tagged’ with polyubiquitin
- The cap complex contains a ubiquitin receptor, a ubiquitin hydrolase, and unfoldases
- 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?
- eIF2-GTP binds tRNA Met and small ribosomal subunit, forming the 43S pre-initiation complex
- Multiple IFs bind mRNA
- 43S complex binds mRNA at 5’ end
- 43S complex scans in 5’ to 3’ direction
- 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?
- 60S ribosomal subunit binds along with eIF5B-GTP to form 80s initiation complex
- 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 ___
VI
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?
- Disulfide bond formation
- Proteolytic cleavage
- GPI anchoring
- N-linked glycosylation
- 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?
- Cargo and cargo receptors
- Adaptor proteins
- 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?
- Modification of phospholipid composition
- Insertion of amphipathic a-helices
- Scaffolding by peripheral membrane proteins
- Cytoskeletal changes
- 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)
