Lectures 20-37 Flashcards
What are the phases of protein development?
The burst phase (0-5ms), the intermediate phase (5-100ms) and the final rate limiting step.
What is the burst phase?
It involves formation of secondary structure and collapse of the hydrophobic core.
What is the intermediate phase?
It involves formation of a molten globule intermediate, which has characteristics of both folded and unfolded proteins.
What is the rate limiting step?
The attainment of native structure. The final transition is marked by conversion of the molten globule via global repacking of hydrophobic side chains and the association of domains that were folded independently in the intermediate stages.
What is a molecular chaperone?
Molecular chaperones are proteins that bind to and stabilize an otherwise unstable conformer of another protein, and facilitate its correct fate in vivo: be it folding, oligomeric assembly, transport to a particular subcellular compartment, or controlled switching between active and inactive conformations.
What is the structure of chaperonin?
Homo-oligomer of 14 subunits (each of 60 kDa) arranged into 2 stacked rings each of 7 subunits. Each ring is structured like a donut with a 7-fold axis and a chamber.
A smaller lid structure, also comprising 7 subunits, sits on top of one of the barrels. Unfolded proteins (orange) bind to the rim of the barrel and are displaced into the cavity by the lid structure. The protein can then fold in a sequestered and protected environment of the chamber. The lid dissociates due to changes in the conformation of the large subunit as ATP is hydrolyzed.
Discuss the function of heat schlock transcription factor.
Molecular chaperone gene transcription is controlled by Hsf which responds to the presence of unfolded protein or heat shock or other types of proteotoxic stress.
What degrades proteins?
Protein degradation in the cytosol and nucleus is largely accomplished by the proteasome, a large gated protease. Proteasome substrates are targeted via covalent linkage to multiple copies of ubiquitin, a 7 kDa protein.
What is the structure of the proteasome?
The proteasome comprises a central catalytic core (20S proteasome) and a regulatory cap (19S) that together are called the 26S proteasome. The 20S core of eukaryotes is comprised of 2 copies each of 14 different subunits, although these fall into two categories of α-type and ß-type. Access to the channel is via the tunnel formed at the ends of the α-subunit rings. It is thought that a single unfolded polypeptide transits into the proteasome at one end and is degraded progressively in the central chamber.
What are the different activities of proteasomes?
The three activities of eukaryotic proteasomes are chymotrypsin-like (cleaves after hydrophobic amino acids), trypsin-like (cleaves after basic amino acids), and peptidyl-glutamyl peptide hydrolyzing activity (cleaves after acidic amino acids). Two additional activities have been described in mammalian proteasomes (cleaving after branched chain amino acids and between small neutral amino acids). The products are peptides in the 7-9 amino acid range.
What does the 19S regulatory subunit do?
The 19S regulatory complex contains subunits that: recognize ubiquitinylated substrates, deubiquitinylate the substrates; and prepare them for proteolysis via protein unfolding if necessary. The 19S complex is comprised of at least 15 subunits, six of which are AAA-ATPases that can perform unfolding.
What does ubiquitin do?
Ubiquitin is a 76-amino-acid protein that becomes covalently attached to polypeptides
that are substrates for degradation. Ubiquitin is linked in linear chains where the carboxyl end of the terminal glycine becomes covalently attached to the epsilon amino group of lysine 48. The attachment of ubiquitin to proteins requires the action of three different types of enzymes called E1, E2 and E3.
What does E1 do?
E1 is an enzyme that carries out ATP-dependent activation of the C-terminal glycine in a two-step reaction. First a ubiquitin-adenylate is formed, followed by transfer of activated ubiquitin to a thiol site in E1. The high-energy thiol bond is important for Ub transfer to E2 enzymes. There are only a small number of E1 enzymes.
What does E2 do?
The E2 enzymes, or ubiquitin conjugating enzymes, accept the ubiquitin from E1 and transfer it to the protein substrate in a reaction that requires E3, the ubiquitin protein ligase.
What does E3 do?
E3 enzymes, or ubiquitin ligases, are a large and diverse protein family that specifies substrate selection.
Draw the ubiquitin/proteasome pathway.
See screenshot 8.
What does CHIP do?
It binds directly to Hsp70 and catalyzes ubiquitinylation of misfiled proteins. Imperative for quality control.
What are aggregates?
Aggregates occur when misfolded proteins overwhelm the ubiquitin/proteasome pathway. Aggregates represent amorphous assemblies of misfolded proteins bound together via hydrophobic interactions or ordered assemblies of amyloid fibres. Both types of aggregates are inaccessible to the proteasome and must be cleared by the autophagic system.
What is the aggregate in Alz and amyloid disease?
Amyloid beta extracellularly and hyperphosphorylated tau intracellularly.
What is the aggregate in Parkinson’s?
Lewy Bodies.
What are some polyglutamine repeat diseases?
Huntingtons, SMBA, DRPPLA etc. In some cases, the triplet encoding Q (CAG) expands during replication and reaches many more residues than it was supposed to have which can be pathogenic depending on the protein.
What are prion diseases?
Prions are transmissible amyloids and cause disease in humans and other
mammals. Transmission can occur by eating contaminated food. Human prion diseases include Creutzfeldt-Jakob disease and fatal familial insomnia. All known prion diseases attack the brain.
What does the ER do?
The Endoplasmic Reticulum is a protein folding and quality control compartment
What does the Golgi apparatus do?
Golgi Apparatus is important for sorting of proteins towards different parts of the cell.
What does the rough ER do?
The rough ER contains ribosomes and is important as a protein- folding compartment.
That does the smooth ER do?
The smooth ER contains membrane- bound enzymes important for lipid synthesis and metabolism, as well as detoxifying enzymes such as cytochrome p450s.
How are newly made proteins targeted to the ER?
Newly-made proteins are targeted to the ER membrane by an N-terminal signal
peptide. The signal peptide is 8-20 residues and enriched in hydrophobic amino acids, and is often cleaved after import into the ER. Some proteins have internal targeting sequences that are not cleaved after import. The signal peptide binds to the Signal Recognition Particle (SRP), a ribonucleoprotein complex that attaches to newly- synthesized proteins while they are still being translated.
What happens to the protein once SRP binds to the N-terminal signal peptide?
Once SRP binds to a signal peptide, translation is arrested and the complex of translating ribosome and SRP bind to the ER membrane via an SRP receptor complex. SRP receptor sits adjacent to the translocation channel called the Translocon. This binding is positioned so that
translation can resume in the context of translocation through the aqueous channel and into the lumen of the ER. For membrane proteins, this channel opens sideways, into the plane of the membrane, and membrane-spanning domains of proteins become inserted into the membrane itself.
How does the ER lumen differ from the cytosol?
The ER is an oxidizing environment and therefore distinct from the reducing
environment of the cytosol. The oxidizing environment helps to facilitate folding of proteins that must exist outside of the cell, which is also an oxidizing environment.
Proteins that enter the ER lumen use?
Fold in association with molecular chaperones, including an ER-specific form of Hsp70 and many others.
What important step for protein folding occurs in the ER lumen?
Proteins entering the ER are also glycosylated on asparagine (N- linked glycosylation) via a 14 residue unit that includes 3 residues of glucose, 9 residues of mannose and 2 residues of N-acetyl gluosoamine (GlcNac). These are transferred from a dolichol anchor to the substrate protein. The three glucose residues have important roles in protein folding.
What are the three types of membrane proteins?
Type I= N terminus in the ER lumen
Type II= C terminus in ER lumen
Or, they may be topologically complex and have multiple membrane-spanning domains comprising hydrophobic amino acids that form alpha helices.
What is the unfolded protein response (UPR)?
When the amount of unfolded proteins in the ER exceeds the chaperone apparatus to facilitate folding, the UPR signaling pathway is activated. Unfolded proteins activate receptors on the ER membrane that then activate transcription regulators. These travel into the nucleus of the cell and increase the transcription of chaperone molecule mRNA (same with ubiq/prot pathway stuff too). This increased mRNA output is transported back to the ER, is translated with the help of a ribosome and raises the amount of chaperones available to help fold protein in the ER.
What is a route other than UPR that allows the ER to avoid the aggregation of misfiled proteins?
Increased expression from the UPR is accompanied by ER-associated degradation (ERAD) where luminal and membrane proteins are retranslated from the ER to the cytosol for degradation by the proteasome.
What is the structure of the Golgi apparatus?
The Golgi apparatus comprises a stack of flattened membranous disks that have a distinct curved appearance. The curvature creates polarity with a ‘cis’ face and a ‘trans’ face at the concave end.
What is the function of the Golgi apparatus?
The Golgi receives all proteins that leave the ER. Once inside the Golgi, these proteins are modified by post-translation modification, such as trimming of carbohydrates, phosphorylation and sulfation. These modifications increase the complexity of the proteins traveling through the Golgi and also function to specify subsequent localization to other membrane systems.
How do proteins get from the ER to the Golgi?
Proteins exit the ER in lipid vesicles that bud from the ER membrane (the formation of vesicles will be described in more detail below). They are targeted to the Golgi where they fuse at the Cis side of the organelle.
What happens to ER proteins that accidentally get transported to the golgi.
They are transported back by a retrieval pathway. This pathway uses a receptor that recognizes a special peptide sequence (KDEL) at the C-terminus of resident ER proteins.
How are lysosomal enzymes recognized and sequestered for transport?
Lysosomal enzymes are identified in the cis-Golgi by an enzyme that phosphorylates on one of their mannose residues of the core carbohydrate unit that was added in the ER. This phosphorylation is recognized in the trans-Golgi by the mannose-6-phosphate receptor that helps sequester lysosomal enzymes into specific vesicles for transport to lysosomes.
Describe the process of endocytosis
Some species, let’s say in this case LDL, blinds to its receptor on the cell membrane. The membrane then pinches off forming a vesicle containing both the species and the receptor with a clathrin coat. The vesicle undergoes “uncoating” in the cytosol and then fuses with an endosome. The receptor then buds off from the endosome into a transport vesicle that is transported back to the membrane and the endosome transfers the LDL to a lysosome.
What is autophagy?
Autophagy is a mechanism of delivering intracellular components to the
lysosome for destruction and recycling. Organelles or protein aggregates are engulfed by a double membrane system for delivery to the lysosome. In some cases, molecular chaperones such as Hsp70 can directly deliver proteins to the lysosome for destruction.
What are the 3 types of coat proteins?
Clathrin, COPI, and COPII
Where do COPII-coated vesicles go?
COPII coated vesicles serve transport from the ER to the Golgi.
Where do clathrin coated vesicles go?
Clathrin coated vesicles serve transport from the trans-Golgi network to the plasma membrane and also to endosomes.
Where do COP-1 coated vesicles go?
They transport proteins from the cis end of the Golgi complex back to the rough endoplasmic reticulum (ER)
What is the name of the protein that uncoats clathrins?
Dynamin
What are rabs and what do they do?
Small GTP binding proteins that target vesicles to certain membranes using proof reading and interacting with specific tethering proteins on target membranes.
Snare proteins work on what two processes?
Vesicle targeting to membranes and vesicle fusion.
Why does vesicle fusion need help?
Fusion of a vesicle with a target membrane is energetically unfavorable because
the hydrophilic head groups and their associated water molecules represent a barrier to the mixing of hydrophobic hydrocarbons.
How do SNARE proteins work?
The energy barrier for vesicle fusion is overcome by interactions of specific SNARE proteins on the vesicle (v-SNARE) and target membrane (t-SNARE). The SNARE proteins interact with each other in such as way as to bring the vesicle into very close apposition to the target membrane, squeezing out water molecules and reducing the thermodynamic barriers to lipid mixing. Similar mechanisms are employed when viruses enter cells. SNARE proteins are disassembled after fusion with the help of a specific chaperone. They are then recycled to their respective membrane systems.
Describe the outer membrane of a mitochondria
The outer membrane is porous to molecules up to 5-10 kDa and contains a translocation apparatus called the TOM complex for translocation of proteins.
Describe the inner membrane of the mitochondria.
The inner membrane is 70% protein and impermeable even to protons. It is folded into many christae to increase surface area. The inner membrane contains the protein complexes of the electron transport chain and the ATP synthase complex that catalyzes formation of ATP from ADP. Movement of protons across the inner membrane creates a membrane potential. The inner membrane also contains several channel proteins for the translocation of metabolites (eg. pyruvate, malate, acyl-CoA, amino acids), ions and the ADP/ATP transporter. Finally, the inner membrane is also the site of a translocation complex for proteins entering the mitochondrial matrix.
What is in the inter membrane space?
An inter membrane space exists between the two membranes and contains enzymes that phosphorylate other nucleotides apart from ADP eg. nucleoside diphosphate kinase which converts GDP to GTPWhat is inside the mitochondr
What’s inside the mitochondrial matrix?
Hundreds of enzymes, including those required for oxidation of pyruvate, fatty acids, ketone bodies to acetyl-CoA. The matrix is also home to enzymes that catalyze amino acid oxidation and enzymes of the tricarboxylic acid cycle and urea cycle. The mitochondrial matrix is also contains the mitochondrial genome, ribosomes, tRNAs and molecular chaperones for folding of newly synthesized and newly imported proteins.
What is different about the codons in mitochondria?
The mitochondrial genome uses a distinct genetic code. For example, UGA is a universal STOP codon, but in the mammalian mitochondrial genome it encodes Tryptophan. AGG encodes arginine in the universal code but is a STOP codon in the mitochondrial genome.
What does the mitochondrial genome contain?
The proteins encoded by the mitochondrial genome are subunits for several components of the respiratory chain including cytochrome c oxidase, NADH dehydrogenase and apocytochrome b. The human mitochondrial genome encodes many of the genes needed for protein synthesis within the matrix including 22 tRNAs as well as 12S and 16S rRNA that are part of the mitochondrial ribosomes.
Where are most mitochondrial proteins made?
Most mitochondrial proteins are synthesized on cytosolic ribosomes
before targeting to the outer membrane and post-translational import. Nuclear encoded mitochondrial genes contain a targeting sequence. These are of two types. The first is an 15-35 residue N-terminal sequence comprising basic amino acids that is cleaved in the matrix by an endoprotease. The second type of targeting sequence is a non-cleaved internal sequence.
What is the role of HSP70 in the mitochondria?
Hsp70 molecular chaperones play key roles in translocation of proteins into mitochondria on the cytosolic and matrix sides of the membranes because proteins are imported in an unfolded conformation.
What mediates import across the mitochondria outer membrane?
The TOM complex. It associates with several import receptors that bind to mitochondrial pre-sequence containing proteins at the outer membrane. These import receptors bring the pre-sequence containing proteins to the translocation channel formed by the protein called TOM40.
What mediates transport across the inner mitochondrial membrane?
Transport across the inner membrane is mediated by the TIM complex. The positively charge pre-sequence plays a role in opening the channel in the TIM complex and the membrane potential is important for subsequent translocation by an electrophoretic effect. Hsp70 is also important for translocation of pre-sequence containing proteins into the matrix.
Draw the mitochondrial protein translocation.
just google it I’m tired of screenshots.
What is a peroxisome?
Small single membrane organelles that get their name from metabolism of hydrogen peroxide in the organelle. Peroxisomes have important roles in fatty acid ß-oxidation. This resembles ß-oxidation in mitochondria except that hydrogen peroxide is the by-product. The hydrogen peroxide is metabolized by the enzyme catalase to water and oxygen.
Where are peroxisome proteins coded?
The nucleus then produced in cytosolic ribosomes. They are imported fully formed and folded using peroxisome target signals (PTS)
What are two peroxisomal diseases?
Zellwegers syndrome, where there is no import of any peroxisomal enzyme.

Adrenoleukodystrophy (ALD), where oxidation of very long chain fatty acids is defective.
Discuss the structure of the nucleus.
The nucleus is surrounded by a double lipid bilayer, the outer one being contiguous with the rough ER. The perinuclear space between the membranes is contiguous with the lumen of the ER.
A distinct structure (10-20 nm diameter) lines the inner surface of the nuclear membrane called the nuclear lamina, comprising a meshwork of intermediate filament type proteins termed lamins (more about lamins in lecture AC7). Transport into and out the nucleus is via aqueous pores called nuclear pore complexes. Inside the nucleus, there are distinct structures that comprise the nucleolus, where rRNA is transcribed and
where ribosomal subunits are assembled.
What is the nucleolus?
The nucleolus is the most prominent structure within the nucleus and is the site of rRNA synthesis, processing and ribosome assembly.
What does RNA PolymeraseII do?
Transcribes ribosomal protein genes.
What happens when ribosomal protein genes are transcribed?
They exit the nucleus and are translated on cytoplasmic ribosomes. The ribosomal proteins then reenter the nucleus and are transported to the nucleolus, as does 5S rRNA, where they assemble with rRNA. Individual 40S and 60S pre-ribosomal particles are then delivered to the cytosol.
Nuclear Localization Sequences (NLS)?
An NLS is typically 4-8 amino acids, rich in Arg and Lys and usually contains Pro. Nuclear export signals (NES) exist that are distinct from NLS import signals. NLSs are not removed because proteins shuttle between the nucleus and cytoplasm or have to reenter the nucleus as it reforms after mitosis.
What is the general structure of a Nuclear Pore Complex (NPC)?
The NPC is composed of several ring assemblies that occupy the cytoplasmic face, inner core and nucleoplasmic face of the structure. Filamentous assemblies radiate out from both cytoplasmic and nucleoplasmic sides.
Draw the nuclear transport mechanism
Screenshot 9
What do karyopherins do?
Transport into and out of the nucleus depends on specific transporters, called karyopherins, that recognize the NLS or NES sequences.
What is Ran and what does it do?
Ran is a small GTPase with co-factors that regulate nucleotide hydrolysis (Ran GAP) and nucleotide exchange (Ran GEF). Ran GAP exists primarily in the cytosol whereas the Ran GEF is nuclear. Therefore, Ran is in the GDP form in the cytosol and in the GTP form in the nucleus.
Describe the structure of Intermediate filaments.
IF monomer is elongated and alpha-helical with a globular N-terminus head and globular C-terminal tail. IF forms a dimer that is a coiled coil.** The dimers associate to form a staggered tetramer. Each of the tetramer forms associates with 7 others to form a filament containing 8 tetramers. IFs have no polarity because the tetramers form head to tail.
What do IF’s do?
Ifs have rope-like character – easily bent but not broken. Ifs form network in cell and often surround nucleus, forming a network towards the cell periphery. They interact with junction proteins called desmosomes or hemidesmosomes.
Name the four classes of IF’s.
Keratins, Vimentins, Nucleofilaments and Nuclear Lamins
What IF’s assembly is reversible and why?
Most intermediate filaments do not have reversible assembly with the exception of nuclear lamins. In this case, assembly is reversible due to phosphorylation that is cell cycle dependent. This allows for nuclear breakdown during mitosis. Mutations in lamin proteins lead to premature ageing.
What are microtubules?
Long hollow tubes that are very dynamic. They function in intracellular organization and intracellular transport. They form the mitotic spindle and also cilia and flagella. Cellular microtubules (MTs) grow out of the centrosome.
What is the structure of microtubules?
MTs grow from a tubulin dimer of α-tubulin and ß-tubulin. Both bind GTP. MTs form a linear protofilament that organizes laterally into a tube containing 13 protofilaments.
MTs have polarity because dimers prefer to bind to exposed ß-tubulin surface rather than α-tubulin in a protofilament. Exposed ß-tubulin surface is the plus end while the exposed α-tubulin is the minus end. In cells, the MTs grow out of the centrosome from the minus end, which binds to structures composed of γ-tubulin.
Explain MT growth and dynamic instability.
Only ß-tubulin hydrolyzes its bound GTP, and this occurs when the tubulin dimer binds to an MT. Under conditions where there are plentiful free dimers, this hydrolysis occurs only after addition of further tubulin to the plus end of a growing MT. GDP-bound tubulin binds to the growing MT more weakly, but does not destabilize it if additional dimers have bound. Under conditions of limiting tubulin dimers, the GDP bound form is at the cap, and this has a destabilizing effect.
What do Colchicine/vinblastine do?
They are drugs that prevent MT polymerization.
What does taxol do?
It’s a chemotherapeutics drug that inhibits MT depolymerization.
What do MT’s do?
Microtubules form the mitotic spindle for chromosome separation during mitosis.
Microtubules are important for transport of organelles in all cell types – including long axons. Transport occurs using motor proteins which move their cargo towards the plus end (known as kinesins) or the minus end (known as dyneins)
