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78 Cards in this Set
- Front
- Back
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What is the nucleus topologically equivalent to? What about inside the ER and Golgi?
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Nucleus is topologically equivalent to the cytosol.
ER and Golgi is topologically equivalent to the outside of the cell. |
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What are the four distinct intracellular compartment families?
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1. Nucleus and cytosol
2. Secretory and endocytic pathway (ER, Golgi, Vesicles, etc) 3. Mitochondria 4. Chloroplasts/Plastids |
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What are the three distinct protein transport pathways?
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1. Gated transport (from the cytosol to the nucleus and vice versa)
2. Transmembrane transport (from the cytosol to the mitochondria, ER and plastids) 3. Vesicular transport - endocytic pathway - note that all protein synthesis starts in the cytosol |
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What are protein signal sequences? How do they work?
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- the address that tells where the protein should go
- contained in the amino acid sequence of each protein at the N-terminus - recognized by sorting receptors and signal sequence is cleaved upon delivery - if there is no signal they stay in the cytosol |
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The nuclear envelope is continuous with what other membrane?
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ER membrane
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How many different proteins are nuclear pore complexes composed of in animal cells? And how many NPCs are present in the nuclear enveloped of a mammalian cell?
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30 and 3000-4000 respectively
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How fast do NPCs transport macromolecules?
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500 macromolecules/second in and out simultaneously through a single pore
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What are the five main components of a NPC? And what sort of transport is allowed through an NPC?
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1. Column subunit - forms bulk of pore wall
2. Annular subunit - centrally located 3. Lumenal subunit - contains transmembrane proteins that anchor complex 4. Ring subunit - form cytosolic and nuclear face 5. Nuclear fibril - extend from the NPC in either direction, form basket-like structure on the nucleus side - together the annular and ring subunits form a tangled mesh of proteins that allows diffusion of small molecules (<5000Daltons) but blocks diffusion of large molecules - active transport can be used for larger molecules up to 39 nm |
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What are Nuclear Localization Signals?
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- sorting signals responsible for selectivity of nuclear import process
- every protein that wants to enter the nucleus must have a nuclear localizaton signal on the outside of the folden protein |
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What are Nuclear Important Receptors?
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- bind to nuclear localization signals and NPC proteins
- every receptor has a specific set of cargo proteins that it transports |
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What are FG repeats?
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short amino acid repeats with phenylalanine and glycine on NPC
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how does NPC active transport work for import ?
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- nuclear import receptor binds to protein of pore and then to FG repeats
Cytosol: protein with nuclear localization signal binds to nuclear import receptor - receptor continuously binds to FG sites and dissociates, moving down one by one to the nucleus Nucleus: Ran-GTP binds to receptor and cargo is released and the receptor returns to the cytosol Cytosol: GAP turns GTP to GDP and Ran-GDP dissociates from receptor and process repeats |
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What is a monomeric GTPase?
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- provides energy for active transport
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What is GEF and what does it do?
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Guanine Exchange Factor
- removes GDP from a monomeric GTPase creating an open nucleotide binding site which GTP quickly fills since it is always in surplus |
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What is GAP and what does it do?
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GTPase Activiating Protein
- inactivates protein by stimulating it to hydrolyze its GTP to GDP |
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What is the cycle of regulation of a monomeric GTPase?
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- inactive monomeric GTPase with GDP
- GEF removes GDP and fills it with GTP - active monomeric GTPase with GTP - GAP inactivates by hydrolyzing GTP to GDP - cycle continues |
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What is RanGTPase and how does it work?
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Ran GTPase is one group of monomeric GTPases
- cell obtains energy it needs through hydrolysis of GTP by Ran GTPase - in cytosol GAP converts RAN-GTP to Ran-GDP - moves to nucleus - in nucleus GEF converts Ran-GDP to Ran-GTP - moves to cytosol and cycle repeats |
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Where are GAP and GEF present?
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GAP is only in the cytosol
GEF is only in the nucleus |
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How is RNA exported out of the NPC?
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- cap-binding complex and poly-A-binding proteins at the end of mRNA help mark when the mRNA is complete, as well the exon junction complex is only deposited on the mRNA after proper splicing to ensure it is finished before transport
- nuclear export receptor attaches to mRNA and then it goes to the cytosol for translation |
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What is the role of Ran-GTP in active transport through an NPC? Describe the process of export
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- same as import except Ran-GDP promotes cargo binding instead of cargo release
Nucleus: Ran-GTP binds to receptor and so does cargo protein with nuclear export signal Cytosol: Ran-GDP and phosphate released and nuclear export receptor back to nucleus |
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How is the NPC regulated?
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- transport is regulated by controlling access to transport machinery
- gene regulatory proteins are kept in the cytosol until they are needed, and export/import signals are turned on and off by phosphorylation as needed - high calcium concentration in the cytosol activates protein phosphatase which removes phosphates from the gene regulatory protein in order to expose the nuclear import signal - gene regulatory protein is then imported to the nucleus and activates gene transcription - ATP and protein kinase phosphorylate the protein and expose the nuclear export signal and the protein is returned to the cytosol |
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What is important for the successful import of molecules through the NPC?
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Ran
- if Ran is taken away the cargo is much more likely to abort |
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How many cargos actually make it into the pore? and how many of these are early and late aborts?
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Only 25% of cargos actually even make it into the pore
- of these 20% are early aborts and 50% are late aborts - only the last 30% successfully make it all the way through |
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What is a proplastid?
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- in a plant cell, can develop into a chloroplast, chromoplast or storage plastid
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What does a chromoplast do?
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- stores pigments, responsible for bright colours of a flower, etc
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What does a storage plastid do?
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- stores fat, oil or starch. ex. seeds
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Can a cell synthesize new mitochondria?
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No, mitochondria can only replicate themselves
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What are mitochondrial precursor proteins?
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- pre-mitochondrial proteins
- fully synthesized in the cytosol - only transported into mitochondria once fully transported |
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How are mitochondrial precursor proteins translocated into the mitochondria?
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- they have a signal sequence for import and localization to the appropriate mitochondrial subcompartment
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What is the signal sequence that directs proteins to the mitochondrial matrix?
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it is always an alpha-helix with positive charges on one side and uncharged amino acids on the other side
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What do protein translocators do?
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- mediate protein translocation across mitochondrial membrane
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What do TOM complexes do?
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- transfer proteins across the outer membrane of mitochondria
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What do the two TIM complexes do?
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- transfer proteins across the inner membrane of the mitochondria
TIM 23 - transports soluble proteins into matrix and integrates proteins into inner membrane TIM 22 - mediates insertion of inner membrane proteins from intermembrane |
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What does the SAM complex do?
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- helps to integrate beta-barrel proteins into the mitochondrial membrane
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What does the OXA complex do?
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- mediates insertion of inner membrane proteins that are synthesized within the mitochondrial membrane
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What does Hsp70 do?
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- heat shock protein that helps to pull proteins into the matrix of the mitochondria
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What are the steps in Translocation into the mitochondrial matrix?
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1. Chaperone proteins are attached to precursor proteins to prevent folding
2. Signal sequence binds to N-terminus of precursor protein which in turn binds to receptor protein in TOM complex 3. chaperones are removed and the protein is inserted into the TOM complex 4. Protein goes through the intermembrane space and is inserted into the TIM 23 complex in the inner membrane 5. Once inside the matrix the signal peptide is cleaved from the protein and degraded, leaving a mature mitochondrial protein |
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What does Hsp60 do?
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- uses ATP to help the protein refold once it is in the mitochondrial matrix
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Which two processes drive the energy required for transport into the mitochondria?
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ATP hydrolysis (driven by proton gradient caused by oxidative phoshorylation) and membrane potential
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How can a protein be kept in the intermembrane space or in the membrane instead of going to the matrix of the mitoochondria?
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- as protein is starting to go through the TIM 23 complex the signal sequence can be cleaved off and replaced with a stop transfer sequence. this prevents protein from being pulled through TIM 22 and it stays in the intermembrane space
- protein can also be integrated into inner membrane by way of OXA and can either stay in the membrane or be cleaved by a protease and released to the intermembrane |
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What are porins?
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- freely permeable for small molecules to travel across membranes
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What are methods for keeping proteins in the outer membrane of the mitochondria?
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- after translocation through the TOM complex, chaperones bind to protein in the intermembrane space.
- SAM complex then inserts the unfolded polypeptide chain into the outer membrane and turns it into a full folded membrane protein. |
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What is an experiment to tell if the protein of interest is located in the mitochondria?
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- use fluorescently labeled antibodies against the protein, and the mitochondria.
- if the two overlap you will know the protein is inside the mitochondria |
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What is PK?
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Proteinase K - enzyme that destroys and degrades proteins
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Can PK destroy protein when there are mitochondria? What if you affect the TOM complex of the mitochondria
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- No.
- However, if the TOM complex is affected the protein can no longer go into the mitochondria and therefore is degraded by PK and is not detected. |
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How are proteins translocated into the thylakoids of the chloroplast?
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- precursor protein contains an N-terminus chloroplast signal sequence followed immediately by a thylakoid signal sequence
- chloroplast signal sequence initiates translocation into the stroma by a mechanism similar to translocation into mitochondria - signal sequence is then cleaved off, unmasking the thylakoid signal sequence which then initiates translocation into the thylakoid |
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What are the similarities between translocation into mitochondria and translocation into chloroplasts?
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Similarities:
- different transport complex for inner and outer membrane - use energy for active transport - post-translational - have N-terminal signal sequence |
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What are the differences between translocation into mitochondria and translocation into chloroplasts?
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- transport complexes are different (in chloroplasts they are called TOC and TIC)
- no electrochemical gradient across inner membrane of chloroplasts so GTP and ATP are used to power transport |
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Are mitochondrial signal sequences or chloroplast signal sequences shorter?
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- mitochondrial sequences are shorter
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Which has more arginine? Chloroplasts or Mitochondria?
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Chloroplasts have about 50% of the arginine that mitochondria have
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What are the three main functions of the ER?
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- lipid biosynthesis - the majority of lipids are made in the ER
- protein biosynthesis - only transmembrane or secretory proteins - intracellular calcium storage - especially in the SR |
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How do you isolate the smooth and rough ER?
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1. Homogenize all of the cell
2. Microsomes, small vesicles of rough and smooth ER, will form 3. Centrifuge these with sucrose- to separate the rough and smooth ER centrifuge in a tube with a sugar concentration gradient - the SER float at low concentration of sucrose because they are low density - the RER will float at more deep higher concentration of sucrose because they are more dense |
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What is co-translational protein translocation? And where is it used?
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Always in the ER:
- for ER, do not finish protein synthesis in the cytosol - released into ER while the protein is being synthesized - brings ribosome to ER making rough ER |
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What is post-translational protein translocation? And where is it used?
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- translocation after translation
- for mitochondria and chloroplasts |
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Are chaperones present in both co-translational and post-translational protein translocation?
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only in post translational transloation because there cannot be folding
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Why are proteins without microsomes slightly longer?
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Because there is no signal peptidase to cleave the signal sequence
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What is SRP and what does it do?
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Signal Recognition Particle - recognizes the 8 non-polar hydrophobic amino acids in the signal sequence
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What is the SRP receptor and what does it do?
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- provides area for signal sequence to bind (hydrophobic pocked lined by methionines)
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What is the process for the SRP and receptor co-translation into the ER?
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- signal sequence on polypeptide binds to SRP causing a pause in translation (ribosome still attached)
- SRP-bound ribosome with polypeptide attaches to SRP receptor int he ER membrane with the help of a GTP-binding domain - translation continues and translocation begins - SRP and SRP receptor are displaced and recycled |
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What does the signal peptidase do?
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cleaves off the signal sequence once translocation is complete
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In what two directions is a translocator gated?
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1. forms pore for protein to go to lumen
2. opens laterally to release hydrophobic part to lipid bilayer |
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How do ER resident proteins stay in the ER?
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They have an ER retention signal
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How does the translocation of transmembrane proteins work?
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- start transfer sequence on polypeptide binds and initiates translocation process
- pull polypeptide into lumen until the stop-peptide sequence binds in the pore which then anchors the protein into the membrane - the C-terminus is in the cytosol and the N-terminus is in the ER |
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How are multi-transmembrane proteins formed?
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- have alternating start and stop sequences allowing a polypeptide to have multiple transmembrane domains
- stop and start sequences are the same, it only depends on which comes first |
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What complex do bacteria, eukarya and archaea use for co-translational translocation?
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Sec 61 complex
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What complexes do eukaryotes use for post-translational translocation?
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Sec 61 complex and also Sec 62,63,71,72 complex which helps to bind BiP to the growing polypeptide
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What does BiP do?
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- pulls polypeptide into lumen
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What complex does bacteria use for post-translational translocation?
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Sec A
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Which groups take part in co-translational translocation and which use post-translational?
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Bacteria, archaea and eukarya do co-translational
only bacteria and eukarya do post-translational |
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How many amino acids are pulled through by the polypeptide with every ATP hydrolysis?
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about 20
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What is glycosylation?
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- major biosynthetic function of the ER = adding sugar to proteins
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How does glycosylation happen?
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- membrane bound oligosaccharyl transferace enzyme transfers the precursor oligosaccharide to the asparagine
- oligosaccharide has N-acetylglucosamine, mannos and glucose which may be modified |
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What percentage of eukaryotic proteins are glycosylated?
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50%
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What do calnexin and calreticulin do?
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- chaperones that bind to oligosaccharides on incompletely folded proteins and keep them in the ER (recognize proteins which have only one terminal glucose)
- a protein undergoes continuous cycles of glucose trimming by glucosidase and glucose addition by glycosyl transferace, maintaining an affinity for calnexin and calreticulin until it has achieved its fully folded state |
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What is the process for ensuring proper folding of glycosylated proteins?
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1. Calnexin or calreticulin binds to incompletely folded proteins containing one glucose
2. Glucosidase removes glucose and protein is released 3. Glucosyl transferace determines if proper folding has occured - if yes, then it leaves the ER - if not, then enzyme transfers a new glucose from UDP-glucose and it rebinds to calnexin and the cycle repeats |
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How are improperly folded proteins degraded?
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- Retrotranslocation
- misfolded soluble ER proteins are translocated back to the cytosol via translocator using ATPase p97 for energy - in the cytosol they are deglycosylated (by N-glycanase), ubiquitylated (by ubiquitin) and degraded in proteasomes |
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What is ERdj5 and what does it do?
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- it is an enzyme important for degradation of misfolded proteins in the ER
- it is a disulfide reductase, so it degrades the disulfide bonds in order to unfold proteins for re-translocation |
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What is the process in the unfold protein response?
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1. misfolded proteins in the ER signal the need for more ER chaperones y activating a transmembrane kinase
2. Activated kinase turns into an endoribonuclease 3. Endoribonuclease cuts specific RNA molecules at 2 positions, removing an intron 4. 2 exons are ligated to form an active mRNA 5. mRNA is translated to make a gene regulatory protein 6. Gene regulatory protein eneters nucleus and activates genes encoding ER chaperones 7. Chaperones are made in ER where they help to fold proteins |
