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101 Cards in this Set
- Front
- Back
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What kind of molecules can diffuse across the membrane?
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-Hydrophobic molecules (O2,CO2,N2,Benzene)
-Small uncharged polar molecules (some H2O, Urea, Glycerol) -Some stuff(most) need to be transported (large uncharged polar molecules, ions) |
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What are the types of membrane transport proteins?
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Carriers
Channels |
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What is a carrier? How does it work?
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-a family of membrane transport protein
- bind solutes specifically and translocate solute across membrane - triggers confromatinal change->translocation of solid - selective for one or two solutes (if two, then two binding sites) |
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What is a channel? How does it work?
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-a family of membrane transport proteins
- form hydrophilic pores through the membrane - solutes (particularly ions) diffuse through the pores; no binding sites - still selective |
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Is the negative charge usually intra cellular or extra cellular? What's one reason for this?
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intracellular; because of the electrochemical gradient formed by pumps like the Na+/K+ pump, that pumps 3 Na+ out, and 2 K+ in
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Compare and contrast
1) simple diffusion 2) facilitated diffusion 3) Active transport give an example of each |
- All are mechanisms of membrane transport
1) Simple diffusion - passive,exergonic -Hydrophobic molecules (O2, CO2), small uncharged polar molecules 2)Facilitated diffusion -passive, exergonic -needs channel or carrier -Bacterial K+ channel, Porins 3) Active transport -active, endergonic -requires carrier -Transport ATPases like the Na+/K+ pump, light-driven pumps, and coupled transporters |
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What is a transport ATPase?
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an example of an active transporter, a Transport ATPase is something like a pump that goes against a gradient to transfer molecules.
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Describe the Na+/K+ pump. What does it do? What does it create?
what's the order of what takes place? |
-Hydrolyzes ATP (1 ATP required)
- transports 3 Na+ out of cell, 2K+ in. since this goes against the Na gradient, it is active transport - increases and strengthens/creates the Na+/K+ gradient, which becomes a source of energy - also creates a negative membrane potential (electrogenic) - consumes 30% of cellular energy (more in neurons) - contributes to cell volume regulation Order -3na bind to pump (no place for k to bind) -ATP binds P to pump AKA phosphorylation -pump changes conformation, faces out of cell: new binding site for K+, no place for Na+ to bind -3na+ released -2k+ attach -causes release of p -release of p returns pump to original conformation -2 k released into cytosol |
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How dows the Na+/K+ pump control cell volume?
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because natural transport of water is in the cell, if there was no mechanism to inhibit this the cell would burst.
-the pump translocated ions to right outside of the cell, changing the water gradient and creating isotonic conditions |
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What are the 3 type of transport ATPases? say something about each kind (give an example, describe its characteristic trait, etc)
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1) P-type ATPases
-cation transporters -ex. Na+/K+ pump, Ca pump -reversibly phosphorylated so that p can be removed 2)v-type +f-type -bacterial H+ pump and organelles (ex ATP synthase) -can work in reverse, to synthesize or hydrolyze atp 3) ABC transporters - Atp Binding Casserres -transport dirven by 2 ATP hydrolysis - either a Bacterial ABC transporter (for nutrients into cytosol) - or a eukaryotic ABC transporter (waste Out of cell) -ex Multi-Drug Resistance protein pumps what it thinks are toxins (medicine, often) out of cell) |
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Describe coupled transporters, What are the two examples we learned about?
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-use energy of ion gradients
ex Na+/Glucose co-transporter -symport of both from outside to inside cell - glucose against concentration gradient, Na+ with gradient -when Na+ binds, allows for glucose to bind more effectively - called cooperative binding -when both are bound, triggers translocation ex bacterial H+/Lactose permease - active transport of lactose is driven by dowhill transport of H+ - the structure is the two C-shaped sides (6 domains each) that swivel back and forth freely between two states until H+ binds, at which point the Lactose can bind and the sides swivel to the other state |
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How can you determine if something is secondary transport?
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doesn't require ATP
- if you shut off a different pump, then it will stop even though ATP is present |
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What are the two types of passive transport protein channels?
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Ion channels
porins |
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What is an Ion channel? What are its rates of transport?
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a passive transport protein channel
- it is a large oligomeric protein that is an integral membrane protein - facilitated diffusion of small inorganic ions like K, Na, Cl etc. - fast transport |
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What allows an ion channel to be selective?
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1) width of pore
- size selection 2) negatively charged residues like Glutamine and Aspartate at the mouth of the pore repel anions, facilitate cation entry 3) SELECTIVITY FILTER - a constriction inside the pore that by virtue of its structure, allows for a specific cation (like K+ in the example of bacterial K+ channel) to pass |
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What is the structure of a Bacterial K+ channel?
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-4 identical subunits that surround the pore = homotetramer
- pore has a vestibule and selectivity filter - vestibule: water-filler cavity, wider portion at entry of channel -selectivity filter: narrow constriction - alpha helices connect subunits -transmembrane domains connected by pore helix, which are both connected by selectivity loop |
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What surrounds a K+ when it enters the bacterial K+ channel? what happens to K+ as it goes through the channel?
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it is solvated, so surrounded by water molecules which dissociate when the K+ is attracted to the carbonyl in the selectivity filter
K+ moves one a t atime |
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What kind of signals to ligand-gated channels respond to?
voltage gated channels? |
ligand-gated respond to chemical signals
voltage gated respons to electric signals |
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What molecules do porins move?
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small polar organic molecules like H2O
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Where would you find an Aquaporin?
where would you find a beta-barrel porin? |
Aqua porin: on the eukaryotic plasma membrane
Beta-barrel porin: outer membrane of bacteria, or in the outer membrane of mitochondria and choloroplasts |
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What are the three types of sorting signals in protein sorting?
which are peptides, which are not? where are they located? |
1. signal sequence/peptide
- peptide - on surface 2. signal patch - peptide - on surface 3. Phosphorylated sugar - not a peptide -signal added after protein synthesis - does not have to be on surface |
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What must all macromolecules pass through to get in and out of the nucleus?
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Nuclear pores
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What kind of signal is required for a protein to be imported into the nucleus? Where is the signal located on the protein?
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The nuclear localization signal
- always internally, never at either of the terminals |
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What drives gated nuclear transport?
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GTP hydrolysis
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What are importins?
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Nuclear import receptors
- bind specific cargo proteins via the NLS |
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What is a Ran protein?
Where is it? What does it do? |
- small GTP-binding protein w/ GTPase activity
- in nucleus and cytosol - binds GTP in the nucleus (Ran-GTP) - hydrolyzes GTP-> GDP in cytosol (Ran-GDP) -GDP stays attached to Ran |
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How does Ran interact with the GTPase activating protein (GAP)
with the Guanine exchange factor (GEF)? |
GAP interacts with Ran-GTP to from Ran-GDP in cytosol
GEF interacts with Ran-GDP to form Ran-GTP in nucleus |
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How does a nuclear import receptor make its way into the nucleus?
how does the bound cargo become dissociated from import receptor? What happens to receptor after it dissociated from its cargo? |
-Import receptor is loaded in the cytosol
- loaded receptor moves through the pore along a tract of "FG nucleoporins" - pore widens as a result of conformational change due to binding interactions - binding to Ran GTP(and only Ran-GTP) inside nucleus causes cargo to dissociate - Ran-GTP importin complex returns to cytosol - Ran hydrolyzes GTP in cytosol, -energy released drives disassembly of the complex (importin and Ran-GDP are released into cytosol) - Ran GDP is imported into the nucleus and GDP is replaced with GTP |
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How does Nuclear import compare to Nuclear Export?
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They're pretty much the same, except for Nuclear Export is in reverse
- Ran-GTP promotes loading of the export receptor, increasing the affinity for cargo protein |
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How does Ran drive the direction of nuclear transport?
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Ran-GTP causes the cargo protein to dissociate from the import receptor (Nuclear import)
Ran-GTP causes teh cargo to bind to the export receptor (Nuclear Export) |
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How can nuclear transport be stopped?
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by modifying the sorting signal
- either by phosphorylation or binding to another protein, so that the receptor can't recognize the protein effect is reversible |
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What is an example of regulated nuclear transport?
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NF-AT (gene regulatory protein)
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Describe regulated nuclear transport with regards to NF-AT.
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-NF-AT has both an NLS and an export signal
- NF-AT is usually in cytosol, inactive - goes into nucleus when cells are activated to stimulate gene expression (Calcium increases in cytosol) -NF-AT moves back into cytosol when cells return to normal (cytosolic Calcium decreases -NF-AT is inactive as long as it has a phosphorus on it, but when it is phosphorylated P is released and the nuclear import signal acts -NF-AT phosphorus removed by Calcineurin! |
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What is calcineurin? What does it do? how is it activated
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Calcineurin is the protein phosphatase that removes NF-ATs phosphorus, causing NF-ATs nuclear import signal to show, allowing it to act
- can act in reverse to mask the NF-AT nuc. import signal by binding P when low Calcium - it is activated by calcium |
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Is mitochondiral protein sorting uni-directional or bi-directional?
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Unidirectional, only in the direction of Cytosol-> mitochondria
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What encodes most mitochondrial proteins?
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Nuclear genes!
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What transport mechanism transports the mitochondrial proteins into the mitochondria?
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Post-translational membrane transport
- active transport mechanism |
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are mitochondrial protein synthesized in the cytosol or in the mitochondria?
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they undergo postranslational membrane transport, so the protein are fully synthesized before they get in the mitochondria
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What proteins might have a mitochondrial import signal?
Where would the signal be located on the protein? |
Matrix and membrane proteins
- signal always located at N-Terminal, then removed by protease |
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Where is the mitochondrial protein translocator located? What is it?
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It is located on the mitochondrial membrane
can function as an anchored receptor AND a channel to allow proteins to from into the Mitochondria |
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What are important Mitochondrial Protein Translocators?
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TOM complex
TIM23 complex |
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What does a TOM complex do? where is it?
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It is the translocator of the outer membrane
- interacts with inner membrane complexes - always involved (can interact with different TIMs) |
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What is a TIM complex? what's an important one?
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TIM complexes are inner membrane translocators
TIM 23 complex - used to import all protein going into mitochondrial matrix |
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What are the three things necessary for a protein to enter the matrix of a mitochondria?
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1. Mitochondrial import signal
2. Receptor TOM and TIM 3. Heat-shock proteins that pull proteins through, into matrix |
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When is a mitochondrial protein folded? unfolded?
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Proteins are unfolded in export
- must remain unfolded because pore on translocator complexes are narrow folded in matrix |
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what is a molecular chaperone?
What does it do? What is the role of molecular chaperones in the mitochondria? what is an example of a mitochondrial chaperone? |
-Molecular chaperone is a protein that drags/ pulls proteins along or helps it fold properly
- in the mitochondria, molecular chaperones pull proteins through the TIM 23 complex and bring them into the mitochondrial matrix - an example is Heatshock protein 70 (Hsp70) - also contributes to folding and unfolding |
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what does Hsp 70 do in the Mitochondria? Why is it needed
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Hsp70 pulls protein through TIM 23 complex into matrix
- needed because when a protein is synthesized, it has hydrophobic parts that cause it to want to aggregate and misfold -Hsp binds to hydrophobic parts and allow proper folding - uses energy to dissociate one at a time, which allows for gradual, proper folding |
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What happens if Hsp 70 attaches to a protein, begins to be removed, and the protein still folds incorrectly?
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Another hsp70 will try
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What role do chaperones play in cytosolic delivery of a protein to the mitochondria?
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Chaperons hold the protein and prevent folding, feeding it into the TOM complex
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What are Membrane insertion signals?
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they are sequences, called "stop-transfer sequences" on the inner mitochondrial membrane.
-act as membrane insertion sequences, - hydrophobic alpha-helical segment that anchors the protein onto the membrane and make it a trans-membrane domain |
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What is the space inside the ER called?
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ER lumen
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What occurs at Rough ER?
at Smooth ER? |
at rough ER, protein synthesis
at smooth ER, lipid synthesis |
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What cation is stored in large quantities in the er lumen? Why?
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Calcium, in order to keep cytosolic calcium levels low
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is the ER static or dynamic? why?
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Dynamic, because it moves along tracks of microtubules
- connections fromed/broken constantly |
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What kind of proteins are synthesized by free cytosolic ribosomes?
by ER -bound ribosomes? |
free cytosolic ribosomes bind nuclear and mitochondrial proteins
E.R. ribosomes syntehsize plasma membrane proteins and other proteins |
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What are the components of the ER signaling system?
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1) ER signaling sequence(s)
2) Import receptor 3) SRP receptor 4) Protein translocator complex |
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What are the properties of ER signal sequences?
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Hydrophobic
alpha helical Nterminal, never C-terminal |
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What is another name for an ER Import receptor?
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a signal-recognition complex (SRP)
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What is an example of a protein translocator complex?
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Sec-61
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what are the 2 critical, functional domains of an SRP for?
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One is for binding to a protein signal sequence
One is for ribosome binding |
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What is the process of an SRP recognizing and binding to a signal in ER importing?
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1. SRP recognizes peptide signal sequence as it peaks out of cytosolic ribosome
2. SRP binds to signal peptide 3. other end of SRP binds to ribosome itslef 4. Once it is bound, no more translation 5. SRP holds it like that until it can bring the ribosome and peptide to the ER |
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How does an SRP receptor interact with an SRP?
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SRP receptor on ER membrane binds to SRP that is attached to ribosome+signal peptide sequence and halting the translation
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What is the process of protein import into ER?
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1. SRP receptor on ER membrane recognizes SRP, binds to it
2. Ribosome binds to translocator complex so that the ribosomal exit site aligns with the translocator pore 3. signal peptide sequence binds to the translocator on the ER 4. this causes a conformation change, causing pore to open 5. SRP dissociates, translation resumes 6. Protein is translocated as it is synthesized - called co-translational membrane transport |
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What does co-translational membrane transport?
post-translational transport? |
Co translational done by ER import
post-translational done by mitochondrial protein import |
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Describe the Sec 61 translocator protein.
What system is it a part of? |
Part of the ER import system
- it is a v-shaped protein with a plug in the middle of its pore - when plug in place, pore closed - binding of signal peptide causes pore to open |
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When does the Sec61 pore open?
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binding of signal peptide causes pore to open
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Post-translation, where do ER proteins go?
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Depends on type
-Some just go into lumen -Some go into ER membrane and become transmembrane proteins -some go into ER membrane and become transmembrane proteins, then go to other places like plasma membrane or golgi (in same orientation) |
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what would determine if an ER protein stays in the Lumen?
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When an N-terminal signal peptide is recognized by ERs signal peptidase
- Peptidase cleaves it off, releasing mature protein into ER lumen - Signal peptide remains stuck in translocator until translocator opens up and the peptide released for degradation |
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What are the two important signal sequences of transmembrane proteins in the ER
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a ER import sequence
- acts like a start transfer sequence - may be n-terminal (cleaved) or internal (never cleaved a stop-transfer sequence - stops translocation (proteins remain in the membrane - always internal (never cleaved) |
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If a transmembrane ER protein is being translated with a start-sequence AT THE N-TERMINAL and a stop sequence is hit by translocator, which end is in the cytosol and which end is in the lumen?
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C-terminal in cytosol
N-terminal in Lumen |
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if a transmembrane has a start sequence INTERNALLY, what is the conformation of the protein once a stop sequence is hit (and let's say, just one stop sequence)
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1. since translocator let protein "go" until it saw the start sequence, the N-terminal end is sticking up into the cytosol
2. then a loop begins to form in the lumen 3. translocator hits stop sequence, gets stuck: both ends are in cytosol 4. conformational change causes N-terminal to be in cytosol |
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What is the exception to the rule that says in the ER, when a protein has an internal start sequence it will have the N-terminal in the cytosol?
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two exceptions
1. if there are positively charged amino acids near the start sequence, then the positively charged end stays in the cytosol 2. if there is a cluster of positively charged residues at the end of the signal sequence, causing the orientation to be reversed ^ not sure if #2 is an exception to the rule, or an exception to the exception stated in #1... |
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What is a peripheral ER membrane protein?
How is it attached? |
a protein with no trans-membrane domains
- attached covalently to a component of the membrane or to a protein |
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what's a GPI-anchored protein
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a peripheral ER membrane protein
- precursor has a C-Terminal GPI attachment sequence |
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What are the two types of covalent modification of protein in the rough ER?
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1) N-glycosylation of ER proteins
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What is N-glycosylation of ER proteins an example of?
what does it do? how does it do it? why? |
- an example of covalent modifications of proteins in rough ER
- it is an oligosaccharide added to a side-chain -NH2 of an Asn residue -only in lumen -because an N-linked oligosaccharide is needed for proper folding in ER |
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What is Calmexin?
how does it do what it does? |
membrane chaperone protein that helps folding
-it recognizes a glucose monosaccharide on a protein, and binds to glucose -changes polysaccharide glucose to monosaccharide by GLUCOSE TRIMMING |
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what is glucose trimming, and what does it?
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when a polysaccharideglucose becomes a mono saccharide glucose
- Calmexin does it to do hold protein in the ER for proper folding |
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What happens in the ER if a protein isn't properly folded?
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1. Calmexin can try to hold it again for refolding
2.can be tagged by ubiquitylization (multiple ubiquitins) 3.will go to a proteosome for degradation |
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what are the three directions of vesicular transport
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1. Biosynthetic-secretory pathway
- building/secreting proteins, -forward transport from ER->out 2. Endocytic pathway - for eats 3. Retrieval pathways |
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What is the formation of a clathrin-coated vesicle?
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1. membrane buds
- energy given by proteins that coat vesicle 2. Curvature of clounds the vesicle membrane causing it to bend and (eventually) pinch off |
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Where do vesicles form on a membrane?
-how does cargo get to these spots? |
at specialized regions rich in inositol phospholipids
- cargo molecules loaded onto specific membrane receptors |
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What induces budding of a vesicle?
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Clathrin assembly
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What does Dynamin do?
What is it? |
Wraps itself around the stock of a vesicle and promotes membrane fusion
- a GTPase |
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What happens to clathrin coat after budding?
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it dissociates after vesicle is formed (vesicle pinches off before it dissociates)
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What are the different types of vesicle coats?
what do the coats tell you about the vesicle origins? |
Clathrin coat:
- predominantly for endocytic vesicles COP1 - Golgi derived vesicles COP2 - ER-derived vesicles |
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What do the different Inositol phospholipids (AKA PIPs) determine?
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they determine where the vesicle will localize to
- PIPs interact with proteins involved in vesicular transport |
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How do vesicles find their destination?
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Transport vesicles have surface protein markers
- V-SNARES if on vesicle - complimentary t-SNARE on target organelle |
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What happens after fusion of t-SNARE and v-SNARE?
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they are pried apart (endergonic)
- V-SNARE returned to organelle of origin by retrieval pathway |
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What does an RAB protein do?
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Helps vesicles find their right targed
- brings vesicles close enough to a taret organelle for the SNAREs to bind -promotes docking |
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What does an RAB bind to?
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an RAB effector on the target organelle
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what happens after an RAB binds?
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GTp is hydrolyzed, RAB-GDP dissociates
- returns to origin after nucleotide exchange |
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What do different RABs determine?
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Different RAB proteins mediate vesicular traffic to different regions of the cell
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What are cisternae?
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Golgi apparatus compartments
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What is a cis-golgi face compared to the trans-golgi face?
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cis- entry face
trans- exit face |
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What kind of modifications occur to proteins and lipids as they move through the golgi body?
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Glycosylations
- N-linked oligosaccharides may be removed ormodified - some proteins are O-glycosylated |
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What coats ER vesicles?
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COPII
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What prevents incorrectly folded protein from entering a vesicle in the ER?
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chaperone proteins
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What is the vesicular tubular cluster? When is it formed?
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- it is the fusion of many vesicles from one (homotypic) origin
- formed when the vesicles are released and get to the Golgi |
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how does the vesicular tubular cluster move from the ER to the golgi? To what face of the golgi?
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along microtubules tracks and by motor proteins, to the cis-golgi
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What is the transport/pathway that returns ER proteins from the golgi to the ER?
how can a cell know which ones to return? What would coat the vesicle? |
the retrieval (retrograde transport/pathway
- there is a special C-terminal signal sequence carried by a protein that needs to be returned -returned in a COPI vesicle |
