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129 Cards in this Set
- Front
- Back
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What are the two protein sorting pathways?
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1) Secretory Pathway
2) Non-Secretory Pathway |
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What is the difference between the secretory and the non secretory pathway
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Secretory: Proteins have a signal sequence for ER
- Proteins are transported out of cell Non secretory: Proteins have no signal sequence for ER - Proteins are directed to a specific organelle |
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Where do proteins in the secretory pathway end up
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In ER, golgi, lysosomes, plasma membrane or are secreted from cell
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What directs a protein to the ER
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An ER signalling sequence
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What are traits of the ER signal sequence
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- 16-30 AA sequence
- Located at the N terminus - One or more positively charged AA next to hydrophobic residues |
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Define cotranslational translocation
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Translation occurs across the ER membrane once a ribosome attaches to the ER membrane
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How did pulse labelling experiments prove that proteins enter the ER lumen
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- radioactively labelled AA are added during protein synthesis (creates labelled proteins). After time, cells are homogenized (ground up), and Er gets broken in microsomes
- microsomes with ribosomes attached are heavier and can be purified by centrifugation. microsomes are then treated with a protease in the abscence or prescence of a detergent (disrupts the ER membrane) - Proteins protected by ER membrane from protease therefore are located in the ER |
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How do we know protein translocation is cotranslational
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Microsomes are treated with EDTA (makes ribosomes fall off)
- Proteins are not incorporated into ER when EDTA is present and are incorporated into ER when EDTA is present therefore translocation is cotranslational |
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Define SRP and describe its function
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Signal recognition particle
- Binds to ER signal sequence, blocks further translation and takes ribosome to a SRP receptor on ER membrane |
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How does SRP attach a ribosome to the ER membrane
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1) SRP directs ribosome to SRP receptor in ER membrane
2) GTP binds to both SRP and receptor, opening a pore 3) GTP is hydrolyzed and SRP detaches 4) The polypeptide elongates |
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How are integral membrane proteins inserted into the ER
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1) Once the ribosome is attached the polypeptide elongates until a stop-transfer sequence is reached.
2) The rest of the protein is synthesized on the cytoplasmic face of the ER 3) A signal peptidase cleaves the signal sequence and the stop transfer sequence diffuses laterally into the membrane |
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How can the stop transfer sequence diffuse laterally, through the translocon, into the membrane during the insertion of a protein into the ER membrane?
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The stop transfer sequence is hydrophobic
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Describe two benefits of GPI anchored proteins
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1) They diffuse easier in the plane of the membrane
2) They can target specific locations |
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How do proteins attach to the GPI anchors
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1) Proteins bound for a GPI anchor have a short sequence in the ER luminal domain that a GPI transamidase recognizes
2) GPI transamidase cleaves off the stop transfer anchor and moves the rest of the protein onto a GPI anchor |
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Define GPI transamidase
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An enzyme that cleaves the stop transfer sequence off a ER membrane protein and attaches the rest of the protein to a GPI anchor
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What happens to a protein with an internal start transfer sequence?
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1) The protein synthesizes in the cytoplasm until the start transfer sequence is recognized by a SRP and taken to the ER membrane
2) The rest of the protein is synthesized into the luminal side of the ER 3) The start transfer sequence moves laterally into the membrane |
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How is the orientation of a integral membrane protein determined in the ER
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If the positively charged AA are located on the N-term side of the start transfer sequence, the N-term will be in the cytoplasm.
If they are on the C-term side, the C-term will be in the cytoplasm If there is a start and stop transfer sequence, both termini will be in the cytoplasm or in the lumen |
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Where do proteins in the non - secretory pathway end up
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Into specific organelles (i.e. mitochondria or peroxisome)
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How are proteins imported into the mitochondrial intermembrane space
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1) Protein is synthesized in the cytoplasm and held in an unfolded state by chaperones
2) The mitochondrial targeting sequence binds to a import receptor on the outer mitochondrial membrane 3) The import receptor moves the protein into a TOM40 general import channel, which the protein moves through |
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What is a TOM
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Translocon of the outermembrane
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What is a contact site?
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A site on the mitochondria where both membranes are close
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What is a TIM protein
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Translocon of the inner membrane
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Describe a general model for the import of a protein into the mitochondrial matrix
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1) Protein is synthesized in the cytoplasm and held in an unfolded state by chaperones
2) The mitochondrial targeting sequence binds to a import receptor on the outer mitochondrial membrane 3) The import receptor moves the protein into a TOM40 general import channel, which the protein moves through 4) The protein simultaneously moves through a TIM channel 5) A protease cleaves off the targeting sequence and Hsp70 binds the protein, and hydrolyzes ATP to move the protein into the matrix |
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How do we know that proteins are translocated into the mitochondria post translationally
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1) Proteins with a signal was fully synthesized in a test tube
2) One group was treated with a protease (trypsin) and the other has mitochondria added to it before trysin treatment 3) The first protein group was degraded but the second group was protected by the mitochondria 4) Therefore the protein was moved into the cell post translationally |
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How do we know that the targeting sequence actually directs proteins to the mitochondria?
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A protein that is normally in the cytoplasm is tagged with a matrix targeting sequence
When the protein is synthesized, the protein is moved into the mitochondria |
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How do we know that proteins are translocated in an unfolded state?
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- A spacer sequence is added to a synthetic DHFR protein between matrix targeting signal and the the normal sequence
- Methontrexate is added to the solution which causes DHFR to fold. - This causes it to fold before it completely translocates - This resulted in halted translocation |
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Describe a pathway for targeting the inner membrane (Path A)
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1) Protein has a N-term signal that import receptor TIM44 recognizes
2) The protein is moved through a TOM40 channel and through a TIM23/17 channel simultaneously. 3) Once a stop sequence reached, translocation is blocked and the protein moves laterally into the membrane |
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Describe a pathway for targeting the inner membrane (Path B)
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1) Protein has a matrix targeting sequence (recognized by TOM22 and TOM20) And a internal hydrophobic domain (recognized by Oxa1)
2) TOM40 -> TIM23/17 -> Hsp70 3) Oxa1 moves protein into inner membrane |
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Describe a pathway for targeting the inner membrane (Path C)
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1) Protein has no matrix targeting sequence but many internal mitochondrial targeting sequences (therefore will be a multipass protein)
2) TOM70 recognizes these sequences -> TOM22 -> TOM40 -> intermembrane space 3) TIM9/10 guides protein to pore made up of TIM22 and TIM54 which move the protein into the membrane |
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Describe a pathway for targeting the intermembrane space (Path A)
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1) Protein has a matrix and intermembrane targeting sequence
2) Recognized by TOM20 -> TOM22 -> TOM40 -> TIM44 -> TIM23/17 3) The second targeting sequence stops translocation and the protein moves into the membrane 4) A protease cleaves the protein near the transmembrane segment and the protein is released into the intermembrane space |
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Describe a pathway for targeting the intermembrane space (Path B)
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1) The protein has a intermembrane targeting sequence
2) It is recognized and transported into the intermembrane space through TOM40 |
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How do proteins get into the outer mitochondrial membrane
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Most TOM proteins move through TOM40
- It is thought that sorting & assembly machinery (SAM) translocate β-barrel proteins |
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How are proteins moved into peroxisomes?
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1) Protein contains peroxisomal targeting sequence 1 (PTS1 or PTS2) on its C-terminus
2) PTS1 binds to Pex5 receptor in the cytoplasm 3) Pex5-cargo complex moves to PEX14 receptor on the peroxisomal membrane 4) Pex5-cargo moves through a PEX2, PEX10, PEX12 complex into the peroxisome and dissociate 5) PEX5 moves back into the cytoplasm |
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Are proteins moving into a peroxisome folded or unfolded
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Folded
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What is a HnRNP and what does it do
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Heterogenous nuclear ribonucleoprotein particle
- Shuttles mRNA, tRNA and ribosomal subunits from the nucleus to the cytoplasm |
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Where does transport into/from the nuclear envelope pass through?
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Nuclear pores
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What is a NPC and what is its function
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Nuclear Pore Complex
- Passage of molecules into and out of the nuclear envelope |
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What are defining features of the NPC
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- 30X larger than a ribosome
- Basket-like structure - Ions/small molecules can diffuse through NPC - Larger proteins are selectively transported |
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What is a NLS and what is its function
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Nuclear localization signal
- Directs protein into the nucleus |
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What is a NES and what is its function
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Nuclear Export Signal
- Directs protein out of the nucleus - Short sequence of positive lys or arg |
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How are proteins imported into the nucleus
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1) Cargo binds onto importin molecule = cargo complex
2) Cargo diffuses through the NPC 3) Ran-GDP is bound by a nuclear transport factor in the cytoplasm and moved into the nucleus 4) A GEF changes Ran-GDP -> Ran-GTP 5) importin and ran-GTP interact, causing a conformational change in importin and a release of the cargo 5) importin-ran-GTP complex goes back into the cytoplasm 6) A GAP stimulates the hydrolysis of GTP and release of importin |
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What is a GTPase
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A molecular switch that is turned on when associated with GTP and turned off when associated with GDP
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What is a GAP
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A protein that accelerates the hydrolysis of GTP and turn GTPases off
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Why does the cargo complex diffuse into the nucleus?
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The low [cargo complex] in the nucleus due to rapid dissociation of importin-cargo (caused by Ran-GTP) creates a concentration gradient
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What does a high [GEF] in the nucleus cause?
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High [GEF] causes all the Ran-GDP to be converted to Ran-GTP in the nucleus, which dissociates the importin-cargo complexes, causing a low [cargo complex] in the nucleus
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What does a high [GAP] in the cytoplasm cause?
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High [GAP] in the cytoplasm causes all the importin-Ran-GTP to dissociate by hydrolyzing GTP to GDP, therfore increasing the [importin] in the cytoplasm and by that the [cargo complex]
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Describe what a cell fusion experiment is and what it proves
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- Human cells and frog cells are fused with treatment with polyethylene glycol
- Protein synthesis is stopped by treatment with cycloheximide - After time, human hnRNP C and hnRNP A1 ab are added - hnRNP A1 protein was seen to have moved into the frog nucleus, which shows that proteins can shuttle in and out of the nucleus |
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How are proteins moved out of the nucleus?
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1) A nuclear export receptor (exportin 1) binds to Ran-GTP, causing an increase in affinity for NES on cargo
2) This forms a trimolecular complex that moves out of the nucleus through a NPC 3) GAP stimulates GTP hydrolysis and the release of the cargo 4) Exportin1-Ran-GDP imported back into nucleus |
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What happens to proteins after synthesis in the ER?
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They are further modified and folded in the ER, then transported via vesicles to the golgi
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Define CGN
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Cis Golgi Network
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Define TGN
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Trans Golgi Network
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What happens during glycosylation of proteins in the ER
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One or more carbohydrate chains are added to proteins.
- Attached to hydroxyl groups on serine/threonine (O-linked) - Attatched to amide N on asparagine (N-linked) |
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Which is more common, O-linked or N-linked olgiosaccharides?
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N-linked
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What is the mechanism for N-linked glycosylation?
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1) Sugar units are added to a dolichol phosphate embedded in the ER membrane
2) N-acetylglucosamine (GlcNAc), and mannose residues are added to oligosaccharide chain 3) A flippase translocates the dolichol phosphate molecule onto the ER lumen side of the membrane 4) More glucose and mannose units are added 5) Oligosaccharide transferase catalyzes the movement of the oligosaccharide onto a residue on a protein |
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What are two functions of an olgiosaccharide?
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1) Protein folding/stabilization
2) Cell surface interactions |
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How are proteins folded after they are modified in the ER
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1) Lectins (calnexin and calreticullin) bind to certain N-linked olgiosaccharides and promote disulfide bond formation
2) Proteub disulfide isomerase (PPI) catalyzes the formation of disulfide bonds 3) Chaperone BiP binds to the polypeptide chains and stabilizes |
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How are improperly folded proteins dealt with in the ER?
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1) Unfolded protein response
2) ER-associated degredation (ERAD) |
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Define the unfolded protein response
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Sensor molecules in the ER membrane activate signalling pathways when misfolded proteins are detected. This stimulates increased production of proteins that are functionally associated with protein folding and/or degredation
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Define ER-associated degredation (ERAD)
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Misfolded proteins are retrotranslated out of the ER, tagged with ubiquitin and degraded by a proteosome
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Define anterograde movement
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CGN -> medial cisternae -> TGN
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How do we know which direction proteins move through the endomembrane system?
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1) Temperature mutant proteins are grown so at base temperature they remain in the ER
2) At other temps they move to the golgi = modification 3) Proteins are treated with endoglycosidase D 4) Modified proteins have carbohydrate chains cleaved while those that remained in ER have their carbohydrate chain attached |
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How do vesicles bud off their parent membranes
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1) Budding is initiated by recruitment of a small GTP binding protein to a patch of membrane
2) Coat proteins then bind to cytoplasmic domain of cargo proteins 3) Membrane cargo proteins also contain sorting signals specific to a polymerized coat 4) Coat proteins provide curvature and the polymerization of the coat drives budding |
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Describe the mechanism of the assembly of vesicle coats
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1) Sar1-GDP approaches membrane
2) ER membrane protein (Sec12) catalyzes the release of GTP 3) GTP binding causes N-term of Sar1 to be exposed and embed in the membrane 4) Sar1-GTP induces assembly of coat 5) Once free vesicle is formed a membrane protein induces GTP hydrolysis and the coat is shed |
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How do vesicles fuse with the appropriate target?
(describe the SNARE hypothesis) |
1) Rab (A GTPase) associates with the vesicle
2) Tethering proteins on the recieving membrane capture the vesicle 3) Rab stimulates the association of t-SNARE with the v-SNARE which promotes membrane formation 4) The binding of NSF and SNAPs promote dissociation of SNARE complex |
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Define NSF
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N-ethylmalemide-sensitive factor
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Define SNAPs
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Soluble NSF attachment proteins
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Describe retrograde transport
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Soluble proteins that function in the ER are passively carried to the golgi and recycled back into the ER
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What sequence does a soluble ER protein carry
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Lys-Asp-Glu-Leu (KDEL)
- C term - Recognized by a KDEL receptor which moves from golgi to ER |
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What causes cystic fibrosis
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The failure of a chloride channel (CTFR) to reach the plasma membrane in epithelial cells.
- a mutated CTFR protein masks the signal that carries it to the membrane |
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How do proteins move through the golgi?
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The golgi cisternae and the proteins in them mature by the movement of the entire cisterna. New cisterna push the old ones forward
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Define Vesicular Transport Stable Compartment
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Each cisterna represents a stable compartment and does not change over time
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Define Cisternal Maturation
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Cisterna form by fusion of ER vesicles and mature from cis to trans then dissapte
- Each cisterna carries material forward |
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What do clathrin coated vesicles do?
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Fuse with the endosome
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Describe clathrin molecules
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3 limbed shape, and can polymerize together to form a lattace
have an light chain and a heavy chain |
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How many layers does a clathrin vesicle have
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Two. An outer clathrin layer and an inner layer made up of adaptor protein complexes
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How do clathrin vesicles determine which cargo proteins to include?
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AP complexes bind to the cytoplasmic face and determine which cargo proteins will be included
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What are the 4 kinds of AP complexes?
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AP1, AP2, AP3 and GGA
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Where does a clathrin coated vesicle with AP1 or GGA go?
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An endosome
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Where does a clathrin coated vesicle with AP3 go?
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Directly to golgi
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How to clathrin vesicles pinch off?
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Dynamin uses GTP hydrolysis to pinch off vesicles
- buds can from without GTP but cannot pinch off - COPI and COPII vesicles do not need dynamin |
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Where do COPI and COPII vesicles go?
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Back to the ER
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What is the signal for a protein to go to a lysosome?
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A carbohydrate: Mannose-6-phosphate (M6P)
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Describe how enzymes bound for a lysosome get there
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1) Protein undergoes N-glycosylation follwed by glucose and mannose removal
2) M6P is phosphorylated by enzymes in CGN 3) M6P finds a receptor in the TGN 4) A clathrin coated vesicle travels to an endosome 5) low endosomal pH causes release of enzymes from receptors |
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How do early endosomes form?
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Merging trans golgi vesicles
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What causes the change from a early endosome to a late endosome?
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Drop in pH from 6 to 5.5
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Describe enzyme replacement therapy
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Cytoplasmic membrane proteins recognize secreted enzymes and reincorporate them back into the cell
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What are the two types of exocytosis?
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1) Constitutive Secretion
2) Regulated Secretion |
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Define Constitutive Secretion
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Secretion occurs continuously
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Define Regulated Secretion
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Secretion only occurs when a signal occurs, otherwise vesicles aggregate and wait in cytoplasm
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Define endocrine signalling
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Hormones target cells far from site of synthesis
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Define paracrine signalling
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Signal molecules only affect nearby cells
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Describe how an action potential occurs and the events around it
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1) Na+/K+ ATPase pumps Na+ out of the cell and K+ into the cell
2) K+ flows down its concentration gradient through K+ channels out of the cell, polarizing the membrane 3) A slight depolarization causes Na+ voltage gated channels to open and the flow of Na+ in causes a postive charge to grow in the membrane 4) Voltage gated K+ channels open and K+ flows out of the cell = negative membrane |
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How do voltage gated Na+ channels open?
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1) In the resting state, protein blocks the pore
2) A small depolarization causes two of the alpha helicies to move towards the outside, causing the pore to open 3) After 1ms the inactivating segment blocks the pore until the membrane is depolarized (refractory period) |
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Where are all action potentials initiated?
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The axon hilock
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What does the myelin sheath do?
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Speeds up the signal by reducing the sites where an action potential can occur
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What are myelin sheaths made of in the CNS?
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Oligodendrocytes
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What are myelin sheaths made of in the PNS?
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Schwann Cells
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What happens to a chemical messenger when it reaches the target tissue?
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It binds to a receptor which results in the production of second messengers
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Define Second Messenger
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Small molecules or ions that amplify a signal
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What are 4 types of secondary messengers?
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1) cAMP
2) cGMP 3) diacylglycerol (DAG) 4) inositol triphosphate (IP3) |
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How does receptor-ligand bonding alter cellular activites?
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Binding of a ligand often activates receptors
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What are two classes of GTPase switches
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1) Trimeric G proteins
2) Monomeric G proteins |
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What is the difference between tirmeric G proteins and monomeric G proteins?
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Trimeric directly bind to and are activated by receptors
Monomeric are indirectly linked to receptors via adaptor proteins |
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What do kinases do?
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Add or remove phosphate groups from other proteins
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Describe the G protein coupled receptor (GPCR) pathway
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1) Resting state: GDP is associated with Gα and Gα is bound to Gβγ
2) Ligand binds to receptor, GPCR binds to G-protein complex and Gα trades GDP for GTP 3) Gα separates from G-protein complex 4) both Gα and Gβγ go and activate target proteins 5) GTP in Gα is hydrolyzed and becomes inactive 6) G-protein complex reforms |
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How do G proteins regulate the release of cAMP
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Gα binding to adenylyl cyclase release cAMP from ATP as a second messenger
- GTP hydrolysis occurs quickly and Gα becomes inactive, and the remaining cAMP is degraded by phosphodiesterase |
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What does the release of cAMP do?
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Stimulates the release of a catalytic subunit of PKA, which phosphorylates and activates a transcription factor CREB
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What happens when acetylcholine binds to GPCR
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Results in Gβγ binding to a K+ channel and opening it in heart muscles. This causes contraction.
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How are IP3 and DAG made?
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IP3 and DAG are produced when phopholipase C cleaves PIP2
- IP3 is released into the cytoplasm and DAG stays in the membrane |
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What is calmodulin and what does it do?
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Calmodulin is a Ca2+ binding protein that modulates the activity of proteins with a calmodulin binding site when calmodulin is associated with Ca2+
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How do protein kinase associated receptors work?
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When bound to a ligand, two receptor kinases cluster in pairs and cross phosphorylate each other over several tyr residues
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Autophosphorylation
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The phosphorylation of a kinase protein catalyzed by its own enzymatic activity
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Describe the receptor tyrosine kinase signal transduction pathway
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1) Receptor is coupled and phosphorylated
2) Proteins (PLC & GRB2) carrying a SH2 domain bind 3) PLC stimulates the release of IP3 4) GRB2 recruits Sos (a GEF) which activates Ras 5) Ras triggers Raf which triggers MEK which triggers MAP 6) MAP enters nucleus where it phosphorylates trascription factors 7) Ras is deactivated by GAP |
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What are the 3 types of filaments
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1) Microtubules
2) Microfilaments 3) Intermediate Filaments |
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Define microtubules and their function
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Hollow tubulin cylinders that function in transport, support and mitotic spindle
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Define Microfilaments and their function
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Polymers of protein actin, functions in cell movement and structure
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Define Intermediate Filaments and their function
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Fibrous protein functioning in tension bearing, and maintenance of shape/rigidity of the cell
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Define desmosomes and their function
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Specialized cell junction indirectly associated with intermediate filaments that mediate cell to cell adhesion
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Define hemidesmosomes and their function
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Specialized cell junction indirectly associated with intermediate filaments that mediate cell to extra cellular matrix adhesion
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List 5 features of intermediate filaments
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1) Many different intermediate filaments exist and are expressed in a tissue dependent manner
2) Have tensile strength 3) No polarity 4) No motors use them 5) Less dynamic |
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How are intermediate filaments assembled?
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α-helical rod domain (dimer) associates with others to form an antiparellel tetramer
tetramers associate end to end to form protofilaments which associate into larger filaments |
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Where do microtubules originate from?
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Originate from γ-tubulin proteins in microtubule organizing centers (MTOCs)
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How do microtubles elongate?
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α-tubulin and β-tubulin add on to the γ-tubulin (γ-α-β) until [tubulin] limits elongation (plateau phase). This inbalance is corrected by disassembly
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Dynamic instability
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The sudden disassembly followed by regrowth of microtubules.
-regulated by the hydrolysis of GTP by β-tubulin |
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Describe the dynamic instability model
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both α-tubulin and β-tubulin must bind to GTP but only β-tubulin hydrolyzes it.
GTP hydrolysis is slower than the addition of dimers so dimers near the beginning have no β-tubulin bound GTP and β-tubulin near the beginning is still GTP bound. as [tubulin] increases, more β-tubulin is added to the end as [tubulin] decreases, less β-tubulin is added and GTP hydrolysis catches up = no GTP cap = castastrophe |
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What regulates microtubules in cells?
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Microtubule associated proteins = MAPs
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What dies kinesin 13 do?
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Curves ends of microtubules to from curved GDP-β-tubulin = catastrophe
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What does stathmin do?
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Curves dimers to from curved GDP-β-tubulin = catastrophe
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What does katanin do?
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Promotes disassembly by breaking parts of the microtubule
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