Vesicular trafficking Flashcards
What are the 3 main trafficking methods within cells?
- Gated transport
- Transmembrane transport
- Vesicular transport
What kind of trafficking occurs between the nucleus and the cytosol?
Gated transport
What is the structure of the nuclear pore? (3)
- Brings together the double nuclear membrane
- Complex consists of nucleoporins
- Each complex is made of 8 subunits with a central plug
What is the function of the nuclear pore? (3)
- Involved in moving substances across the nuclear envelope
- Histones are made at ribosomes in the cytoplasm and need to get into the nucleus to package the DNA
- Ribosomal subunits are made in the nucleolus and have to be transported into the cytoplasm
What are the 2 processes by which substances are transported by the nuclear pore complex?
- Diffusion
- Active transport
What is required for active transport through the nuclear pore? (2)
- Signal
- ATP
What signal is required for active transport through the nuclear pore?
An amino acid sequence that is rich in lysine, arginine and proline
What is an example of a signal in transport through the nuclear pore? (2)
- T-antigen of the SV40 virus contains the sequence Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val
- Mutation of a single amino acid in the sequence prevents nuclear translocation
What experiment would show that an amino acid sequence is necessary and sufficient to cause nuclear transport?
Put the sequence onto a protein that wouldn’t normally enter the nucleus and show that it causes nuclear translocation
What experimental evidence shows that active transport is used in nuclear translocation? (2)
- Cool cells so they can’t make ATP, inhibits mRNA transport out of the nucleus
- In the absence of ATP proteins bind to the pore complex but aren’t translocated, add ATP and they appear in the nucleus
What are the 2 ways in which newly made membrane proteins can be translocated into the ER?
- Co-translational translocation
- Post-translational translocation
What is co-translational translocation?
Protein is being fed into the lumen of the ER as it is being translated by the attached ribosome
What is post-translational translocation?
Proteins are fully made in the cytoplasm by a free ribosome and then transported into the ER after translation
What is the most common way in which secreted/membrane proteins are transported into the ER?
Co-translational translocation
What is the signal hypothesis?
Protein made in the cytoplasm that are targeted to the ER use a signal to direct them to the ER membrane
What are microsomes?
Isolated ER membrane preparation
How is a soluble protein destined for secretion made via co-translational translocation? (5)
- Translocator protein on the ER is usually closed
- Ribosome translating the mRNA is closely associated with the translocator
- Peptide signal in the protein causes the translocator to open and the protein is fed through into the ER lumen as it is being translated
- Peptide signal is cleaved by a signal peptidase
- Full translated polypeptide chain is folded by chaperones in the ER lumen
How are type I membrane proteins inserted into the ER? (3)
- Start-transfer sequence is recognised by the translocator protein
- Protein is fed through the translocator until it reaches a hydrophobic stop-transfer sequence
- Start-transfer sequence is cleaved off and the stop-transfer sequence remains in the membrane so the N-terminus is in the ER and the C-terminus is in the cytosol
What is the difference between type I and type II transmembrane proteins?
Type I transmembrane proteins have a cytoplasmic C-terminus and an extracellular/luminal N-terminus whereas type II transmembrane proteins are the other way round
How are type II membrane proteins inserted into the ER? (3)
- Signal sequence is recognised by the translocator
- N-terminus end remains in the cytosol and the C-terminus end is fed through into the ER lumen
- The signal sequence acts as the transmembrane domain
How is proper protein folded ensured in the ER? (3)
- BiP chaperone associates with newly made proteins until they are folded correctly
- Proteins are retained in the ER lumen until they are correctly folded
- Misfolded proteins are reverse translocated into the cytoplasm and degraded
What is the function of glycosylation in the ER?
Ensures good quality control
How can protein misfolding cause disease?
CFTRdelta508 mutation causes misfolding in CFTR which is recognised by quality control machinery so the mutant CFTR is retained in the ER membrane
What is triggered by a high number of misfolded proteins in the ER?
Unfolded protein response (UPR)
What happens in the UPR? (3)
- Stop translation
- Upregulate synthesis of chaperones
- Cells can recover but if the cell is overwhelmed apoptosis is triggered
Which method is used to get proteins into the mitochondrial matrix?
Post-translational translocation
How do proteins get into the mitochondrial matrix? (3)
- Fully translated proteins have an N-terminal signal sequence which is recognised by the TOM complex on the outer mitochondrial membrane
- Protein translocates into the matrix via the TIM23 complex on the inner mitochondrial membrane
- Signal peptide is cleaved by signal peptidase
What is the signal for mitochondrial translocation? (3)
- Amphipathic alpha helix structure
- One side of the structure is hydrophobic, other is hydrophilic
- The hydrophobic residues bind in a hydrophobic groove in the receptor associated with the TOM complex
How are proteins inserted into the outer mitochondrial membrane? (2)
- Polypeptide translocates into the intermembrane space
- Chaperones in the intermembrane space help to assemble the protein in the outer membrane with the SAM complex
How are proteins inserted into the outer membrane of bacteria? (3)
- Protein is translocated into the periplasmic space and associates with chaperones
- Protein is inserted into the outer membrane via the BAM complex
- Similar process to mitochondria
What are the similarities between insertion of membrane proteins in mitochondria and chloroplasts? (4)
- Both occur post-translationally
- Both require energy
- Use membrane potential as well as energy to drive the process
- Signals are similar but the proteins aren’t mixed up in plant cells
What can vesicles be coated with? (3)
- Clathrin
- COPI
- COPII
What are the essential components for transport vesicle formation? (3)
- GTPase
- Adaptor proteins
- Coat
What is the function of adaptor proteins? (3)
- Recognise and select cargo
- Link the coat to the ER membrane
- Recognise motifs in the cytoplasmic domains of membrane proteins
What GTPases are in the Ras superfamily? (5)
- Ras
- Rabs
- Arfs
- Ran
- Rho
How do small GTPases work? (3)
- Inactive when bound to GDP
- GEFs cause the exchange of GDP for GTP to activate the GTPase
- GAPs cause hydrolysis of GTP to GDP to inactivate the GTPase again
What is a GEF?
Guanine nucleotide exchanging factor
What is a GAP?
GTPase activating protein
Which GTPase is required for nuclear transport?
Ran
How is Ran involved in nuclear transport? (6)
- Ran-GDP is in the cytosol
- Ran-GTP is in the nucleus because the GEF is localised to chromatin
- Cargo binds to nuclear import receptors to enter the nucleus
- Ran-GTP binds to the import receptor causing the cargo to dissociate in the nucleus
- Ran-GTP bound to the receptor exits the nucleus, GAP causes GTP hydrolysis
- Ran-GDP dissociates from the receptor which is free to pick up more cargo
What kind of vesicles form at the exit sites of the ER?
COPII coated vesicles
What are the components of COPII coated vesicles? (3)
- GTPase: Sar1 (Arf family)
- Adaptor: Sec23/24
- Coat: Sec13/31
How are COPII coated vesicles formed? (5)
- Cargo exit signal binds to cargo receptor in the ER membrane
- GEF in the ER membrane activates sar1
- Allows recruitment of adaptor complex, sec23 binds to sar1, sec24 binds to the cargo
- Adaptor sec23/24 complex recruits coat proteins sec13/31
- Bud pinches off to form coated vesicle
How are ER resident proteins excluded from budding vesicles?
High surface area to volume ratio of the bud
What is the function of coat proteins?
Provide a structural scaffold
How can ER be isolated from cells for reconstitution experiments? (4)
- Homogenise cells
- ER vesiculates into rough and smooth microsomes
- Centrifuge the mixture in a tube with a gradient of increasing sucrose concentration
- Smooth and rough microsomes float at different sucrose concentrations so can be separated
How did reconstitution experiments demonstrate the components of COPII vesicles? (4)
- ER membrane preparation containing ribophorin (known ER protein)
- COPII vesicles containing p58 (known vesicle protein)
- Vesicles and ER pellet at different sucrose concentration
- Found that they needed to add cytosol, ATP and GTP to ER membrane in order to form COPII vesicles
How are COPII vesicles disassembled? (3)
- Sec23 acts as a GAP for sar1
- GAP activity of sec23 is enhanced by recruitment of the coat sec13/31
- Causes GTP hydrolysis, inactivation of sar1 and disassembly of the coat
What are the 2 types of GTPase mutants?
- GTP mutants (constitutively active, can’t hydrolyse GTP)
- GDP mutants (can’t exchange GDP for GTP, sequester GEFs)
What are COPII vesicles used for?
ER budding to deliver material to the Golgi complex
What are COPI vesicles used for?
Delivers material in an anterograde and retrograde direction through the Golgi
What are clathrin coated vesicles used for?
Delivers material from the TGN to lysosomes and also bud from the cell surface
What cargo are COPII coated vesicles used for?
Newly synthesised proteins
What cargo are COPI coated vesicles used for?
Retrieved and newly synthesised proteins
What cargo are clathrin coated vesicles used for? (2)
- Lysosomal proteins
- Regulated secretory proteins
Which GTPase is required for COPII vesicle formation?
Sar1
Which GTPase is required for COPI vesicle formation?
Arf1
Which GTPase is required for clathrin vesicle formation?
Arf1
What is the structure of AP2 (adaptor protein)? (3)
- 2 large subunits which bind the coat
- 2 flexible appendage domains
- 2 smaller subunits which recognise signals in transmembrane proteins
Which proteins are important for endocytosis? (2)
- AP2 adaptor
- Clathrin coat
How is large cargo packaged?
COPII coat is modified which sorts the cargo into tubular structures for transportation
Which GTPase superfamily do the Rab GTPases belong to?
Ras superfamily
What are the features of Rab GTPases? (3)
- Cycle between GDP-bound Rab in the cytoplasm and GTP-bound Rab in membranes
- Required for membrane fusion
- Different ones are associated with specific membranes
What is the role of Rabs in fusion?
Rab-GTP on a vesicle recruits a tethering protein which facilitates the formation of SNARE complexes by bringing the membranes close together
What is the role of Rab cascades? (2)
- GAP for the previous Rab can recruit the GEF for the Rab in the next compartment where the cargo is being delivered to
- Allow movement of cargo between organelles
What disease is caused by a Sec23A mutation? (2)
- Cranio-lenticulo-sutural dysplasia
- Problems with the secretory pathway results in issues with ECM formation
What disease is caused by a Rab mutation? (2)
- Charcot-Marie-Tooth 2B
- Causes too much autophagy which inhibits neurite outgrowth
