Microbiology 13: (Prigent) Vesicular traffic and secretion Flashcards
2 approaches which identified the key components of vesicular traffic in the secretory pathway?
Genetic approaches (Shekman)
- generated yeast mutants with defects in secretion
- relatively quick + allows identification of gene responsible for phenotype
Biochemical approaches (Rothman)
- Use of cell-free transport assays
- Allows fractionation of cytosol for precise characterisation of protein complexes
Describe and explain Shekman’s findings in his mutant yeast experiment
Mutant yeast cultured which had temperature sensitive protein -> mutagenis at 23 degrees to allow folding, heating to 36 degrees to express phenotype
Many ‘Sec’ mutants identified -> pheotypes put into classes A - E depending on function in pathway (Vesicular docking, budding etc.)
Describe and explain Rothman’s cell-free transport assays
Golgi cells in 2 batches, one with and one without NAG-transferase enzymes infected with VSV
Transfer of NAG did not occur in mutant medial-golgi to glycoprotein
Cells from mutant were added to isolated, uninfected wild-type cells
- radiolabelled NAG added
- addition of this radiolabelled NAG monitored
- fusion of mutant carrying VSV protein to WT cells allowed addition of NAG, showing NAG-transferase function
What are the 2 vesicular coating proteins and their functions?
COPII
COPII coated vesicles transport newly synthesised proteins containing golgi-targetting sequences in their cytosolic domain to the cis-golgi (anterograde direction)
COPI
COPI coated vesicles transport vesicles carrying ER/golgi-resident proteins in the retrograde direction
What does Brefeldin A block?
anterograde transport
evidence for retrograde transport
Which proteins regulate vesicle coat protein formation and where?
Monomeric GTPases
Arf proteins mediate COPI and clatherin coat assembly at golgi membranes
Sar1 protein mediates COPII assembly at the ER membrane
Function of Rab proteins? Molecule bound in different forms?
Guide vesicle targeting
> 60members in rab family
- Rab-GDI (GTP dissociation inhibitor) keeps rabs soluble and inactive
- Membrane bound Rab-GEF (GTP exchange factor) activates Rabs via GTP binding
- Rab-GTP binds Rab effectors on membranes that mediate vesicular transport, membrane tethering and fusion
2 sorting signals and their purpose? Where are they located? How can recombinant technology be used in this context?
Sorting signals ensure resident proteins are returned to the ER via COPI coated vesicles
- small amino acid sequences
KKXX - found at C-terminus of ER membrane proteins, binds directly to COPI
KDEL - found on soluble resident ER proteins, binds to KDEL receptor
Recombinant technology can be used to add signals (KDEL) to protein’s C-terminus resulting in ER retention
- sequences can also be deleted to manipulate function
What follows after process of coat shedding?
Exposure of Rab and SNARE proteins
- Target vesicles for fusion with specific target membranes
2 types of snare protein?
v-SNARE = vesicle SNARE t-SNARE = target SNARE
After Rab-GTP binding with Rab effector - which proteins form a complex known as the SNARE complex and where are they located? What follows?
VAMP - from vesicle membrane
Syntaxin - from target membrane
SNAP-25 - from target membrane
form tightly coiled coiled-coil complex stabilised by non-covalent interactions
formation of complex drives membrane fusion
What is required for SNARE complex dissociation
NSF and ATP required for SNARE dissociation
Overview of later stages of secretion in trans-golgi
trans-golgi networks sort proteins into vesicles targeted for different destinations
Lysosomal enzymes bear M6P tags recognised by M6P receptors -> delivered by clathrin coated vesicle pathway to lysosomes
proteins stored until secretion is signalled -> once signalled, proteins delivered to plasma membrane
