vesicular traffic and secretion Flashcards
how is the transport of cargo proteins in the secretory pathway governed?
by a series of budding and fusion reactions from the donor compartment to the target compartment
what two types of techniques were used to investigate vesicular transport?
- genetic approaches
- biochemical approaches
what did schekman do in his genetic experiments regarding vesicular transport?
generated yeast mutants with defects in secretion using ionising radiation
what biochemical techniques did Rothman use to measure transport of proteins?
cell free transport assays - this allows for fractionalisation of the cytosol and precise characterisation of protein complexes
how were yeast budding experiments used for investigation of proteins involved in secretion?
- temperature sensitive mutants were created
- these can be transferred from lower to higher temperatures and will accumulate vesicles in the part of the secretory pathway that their mutation blocks
why are yeasts a good model organism for the study of vesicular transport?
they are single celled but do secrete some enzymes
what is the defective function of class A mutants?
- transport into the ER
- these accumulate in the cytosol
what is the defective function of class B mutants?
- budding of vesicles from the rough ER
- vesicles accumulate in the ER
what is the defective function of class C mutants?
- budding of vesicles from rough ER
- accumulation in ER to Golgi transport vesicles
what do yeast budding assays tell us about vesicular transport?
studies confirm that a secreted protein is synthesised and processed, it moves from the cytosol to the rough ER -> transport vesicles -> Golgi cisternae -> secretory vesicles -> protein is exocytosed
what did Rothman measure in cell free assays?
incorporation of radio labelled N-acetylglucosamine into the oligosaccharide on proteins in vesicles in cells with defective or wild type NAG transferase function
what is the function of protein coats on vesicles?
to facilitate curvature of the membrane and selection of cargo through distortion of the membrane
how is budding initiated?
recruitment of small GTP-binding proteins to a patch on the donor membrane, complexes of coat proteins in the cytosol then bind to the cytosolic domain
outline the steps of protein coat formation
1- four active GTP binding proteins associate with the forming bud
2- The GTP binding proteins recruit the components of the protein coat
3- the protein coat components are able to selectively bind to cargo proteins via membrane receptors
4- these receptors can bind to soluble cargo proteins to selectively incorporate these into vesicle formation
what type of transport do COPII coated vesicles mediate?
anteretrograde transport
which type of transport do COPI coated vesicles mediate?
retrograde transport
what is anteretrograde transport?
when newly synthesised proteins are transported from the ER to cis golgi
what does Brefeldin A do?
blocks anteretrograde transport
how can brefeldin A be used experimentally?
provides evidence for retrograde transport - if there was no retrograde transport, when brefeldin A is added to culture, the protein would remain localised to the Golgi
what is Arf2?
a monomeric GTPase that controls COPI and clathrin coat assembly at Golgi membranes
what is Sar1?
a monomeric GTPase that mediates COPII coat assembly at the ER membrane
what is a Rab protein?
a family of monomeric GTPases that are associated with one or more membrane enclosed organelles in the secretory or endocytic pathways
what is the purpose of Rab GDI?
- dissociation inhibitor
- keeps Rab soluble and inactive
which sorting signal is found on ER membrane resident proteins?
KKXX - found at C terminus, binds directly to COPI coats
which sorting signal is found on soluble ER resident proteins?
KDEL - binds to KDEL receptor
what is the purpose of sorting signals?
ensure ER resident proteins are returned to the ER via COPI coated vesicles
how do we know that sorting signals aren’t the only mechanism for ER retention?
mutants with KDEL deletions still secrete proteins, but this is slow
how does the SNARE complex mediate vesicle fusion?
- transport vesicles have a v-SNARE called VAMP
- VAMP carries Rab, which binds to the target effector when in GTP bound form
- in the target membrane, the SNARE is called syntaxin and SNAP-25
- tails of the t-SNARE and v-SNARE associate to form a coiled structure
what is the function of the SNARE complex?
holds the vesicle close to the target membrane and assists fusion
