ENDOCYTOSIS 2 Flashcards
RABs and PIs - what is their main function and where do they function?
Define early endocytic intermediates.
Act as sorting stations at the EARLY ENDOSOME – cargo enters this compartment and is then sorted to the correct place.
Where are RABs and PIs found and how can mutants in Rabs help us?
Found cycling between the cytosol and correct target membrane
Mutants Rabs can help us to properly define their functions
RAB family - what are Rabs, where are they found and what is their function?
- Rab proteins are part of the Ras protein superfamily and are small GTPases that often give rise to cancers when they are mutated
- They are localised to different sub compartments of cells and cycle between the membrane and cytosol. Rabs regulate specific trafficking and are needed for vesicle fusion with the target membrane
Rab cycle – what are the two states of Rabs and what proteins mediate these states?
What is the advantage of this switch mechanism?
Rab/GDP —– G.E.Fs —– > Rab/GTPs
Switch mechanism allows for rapid and regulated response. This is advantageous as Rabs have a low intrinsic rate of GTP —> GDP + Pi
Outline the rab cycle
GEF (often embedded into the plasma membrane) converts GDP–>GTP allowing Rab to bind GTP (active confirmation)
Rab incorporated into vesicle and pinches off to take to target membrane
Rab effectors (activated by active Rab) found in the target membrane aid in membrane fusion
GAPs convert GTP–>GDP, inactivating Rab
GDI scoops GDP out of the membrane and recycles back to cell cytoplasm for activation again – cycle repeats.
RAB5 - what are its major roles?
Where is it localised?
What does it define?
Major roles in endosomal fusion eg tethering, movement of endosomes and cycling of clathrin coated vesicles.
Localises to endocytic pathway and defines membrane domains in the endocytic pathway
Phenotype in cells where RAB5 OVEREXPRESSED?
Formation of enlarged endosomes
Why are different membranes of the area enriched in different Rabs?
Localised areas of the membranes are enriched in particular Rabs so that particular membranes are organised to have particular domains
PHOSPHOINOSITIDES – what are they? where are they found and why are they important?
How are they useful in visualisation experiments?
PIs are low abundant lipids present in the membrane - - they are important for endosomal sorting and define particular organelles by working alongside adaptor proteins.
By tagging with GFP can see which proteins correlate with which PIs to visualise protein localisation
RAB5 PULL DOWN ASSAY - what are pull down assays and why is one particularly useful in the case of Rabs/Rab5?
Pull down assays reveal the physical interactions between 2+ proteins.
Particularly useful in the case of Rab proteins as they are found in active and inactive forms by binding to GTP and GDP respectively.
WHat was revealed in the Rab5 pull down assay – and what other proteins were involved?
Rab5 involved in REGULATION OF EVENTS IN EARLY ENDOCYTIC PATHWAY, such as tethering by tethering proteins (which are often Rab effectors)
Other proteins:
EEA1 - coiled coil protein recognises and tethers incoming vesicle
RABef5 - RAB exchange factor 5 - found bound to GTP activated version
Rapaptin - involved in fusion of the vesicle with the target
How do Rabs select particular proteins to participate in different roles?
Rabs can establish membrane microdomains!!!!
How do Rabs establish microdomains (use Rab5 as an example)
Activate Rab5»_space; Recruits Rabaptin which recruits and activates PI3K»_space; detects Rab5 effectors»_space; recognised by EEA1»_space; tethers incoming vesicle allowing membrane fusion
Sorting material from early to late endosome - how is cargo selected to be sorted into the MVB?
SIGNAL IS REQUIRED - ubiquitin!
Process of sorting vesicles from the early to late endosome?
- Ubiquitin binds to receptor in the early endosome, causing INWARD BUDDING and pinching off (SEQUESTRATION)
- Vesicles forming inside leads to formation of MVB
- Vesicles then either recycled to the plasma membrane or taken to the lysosome to be degraded
