Endocytosis and trafficking (lect7-8) Flashcards
where in the endocytosis pathway are sorting decisions made?
PM, sorting endosome, late endosome
what is pinocytosis?
when the cell “drinks” the ECM randomly, without signal needed
what type of endocytosis is rafts vs non-rafts
clathrin-mediated is associated with non-rafts;
caveolae is associated with rafts
what are the main characters of clathrin-mediated endocytosis?
cargo receptors and molecule, clathrin, adaptin, dynamin, PIP2, Rab5, SNAREs
name cargo proteins of clathrin-mediated endocytosis and where they are most found
- present in coated pits = LDLR, transferrin
- recruited to coated pits = EGFR, PDGF receptor
- excluded from coated pits = ion channels
what is the clathrin coat triskelion made of?
heavy chain (192 kDa) + light chain (36 kDa) organized in trimers via their c-terminus
what does the C vs N terminus of the clathrin triskelion each do?
c-terminus holds the triskelion together;
n-terminus mediates protein-protein interactions
what is the most important adaptor protein for clathrin?
AP2: found in coated pits and selects proteins that concentrate in coated pits
what are the subunits that make AP2? (and others) and what do they each bind?
- alpha and beta2: large subunits that each bind one PIP2
- u2: binds YXXO and PIP2
- o2
what AP2 subunit maintains the configuration open and how?
u2 subunit by being phosphorylated by AAK-1
apart from phosphorylation, what favors AP2 to be in open configuration?
binding to PIP2
what is the actual name for PIP2?
Phosphatidylinositol 4,5 bisphosphate
where is PiP2 found?
in the intracellular leaflet
in what case is PIP2 an important signaling molecule?
when it is a substrate of phospholipase C
what kind of protein is dynamin? function?
a small GTPase that hydrolyzes GTP to constrict the neck of the forming clathrin-coated vesicle
what is the proposed sequence of events of clathrin coat assembly?
1) Binding of AP2 to PIP2:
2) AAK1 phosphorylates u2 subunit: u2 set in “open” configuration
3) Binding of AP2 to an internalization sequence: stabilizes the “open” state
4) Clathrin coat assembly
what causes the invagination of clathrin-coated pit? (2 things)
- actin microfilament polymerization which bind to the coat via Epsin protein
- Epsin binds PIP2 and promotes curvature by inserting an amphipathic alpha-helix in the cytoplasmic leaflet
what causes the budding of clathrin coat vesicle?
- amphiphysin and endophilin associate with a curved-lipid surface and couple with the growing actin to help narrowing the neck
- Dynamin activates & hydrolyzes GTP
what are the steps of uncoating of clathrin-coated vesicles
1) Auxilin protein: associates with the hub region of the triskelion
and recruits Hsc70
2) Hsc70 hydrolyzes ATP and associates with the N-terminus of the coat
what is Hsc70?
a chaperone protein
what is synaptojanin?
an enzyme bound to the membrane that converts PIP2 to PIP
what is most likely the way how clathrin coat disassembles? explain
via phosphoinositides remodeling:
- synaptojanin enzyme converts PI-4,5-P2 to PI-4-P -> no more PIP2, AP2 can’t bind the subunit anymore and closes
what are the 2 other hypothesizes (apart from phosphoinositides remodeling) of how clathrin vesicles uncoat?
- H+ pump (ATP dependent) acidifies the budded vesicle once sealed
or - drastic fall in Ca2+ due to a Ca2+ channel that exports Ca2+ out of the vesicle
what is EEA1?
early endosome antigen 1: binds activated Rab5 on early endosome and PI3P also on early endosomes and endocytic vesicles
how does the now uncoated clathrin vesicle fused with the early endosome?
- EEA1 tethers the 2 membranes via Rab5 and PI3P
- Rab5 then causes EEA1 to collapse and that pulls the two membranes closer together
- membrane docking via SNAREs
remember what is V vs T snare?
v-SNARE = vesicle
t-SNARE = target (in case of clathrin vesicles, it’s the early endosome)
name functions of Rab5
- fusion of uncoated vesicle with endosome via EEA1 and PI3P
- promotes binding of vesicle to microtubules
- Rab5GTP activates SNAREs to promote docking and fusion
- Rab5GTP in sorting endosomes: recruits its own deactivators and activate Rab7GTP -> mature to late endosome
how do snares fuse membranes?
- V and t SNAREs fuse together via their n-terminal
- the bilayers fuse
- fusion pore appears
- SNARE’s four helix bundle rearranges to a more stable conformation, which release energy required for membrane fusion
what is pinocytosis and what is it used for?
bulk-phase uptake: retrieve synaptic vesicles under intense stimulation when clathrin machinery is saturated
what is faster, clathrin-mediated endocytosis of pinocytosis?
clathrin-mediated (15s)
give an example of a marker for clathrin-coated pits
transferrin
how can you test if your protein is internalized by clathrin or by bulk phase?
by measuring the colocalization of your protein with transferrin (clathrin pit marker) vs with a protein that floats around in ECM
caveolin has a high concentration of what domains?
raft domains
describe caveolin
caveolin coat protein with an unusual hairpin-shape membrane interaction domain (TM domain that doesn’t fully go through membrane) and is palmitoylated on the c-terminus
what kind of molecules are often found in caveolin?
signaling molecules (high conc of then in caveolae)
in what type of cells is endocytosis abundant?
in mechsnically stressed cells such a muscle, fibroblasts, endothelial, adipocytes
what can caveolae form when the tension of the membrane decreases? in what case are they formed?
rosette: clusters of caveolae can form when cytoskeleton is disrupted, when cell adhesion is lost
what proteins are involved in caveolae endocytosis?
- filamin = docking protein
- mDia1 = actin polymerization
- Dynamin = seals neck
more precisely, what is mDia1?
an actin nucleation factor that acts as a Rho GTPase effector protein. It accelerates actin elongation by rotating around the long-pitch helix of F-actin
(BASICALLY polarizes actin filaments to drive the caveolin vesicle to the early endosome)
what happens in the sorting endosome?
ph drops to 6, causing cargos to detach from their receptors
why does the pH dropping cause cargo to detach from receptor?
the H+ competes with the hydrophilic interactions
what complex recognizes membrane proteins in sorting endosomes that are destined to the late endosome?
ESCRT
how is the sorting of material in sorting endosomes based on geometry?
- middle of endosome = goes to late endosome/lysosome
- thin tubules extending from endosome = goes back to PM
how is the surface/volume ratio in tubules of sorting endosomes vs middle?
high in the tubules, low in the middle
what is ERC?
endocytic recycling compartments that brings proteins back to the membrane
gives an example of a membrane protein that is recycled via ERC
Glut4: insulin triggers its recruitments to the PM in adipocytes and muscle cells
what is ERC tightly bound to?
cytoskeleton
what type of vesicles transport between trans/cis golgi and ER?
- COPII = anterograde transport from ER to cis-golgi
- COPI = retrograde transport from trans-golgi to cis-golgi to ER
how are COPI and COPII vesicles formes and disassembled?
formed via small GTPases
disassembled via GTP hydrolysis
how does COP II cage form?
like clathrin coat: sequentially
- Sar1GTP recruits Sec23/24 adapter proteins
- Sec13/31 are recruited (coat)
how does COP I form?
coat is preassembled and recruited via GTP-Arf1
what happens to COPI/II vesicles once they arrive at their destination?
- GTP hydrolysis disassembles the coat
- v-/t-SNARE fuse the membranez
