Professor Ugalde Lecture 13 Flashcards
What is the difference between proteins used when going to endosome vs going to lysosome for tethering?
-to endosome–>Rab5 and CORVET
-to lysosome–>Rab7 and HOPS
For lysosome and endosome tethering what is the difference/similarity in complex+subunits?
-complexes have the same core subunit
-different end subunits bind different Rabs
-Also bind SNARES
How does clustering of tethers occur, what protein/activities are involved?
-Rab5 effectors have GEF or PI kinase activity on early endosome
-GEF activity produces more Rab5-GTP in local area of membrane
What do PI-phosphatases do in clustering of tethers?
what do the cluster of tethers form?
-PI-phosphatases provide additional binding sites for vesicle tethers
-cluster of tethers form “landing site” for vesicle (does not involve membrane thickness or lipid content)
What is the importance of the increase of GEF in clustering of tethers?
more GEF–> more Rab5, allowing more production of PI, and more tethers being formed
In endocytosis pathway where do vesicles traffic?
-What does early endosome matuer into, what does membrane switch from?
-vesicles traffic between PM, early endosome and trans-golgi
-early endosome matures into multivesicular body (MVB) an late endosome
-membrane switches from Rab5 to Rab7
What is endocystosis?
what does a late endosome mature into in endocytosis pathway?
-taking cargo from exterior of cell into cell membrane
-late endosome matures into lysosome
-other vesicles traffic to lysosome
What does Rab5 activate?
what does Rab5 effector do?
What does Rab7 effector do
-Rab5 at endosomes activates Rab7
-Rab5 effector and tether (CORVET) is GEF for Rab7
-Rab7 effector is GAP to inactivate Rab5
What happens when Rab5 vesicles fuse with early endosomes?
-more and more Rab7 is activated and less and less Rab5 stays actiave, membrane becomes late endosome
What is a SNARE, what recruits them?
SNARE proteins: family of membrane proteins that carry out vesicle fusion
-Rabs and tethers can recruit SNAREs to fusion site
What can v-SNARES recognize, how does the complex form, how is specificity determine?
-v-SNAREs on vesicles recognize partner t-SNAREs on target membranes
-complexes form after tethering
-unique combination of v and t-SNARES determine targeting specificity
What is the difference between v and t SNARES?
What do v and t SNares form?
-v-snares: are monomers with single TM helical domain
-t-SNAREs: are trimers with combination of TM and peripheral subunits
-correct set of v and t Snares form a stable tetramer
What do SNARE complex induce?
-multiple SNARE complexes form at a target site to induce vesicle fusion
Why is a v and t-SNARE complex very stable?
How are they folded?
-it has a 4 helix bundle
-the folding process pulls the membranes close together, generating physical strain like a spring and it is not dependent on ATP or GTP
What do SNARE complexes form a ring around?
How does the bending of SNARE TM contribute to structure?
What happens to outer+inner layers of membrane, what does this do?
-SNARE forms a ring around the vesicle contact site
-SNARE TM anchors are bent and strained, exerting a force that holds the membrane together
-outer+inner layer of membranes fuse, relieving strain in the SNARE complex
What happens to SNARE after fusion?
What dissociates v and t-SNAREs
after fusion, the SNARE complex is stable, unstrained and inactive
-an AAA-family ATPase (NSF) dissociates the SNAREs which is essential for continution of vesicle traffic
What happens to v and t SNARES after dissociation?
-t-SNAREs become active again (stay in compartment where they belong to bring more vesicle)
-v-SNAREs are recycled back to their dono membrane by vesicles
What happens in homotypic fusion, what is fused?
when does ER and golgi reform?
-donor and target membranes are the same
-the fusion of COP-II vesicles into vesicular-tubular cluster that becomes cis-golgi
-re-formation of ER and golgi after cell division
What type of v and t snares are in homotypic fusion?
What separates the SNAREs and why?
-both membranes have identical v-and t-snares already in complexes and inactive
-SNAREs must be separated by NSF to allow new fusion
What does NSF bind?
What happens to NSF during ATP-hydrolysis?
NSF binds SNARE complex through adaptor protein (alpha SNAP)
-NSF twists and pulls during ATP hydrolysis
What is needed to unwind the SNARE helices?
-multiple cycles of ATPase unwind the SNARE helices
What do CCV (clathin coated vesicles) mediates in endocytosis?
-Where are some PM proteins transported?
-CCVs mediate endocytosis from PM to early endsome
-Some PM proteins are transported to recycling endosome, for exocytosis back to PM (once cargo is in we need to recycle receptors)
Where are lysosomal proteins trafficked from and where?
What does endosome invaginate to form?
-trafficked from trans-golgi to endosomes, their receptors are trafficked back
-invaginates to form multivesicular body (MVB) and late endosome
What does late endosome turn into and what for?
-late endosome matures into lysosome, for degradation of proteins and lipids
