Lecture 28 Flashcards
What is exocytosis (AKA. secretion)?
The process of moving material using membrane-bound secretory vesicles out of the cell
Vesicles must fuse with the membrane to release their components
It is also the way to get membrane proteins from the vesicular membrane to the plasma membrane surface
What are the steps for exocytosis?
1) Vesicles are generated in the Golgi
2) Transported on cytoskeletal tracts (eg. microtubules or actin) to the plasma membrane
3) Get captured by CATCHR family members. Family members have a low sequence identity, but conserved helical bundle structures
4) Binding of the complexes at the membrane brings the vesicle very close to the plasma membrane. This is followed by a SNARE on the vesicle binding to a SNARE on the plasma membrane
5) Membrane fusion
6) Extracellular release of vesicular material
What tethering factors will we talk about today?
Multisubunit tethering factors (the CATCHR family members)
What does the CATCHR family members include?
1) Exocyst complex (~750kDa) (Exocytosis)
2) Conserved oligomeric Golgi (COG) complex
3) Dsl1 complex (At the ER. Involved in ER-ER fusion events and Golgi vesicle fusion at the ER).
4) Golgi-associated retrograde protein (GARP) complex (Trans-GOlgi)
5) Endosome-associated retrograde protein (EARP) complex (Recycling endosomes)
What does the use of these complexes do?
The use of these complexes gives the specificity of binding for each segment of the process
How many subunits are in the protein complex?
An 8 subunit protein complex that tethers secretory vesicles at the plasma membrane to undergo exocytosis
What are the 8 subunits of exocyst?
Sec 3, 5, 6, 8, 10, 15, Exo70, Exo84
Who discovered the Sec proteins?
The Sec proteins (all 6 of them) were discovered by Randy Schekman in a secretory pathway screen they ran in yeast
How are Sec proteins named?
“Sec” proteins are named from “secretion” in yeast as they are known to be needed for secretion
What happens if any one protein is mutated?
If any 1 protein [apart from Sec 3] is mutated (in Yeast), then vesicles accumulate. They are not exocytosed.
Thus, Sec 3 is not essential for secretion in Yeast, it is likely a regulator of the complex
What is the sequence similarity amongst the Exocyst components?
<10%
But all have long helical compositions
What shapes do exocysts form?
Exocyst forms flower (unfixed) or a “Y” shape (fixed)
This suggests that the 2 arms could bind to opposing membranes to facilitate the fusion event
What is fixation supposed to do?
Fixation is supposed to lock protein in their conformations, but here the conformation changed
What happens if you isolate the exocyst complex?
If you freeze cells then isolate out the exocyst complex from yeast you get yet another different physical structure by EM! Importantly a model that combined cryo-EM, crosslinking data and mass spectrometry assembled a structure that looks very similar to that EM
Could the structure be changing their conformation when bound and not bound to a vesicle?
No
Measurements of distances between the exocyst molecules indicate that there is little (if any) changes to the complex when bound or not bound to vesicles
Additionally, there is no disassembly of components when bound to vesicles
This shows that exocyst is likely pre-formed prior to vesicle docking
What is the exocyst made of?
It’s made of 2 modules (4 proteins in each molecule):
MODULE 1: Sec 10,15, Exo70 and Exo84. Sec 15 sits by itself at the bottom of the complex
MODULE 2: Sec 3,5,6,8. Sec 6 makes a cap on the top of the structure
Is it a weak or strong interactions between the exocyst protein bonds?
Exocyst protein bonds between subunits are very weak
This is likely useful for the assembly and disassembly of the complex
Where is the strongest interaction?
The strongest interaction are :
Sec 6-8, Sec 3-5 and Sec 10-15 (in order)
Can exocyst components bind to non-exocyst components?
Yes
Sec 15 can bind to Rab GTPases and Myosin 2 on vesicles
Exo 70 binds to PI (4,5)P2, CDC42 and Rho
Sec 6 binds to SNARES
Sec 3 binds to PI (4,5)P2, CDC42, a t-SNARE and Rho
Sec5 and Exo84 bind to Ral GTPases
What kind of interactions are in exocysts?
Protein-protein interactions within exocysts
AND
GTPase interactions with the exocyst complex
How many copies of exocysts are present at each exocytosis region of the membrane?
~15
Do the 15 complexes coordinate for exocytosis?
Unknown
How many of the 15 are needed to grab a vesicle for membrane fusion?
Unknown
How do you get a vesicle to the plasma membrane?
Remember most vesicles will use microtubules as tracks, but in yeast they use actin
What about microtubule dynamic instability?
You need a microtubule to be stable and tether to exocyst
How can we do that?
So far there is 1 protein that we know of that can do this: IQGAP1
What is IQGAP1?
IQGAP1 is a multifunctional protein
Binds to the + end tracking protein of microtubules (eg. CLASP and CLIP-170) and Exocyst (IQGAP1 is also a component of adherens junctions)
What happens when exocyst is blocked?
Vesicles are still transported along the cytoskeletal tracts, but SNARE and membrane fusions does not occur
Do exocyst proteins do anything apart from exocytosis?
Yes
Remember exocytosis also brings protein to the plasma membrane
Exocyst mediates the delivery of integrins to focal adhesions
Exocyst mediates the delivery of E-cadherin vesicles to the basolateral membrane
What does exocytosis have to do with diabetes?
Exocytosis is used for insulin secretion/glucose uptake. Defects lead to diabetes in rodents
What does exocytosis have to do with Alzheimers disease?
Plaques are deficient in exocytosis proteins
What does exocytosis have to do with Huntington’s disease, schizophrenia and ADHD?
Associated with decreased function (or abundance) of exocytosis proteins
How does von Willebrand disease occur?
Blood vessel endothelial cells need to exocytose blood clotting factors and inflammatory mediators. If they can’t that leads to von Willebrand disease, an inherited bleeding disorder that involved blood not clotting well
What are the non-exocytic roles of exocyst?
It interacts with the Arp2/3 complex at the leading edge of migrating cells.
What does decreased expression of Exo70 do?
Decreased expression of Exo 70 by siRNA blocked Arp2/3 recruitment to the leading edge, thus no lamellipodia formed, decreased cell motility.
What does an overexpression of Exo70 do?
Lots of filopodia and invadopodium (with cancer cells)
What is the conserved oligomeric Golgi (COG) complex?
A multi-subunit tethering factor for intra-Golgi retrograde trafficking
What does COG interact with?
It interacts with ALL of the molecules used for intra Golgi trafficking (vesicle coats, SNAREs, Rabs, and coiled-coiled tethers)
How many subunits does COG have?
Has 8 subunits (COG 1-8)
What are the CATCHR family, COG, separated into?
Separated into 2 components (Lobes) [in yeast]:
Lobe A (COGs 1-4)
Lobe B (COGs 5-8)
COGS 1 and 8 link the two lobes together
What is each cistern of the Golgi used for?
For the post translational modification of proteins
So, different enzymes are in each of the cisternae
What are COGS used for?
COGS are used to recycle the resident proteins of the Golgi (eg. the glycosylation enzymes)
They tether vesicles containing those proteins
What is the Dsl1 complex?
At the ER. Involved in ER-ER fusion events and Golgi vesicle fusion at the ER
Recognizes COPI vesicles
What is the Dsl1 complex made of?
Made of 3 proteins:
Dsl1
Dsl3 (Sec39 in yeast)
Tip20
What do the proteins do?
Dsl1:
Links Tip20 and Dsl3
Recognizes the alpha and delta subunits of COP1
Probably is also used to un-coat the COP1 vesicle, as those binding sites of Dsl1 and the COPI subunits overlap
Likely links the COPI vesicle to a t-SNARE at the ER membrane
Does not interact with SNARES directly
What happens if you deplete Dsl1?
COPI vesicle clustering. Those vesicles can’t fuse with the ER membrane
What does Dsl3/Sec39 do?
Unknown, but likely interacts with SNARES. It is unstable in vitro and this difficult to work with
What does Tip20 do?
Might be involved in SNARE complex formation or COPI coat recognition (or both)
