L13 - Vesicular transport 1 - COPII coated vesicles

what is the donor compartment?

the organelle the vesicle buds from

what is the target compartment?

the organelle/membrane the vesicle fuses with

what happens in vesicle budding?

the vesicle buds off from the donor compartment and gets delivered to the target compartment

during budding of the vesicle what happens to the structures?

• organisation of bilayers stays the same

• fusion is non leaky

• topology/orientation of membrane proteins is maintained post fusion

what are SNAREs?

• integral membrane proteins that help vesicle fusion

what are V-SNAREs?

• SNAREs (like VAMP) embedded in vesicle membrane

what are T-SNAREs?

• SNAREs (like Syntaxin and SNAP-25) on target membrane

what is transport of cargo mediated by?

• vesicles and tubules

what are vesicles characterised by?

• the presence of a coat - transport vesicles are coated

what are some examples of vesicles?

• COPI, COPII, clathrin

what are the 3 components required for transport vesicle formation?

1. GTPase

2. Adaptor protein - connect cargo to the coat

3. Coat protein

what are small GTPases?

• small molecular switches that switch between active and inactive form

• GDP (inactive) → GEFs → GTP (active)

• GTP (active) → GAPs → GDP (inactive)

where is the GTP form and GDP form?

• GTP form is found in membranes

• GDP form is found in cytosol

what is Ras?

• founding member of the superfamily of GTPases

• it is a small GTPase

what can mutations in Ras lead to?

• increased signalling = increased proliferation

• because Ras gets stuck in GTP bound state (ON)

what is the GTPase switch regulated by?

• 2 families of proteins

• GEFs and GAPs

• GEFs = guanine nucleotide exchange factors

• GAPs = GTPase activating proteins

what do GEFs do?

• they kick out GDP (inactive form) allowing GTPases to bind GTP (active form)

• exchange GDP for GTP = activating the GTPase

what do GAPs do?

• stimulate their GTPase activity in order to become inactivated

• speed up GTP hydrolysis

• GTP → GDP = Pi (inactive form)

• inactivating the GTPase

what is the level of active GTPase in the cell determined by?

the balance between the GEFs and GAPs

what are the components of COPII?

1. GTPase: Sar1

2. Adaptor: Sec23/24

3. Coat: Sec13/31

   +GEF on ER membrane

   +ATP and GTP

what keeps proteins in the ER?

association chaperones

why do association chaperones keep proteins in the ER?

to ensure

• proteins conformation is correct

• proteins are correctly folded

what happens once the proteins are folded?

they are sorted into the exit sites = budding sites on ER membrane

steps of formation of COPII coated vesicles

1. Sar1 (GTPase) activation - Sar1-GTP inserted into ER membrane from the cytosol

2. Sar1-GTP recruits inner coat adaptor proteins (Sec23/24) - these bind cargo receptors or transmembrane cargo with exit signals

3. Cargo selection - only proteins with the right exit signals are selected for export, misfolded proteins (resident ER proteins) kept in ER by associated chaperones

4. the outer COPII coat proteins (Sec13/31) are added which bends the membrane and helps form the vesicle shape

5. vesicle buds off carrying properly folded and selected cargo

6. after vesicle budding - Sar1 hydrolyses GTP to GDP which causes coat disassembly

what is the adaptor of COPII and what does each component do?

• Sec23/24

• made of two subunits : Sec23 and Sec24

• when Sar1 is active form (Sar1-GTP) = it can interact with Sec23 = binds to activated GTPase

• Sec24 = recognises the signals on the cargo and binds to the cargo proteins

• together the two subunits act as an adaptor

• once the adaptors have been recruited to the membrane it can then recruit the coat (Sec13/31)

what are the steps in the reconstitution experiment that allows us to understand what makes COPII vesicles?

1. incubate ER with cytosol + ATP + GTP = makes COPII vesicles (in vitro)

2. the product can be separated from the starting substrates by adding the whole mix to the top of a sucrose gradient

3. high sucrose concentration = bottom of tube

   low sucrose concentration = top of tube

4. centrifuge - spin at high speed

5. as a result - the vesicles remain at a certain concentration of sucrose and the ER membrane at a different sucrose concentration

6. allowing them to separate - isolate vesicles and ER membrane from each other

7. run materials on gel - allows to analyse what’s in the fraction

how are GTPases (Sar1) recruited from the cytosol?

1. Inactive form

   in the cytosol - Sar1 is bound to GDP

   amphiphilic helix is hidden

2. Activation

   GDP → GTP exchange via GEFs

   this exposes Sar1’s amphiphilic helix

3. Insertion into membrane

   the amphiphilic helix inserts into the outer leaflet of the ER membrane - this anchors Sar1 to membrane

what does Sec23 act as and what is it’s activity stimulated by?

• acts as a GAP (GTPase activating protein)

• the activity of Sec23 (GAP) is stimulated once the coat forms (Sec13/31)

• once the coat forms - Sec23 stimulates the GTPase activity of Sar1 - which facilitates disassembly of the coat

what is a GTPase GDP mutant?

• a mutant stuck in its GDP form which means it cannot activate so it will gather GEFs

• acts as a dominant negative as it interferes with normal function

what is a GTPase GTP mutant?

• a mutant bound to GTP so cannot hydrolyse GTP so it remains active

why are GTPase activity cycles important?

because if the cycle is disrupted then vesicle trafficking fails → assembly and disassembly disrupted

what does expression of Sar1-GDP inhibit?

COPII formation

what are COPI vesicles responsible for?

• retrieval pathway

• if material has escaped to the Golgi network it can be captured and brought back to the ER by a process of retrieval

what cargo does COPII and COPI transport?

• COPII = transports newly synthesised proteins

• COPI = transports retrieved + newly synthesised proteins

what does clathrin (plasma membrane) transport?

endocytosed material

what does clathrin (trans. Golgi network) transport?

lysosomal proteins + regulated secretory proteins

what do all of the vesicles (COPI, COPII, clathrin (PM), clathrin (TGN) ) have in common?

• they all have:

  1. coat

  2. particular GTPase

  3. form by same principle