Vesicle trafficking one

Question 1

What are the essential components for all transport in cells?

Answer 1

Vesicle formation

Question 2

What are the different features of vesicles?

Answer 2

They have adaptor proteins- These allow proteins coats to bind to the outside of vesicles

They have protein coats - this helps for vesicle attachment to different places like the membrane

GTPases are also bound to to vesicles

Question 3

What are small GTPases? What are examples of them? What forms do these GTPase have?

Answer 3

These interpret extracellular signals from the environment

They are MEMBRANE bound

Examples: RAS, ARF, RAB - in the skeleton, Rho, Cdc42 - also in the skeleton

In the cytoplasm the GTPAse have a GDP inactive form, or they have a GTP active form.

Question 4

What causes the exchange of GDP for GTP for GTPases

Answer 4

GEFs - guanine exchange factors

The change from GDP to GTP is catalysed by exchange factors

Question 5

What is key to note about GTPases?

Answer 5

They are not G proteins

They are enzymes which hydrolyse GTP on G proteins.

When these GTPases(the enzymes) have GTP bound they are active

Question 6

Why are GTPases considered rubbish? And why do you need GTPase activating proteins?

Answer 6

GTPases are considered rubbish because they have a low rate of intrinsic GTP hydrolysis.

So you need GTPase activating proteins in order to make them effective at hydrolysing GTP.

These are proteins which activate GTPase to hydrolyse GTP back to GDP on G proteins

Question 7

What needs to be balanced in terms of GTPases?

Answer 7

Guanine exchange factors GEFS and GTPase activating proteins

Question 8

Why is it useful for cells to have molecular switches such as G proteins?

This doesnt say GTPases it says G proteins.

Answer 8

The cells can respond quickly to extracellular signals

It allows them to regulate different processes in the cell

Question 9

What happens when G proteins (not GTPases) are in the GTP active form?

Answer 9

This means they can interact with other proteins

They can do various jobs in the cell

Remember the alpha sub unit usually comes off and interacts with effectors.

Question 10

What can GTPases interact with? And how can these GTPases detach from the membrane?

Answer 10

These GTPases can work with effectors when they’re in the active form

They become active when they’re assoicated with the membranes. They then dissociate from the membrane, do there thing, and then become inactive (usually after phosphorylation)

Note GTPases can dissociate from the membrane with the help of fatty acids attached to them.

Question 11

How are GTPases converted from one form to another?

Answer 11

GTPases go to the activated form by GEFs (with GTP attached)

GTPases go to the inactive forms by Gtpase activating proteins (GAPs)

Question 12

When are GTPases in the active and inactive form?

Answer 12

Active form when GTP is bound which in the membrane

When in the cytosol, its in the GDP bound state in the cytosol. This is because when it moves into the cytosol it swaps its GTP form GDP in order to activate effectors like the Alpha G protein sub unit of G proteins.

Question 13

What make up COPII vesicles? And what do GTPases have to do with these vesicles? Where were these vesicles originally found?

Answer 13

These are transport vesicles

Note GTPases being to COPII vesicles

GTPases in the COPII components are called Sar 1

They have adaptor proteins attached called sec 23 /24

They have vesicles in the coat called sec 13 /31

Sar 1 is a member of the Arf family.

These vesicles were originally found in yeast.

Question 14

How are COPII vesicles formed first stages? Where does formation happen?

Answer 14

This happens in the endoplasmic reticulum

They sort cargo sent through secretory pathways - into buds

These buds come off of the endoplasmic reticulum.

Question 15

How are cargo proteins formed and what do they attach to in vesicles?

Answer 15

In the endoplasmic reticulum chaperones help to fold these cargo proteins

These then are moved into vesicles to be transported out of the ER

These cargo proteins have exit signals and bind to adaptor proteins which have receptors which stick into the membrane.

Question 16

So what links transmembrane cargo in the vesicle bud from the endoplasmic recticulum to the outer coat of the vesicle? Whats important to note about the protein coat of vesicles.

And what are adaptor proteins?

Answer 16

The adaptor proteins 23 /24 link the transmembrane cargo in the vesicle bud to the outer coat of the vesicle

Its important to note The protein coat doesn’t link to the membrane directly, it needs an adaptor protein to bind to.

The adaptor proteins are peripheral proteins as is the coat of the vesicle.

Question 17

What does the selection of cargo into vesicles exclude?

Answer 17

It excludes resident proteins in the ER

Question 18

What is the endoplasmic reticulum exit site? What binds to this site? How does whatever binds become active

Answer 18

This is where vesicles can bud from the endoplasmic reticulum

For this exit site to be functional the Sar 1 GTPase binds to the endoplasmic recticulum and is converted to its active GTP form

This becomes active as there are GEFS (guanine exchange factors) on the ER membrane

This is important for adaptors and the coat.

Question 19

Why are adaptor proteins on vesicles so important?

Answer 19

They are important for recognising and specificity of proteins

Essentially minimising the wrong material getting into transport proteins.

Question 20

How does endocytosis of cargo proteins occur?

Answer 20

This is the process of moving cargo proteins into a vesicle bud in the ER

Adaptor protein 2 is a major clathrin adaptor protein

Its selects material inside of the cells and allows it to be internalised into the vesicle bud

In endocytosis there are lots of adaptor proteins which bind to cargo to form buds

Question 21

How do adaptor proteins work?

Answer 21

They recognise motifs in the cytoplasmic domains of the membrane bound cargo

The motifs are sequences of amino acids (these motifs can also be post translational modifications)

These are recognised by adaptor proteins

The motifs are in the cytoplasmic domains of membrane proteins.

The cargo then binds to receptors of the adaptor proteins which pierce into the endoplasmic reticulum membrane

Question 22

What causes the endoplasmic membrane to bud?

Answer 22

Cargo proteins binding to the adaptor proteins receptors.

This binding causes the adaptor proteins to pull the membrane

The membrane buds more as more cargo bind.

Question 23

Which adaptor protein in the copII vesicles do cargo bind to? What does the GTPase bind to?

Answer 23

They bind to the sec 24 protein

The sec 23 is what is activated by the GTPase Sar 1 when GTP is bound

The sec 23 adaptor protein and the sar 1 GTPase bind together to form a compelx

Question 24

Remember sec 13 /31 proteins are coat proteins in the COPII vesicles coat

Whats there role?

Answer 24

These sec proteins provide a structural scaffold

This facilitates the pinching off of the bud. Remember these are protein coats

Question 25

What is the experiment that tests what make vesicles form?

First stage of taking out the ER membrane?

And what do these ER membranes have? What are in the vesicles they form?

Answer 25

Take endoplasmic reticulum membranes out of the cell by:

Permeabilising cells by pocking holes in the membrane

Wash out the soluble compartments

Take out the ER membranes through the cells hole!!! This is before they’re forming vesicles!!

These ER membrane have resident proteins such as ribophorin

In this experiment their vesicles have p58 cargo

Question 26

How to know what makes vesicles form from the ER? This is part one of the experiment

And what to do once vesicles form

Answer 26

This is part one of the experiemnt

You incubate the ER membrane with lots of different combos

Such as cytosol to see if cytosol is enough to get vesicles to form

Then add thinks like ATP and GTP

Then carry out centrifuge experiments

When centriguing you separate out materials

The separated materials can be observed and you see if you have vesicles formed.

If vesicles DO form?
- look whats inside of them e.g. if there are and ribophorin resident proteins.

Question 27

So part one of the experiment to test How to know what makes vesicles form from the ER

Summary?

Answer 27

So you do this part one of the experiment on the ER with cytosol, ATP and GTP

These are the minimum requirements for vesicle formation.

GTP and ATP are both energy sources remember.

Question 28

Part TWO of the experiment to test How to know what makes vesicles form from the ER?

This part involves removing certain components

Answer 28

Vesicle formation was then looked at under different components

Incubating the ER with nothing (like no cytosol) causes neither ribophorin to be present or p58 to be present in vesicles

Remember ribophorin is a resident protein and shouldnt be in vesicles. It should be outside of them when the cell is centrifuged.

High concentration of cytosol doesnt show p58 in the vesicle either nor resident proteins being present

But all three of cytosol, ATP and GTP are required for correct vesicle formation.

The presence of robinsphorin and vesicle in this instance tells you before ribosphorin had originally been included into the vesicles. This is incorrect!

Question 29

What in the cytosol is used to form the COPII vesicles?

Answer 29

This is

Sar 1 GTPases

Sec 23 /24 adaptor proteins

Sec 13 / 31 coat proteins

And then ATP and GTP

Question 30

Remember what GEFs do to sar proteins?

Answer 30

They exchange SAR GDP into the active GTP form

These are GTPases!!

Question 31

Why are GTPases easy to study?

Answer 31

They have generalities

I.e. the GTPase catalytic domain.

Question 32

GTP mutant GTPases

Answer 32

Theses mutations make GTPases continuously active

They are always bound to GTP

In RAS mutations in cancer this is whats happening

Question 33

GDP mutations for GTPases

Answer 33

These mutants cant exchange GDP for GTP to become active

This is because these GTPases cant sequester GEFs

This means the GTPases are always inactive

This has a domiant negative effect (this affects downstream system, like COPII cant form due to the GTPases being inactive

Question 34

Other form of GTPase GTP mutant?

Answer 34

Cant hydrolyse GTP

Cycles of GTPase activity are important

This also has a dominant negative effect (affects downstream systems)

Question 35

What is transport through secretory pathways mediated by?

Answer 35

Coated vesicles and tubules

There is a high surface area to volume ratio of these tubules

Question 36

What is CLS dysplasia?

Answer 36

Disease which causes issues with ossification of skulls of babies = cant close properly

There is a issue with connective tissue

This is caused by mutations

Sec 23 mutation is caused. This is a conservative mutation.

You get dilation of the ER

This shows a defect in COPII vesicles.

Question 37

What is the experiment done on a mutant for on sec 23?

This is like the other vesicle experiment

Answer 37

Harvest and incubate liposomes (these mimic vesicles)

Incubate with sec 23 mutant , in presence of GTP ATP and cytosol

Binding of the mutantsec 23 isnt affected ( binding to the endoplasmic reticulum remember)

However COPII vesicle formation

Question 38

Types of cargo which affect bone ossification?

Answer 38

Collagen degradation

We have two paralogues. Sec 23 A and B

Type A might be important in cop2 vesicles

Collagen requires COPII to be secreted

Question 39

Why are only some tissues affected when copII mutates?

Answer 39

SEC 23 B can compensate if sec 23 A is a mutant

Especially if you’re packing things like collagen.