Lecture 6 trafficking 1

Question 1

Why do cells need vesicular transport?

Answer 1

To take materials up from extracellular environment. Communicate with the surrounding environment (through extracellular vesicles). Regulation of protein delivery. Co ordination of protein synthesis, modification and delivery.

Question 2

What are endosomes?

Answer 2

Small vesicular structures. Early, late and recycling types. But difficult to categorise them into these types. Some early endosomes mature into recycling and late by recruiting protein components.

Question 3

What is the direction of anterograde transport?

Answer 3

Outwards e.g., ER, golgi out.

Question 4

What is retrograde transport?

Answer 4

Movement inwards. E.g., Receptor mediated endocytosis. Receptor binds to ligand causing it to be internalised into the cell. Important for getting stuff back from golgi (retrieval pathway) to ER.

Question 5

How much of a cell membrane is internalised every hour?

Answer 5

200%. Shows need for anterograde transport.

Question 6

What is the classic biochemist approach to studying trafficking?

Answer 6

Cell free systems.

Question 7

What is the experiment with the donor and acceptor golgi stacks?

Answer 7

The donor stack contains a viral glycoprotein that has not yet been modified with N-acetylglucosamine (GlcNAc). The acceptor Golgi stack has enzyme capable of adding radiolabeled 3H-GlcNAc to glycoproteins. The glycoprotein can only leave the donor if it has been modified. The donor golgi has a genetic defect so that glycoprotein can’t be modified. So the protein can’t enter the golgi. The acceptor cell can modifiy.

Question 8

What was the hypothesis of this experiment?

Answer 8

That the glycoproteins on the donor stack will be transported by a transport vesicle to the acceptor stack.

Question 9

How is this measured?

Answer 9

The enzymes in the golgi stack transfer radiolabelled 3H-GlcNAc into the glyoprotein serving as a measureable readout. The system is incubated with cytosol and ATP to provide necessary factors and energy for vesicle budding and fusion.

Question 10

What were the results?

Answer 10

Adding cytosol alone and ATP alone nothing happens. If you add both you see an incorporation of modified glycoproteins into the golgi. The data supports the hypothesis. Showed also it is ATP dependent and that there are further factors in the golgi.

Question 11

How could you modify this experiment?

Answer 11

Add an inhibitor of something. Have mutations.

Question 12

What are the different classes of mutations in yeast sec 61 proteins?

Answer 12

Class A accumulate in cytosol, Class B accumulation in RER, class C accumulation in ER to Golgi transport vesicles, accumulation in golgi, accumulation in secretory vesicles.

Question 13

What can you do with temperature sensitive yeast mutations of Sec proteins for example?

Answer 13

Have a lower permissive temperature then a higher restrictive temperature. They denature leaving folding defects. Shows the phenotypic defect.

Question 14

How is GFP used?

Answer 14

Monitor GFP tagged proteins. Build up fluorescent reported protein using a restricted temperature in ER. Then decrease the temperature so they go to their native form and go to golgi and other parts of the cell.

Question 15

What is the shape of vesicles?

Answer 15

Any shape. Lots of heterogeneity.

Question 16

How do you use inhibitors to analyse transport mechanisms?

Answer 16

Brefeldin A. Broad effect on anterograde movement as blocks it but no retrograde. Binds to small GTP binding protein. Nacodazole. Microtubule disrupting agent which affects vesicular tubular clusters (VTCs).

Question 17

What are the features of transport compartments?

Answer 17

Bounded by lipid bilayers. Cargo can be present in both membranes and lumen. Budding requires fission.

Question 18

What are the transport considerations?

Answer 18

Budding vesicle has to correctly form they are coated by a coat complex. Pinching off so fission event must occur so they leave the donor. Cargo selection event. Then movement has to occur through tracks (Cytoskeletal network) and motor proteins (kinesin, dyenin etc). Has to be an addressing mechanism so vesicle goes to correct compartment or moves when in wrong compartment. Coat must then be removed. Regulation of contact for
fusion must occur. Secreted granules are primed and ready for fusion but only occur when signal is there. Finally membrane fusion. Retrieval: cargo does go to wrong place have to be able to get it back.

Question 19

What are the three different coats?

Answer 19

Clathrin. Coats transport vesicles. Involved in coating of budding vesicles on cell surface. Involved in some internal vesicles that are in golgi. COPII exclusively for ER vesicles. COPI for retrograde retrieval pathway when proteins go wrong and go for example into ER instead of golgi to late endosome. At least one other called retromer involved in transport at late endosome.

Question 20

What are the small GTP-binding proteins for?

Answer 20

The correct initiation of budding vesicles. E.g., ARF. Different ARF variations for different functions.

Question 21

What is the structure of COPI?

Answer 21

A complex and has domains that are homologous to COPII and clathrin.

Question 22

What is the structure of clathrin?

Answer 22

Clathrin coat is composed of triskelion (like isle of man flag). It has 3 heavy chains (180kDa) and three light chains (35-40kDa)

Question 23

What is adaptin?

Answer 23

Subunits of the adaptor protein (AP) complexes which help recruit clathrin to membranes and select cargo for transport in vesicles. Do not form the vesicle coat themselves but link clathrin to transmembrane cargo proteins. Composed of alpha, beta and gamma subunits.

Question 24

What does the diagram of the triskelion show?

Answer 24

The ends of the heavy chains point inwards to the structure. At the ends of these AP is bound. Basket formation of clathrin polymerisation on the surface of plasma membrane. Built in curvature. Involved in the deformation of the membrane. Deforms membrane into the vesicle as it buds.

Question 25

What is the structure of COPI?

Answer 25

700kDa coatamer complex comprimising of COP alpha, beta, gamma and other proteins. COPI has subunits that have homology with adaptins so could be involved with cargo recognition too.

Question 26

What is the structure of COPII?

Answer 26

Two sub complexes. Sec23/24 assembles to the tetramer. Recognise cargo with a cargo recognition component for ER cargo. Sec13/31 assembles to produce the curvature.

Question 27

Is there sequence homology between COPI and COPII?

Answer 27

No but have functional similarities.

Question 28

What is the problem with COPII coats?

Answer 28

They can only carry cargo of a maximum size of 25nm.

Question 29

What is the suggested mechanism for procollagen leaving the cell through a COPII coat?

Answer 29

It is bigger than 25nm. This is ongoing research. Their is recruitment of extra packaging proteins. Been some suggestion that ubiquitination of some of the COPII coat can change its packing mechanisms and allow larger vesicular cargo. Suggested it is the Sec31 that is modified.

Question 30

How does clathrin vesicle formation occur?

Answer 30

The adaptor proteins bind first. The adaptin 2 complex is recruited first which is quite transient (constant docking and leaving) as it searches for cargo to bind to. Something similar may happen for sec23/24 in COPII. Statistical probability mechanism happening where if enough AP2s are recruited to a cytovesicle budding that stimulates the accumulation of the clathrin coat and vesicle begins to properly form. Concentration of cargo has to reach a certain treshold to recruit AP2s.

Question 31

What are the steps to clathrin vesicle formation?

Answer 31

Donor membrane where a cargo receptor is where cargo molecules bind to the receptor. Adaptin proteins bind in recognition of the activated receptor. If there is a high enough concentration of receptors the receptors bind to the heavy chains and light chains of the clathrin coat so they can self assemble. This facilitates curvuture. Membrane bending and fission proteins bind as a clathrin triskelion is formed. The coated vesicle membrane is formed and leaves membrane. When arriving in the cytosol chaperon proteins e.g., Hsc70 break down clathrin coat. The Hsc70 protein specific to clathrin is auxilin which is recruited into the apex points where the clathrin triskelion apex points are. Breaks up the matrix. The vesicle is competent to dock.

Question 32

How is coat assembly to and from an organelle balanced?

Answer 32

By monomeric coat recruitment GTPases: ARF controls coat recruitment to clathrin and CPOI vesicles. Sar1 performs the same function for COPII. They have a high concentration of the inactive GDP bound state. The activation of these GTPases is one of the first stages. The process is initiated by interaction of GTPases with membrane bound GEF. SAR1 and ARF have poor GTPase activity- this whole mechanism is very unclear. Loss of GDP and recruitment of GTP by GEF. High concentration of GTP in the cytosol.

Question 33

How is SAR1 activity controlled?

Answer 33

The GEF for SAR1 is sec12. Conformational change in SAR1. Tertiary structure changed. Helix of SAR1 is flipped from the ER lumen to the aqueous environment. The helix is very hydrophobic so doesnt like this. so it is inserted into donor membrane to stabilise this. Is now active and competent for coat recruitment. Interacts with sec23. This is bound to sec24. Sec24 can bind to the cargo receptor which can bind to cargo.

Question 34

What is a key protein that binds to the neck of the forming vesicle in clathrin coat formation?

Answer 34

GTPase protein dynamin. Required for fission away from donor membrane. Polymerises into an oligomeric structure up the neck of the donor membrane. Then GTP leaves the dynamin in a synchronised hydrolysis and causes a structural change in the dynamin constricting the neck, leading to fission.