Lect4: Protein Sortng and Vesicular Transport

Question 1

Genetics to explore the secretory pathway?

Answer 1

1: Yeast was grown in a mutagen. 2: Yeast normally excrete phosphates, so you can grow them in a phosphatase and check if its being cleaved 3: if it stops being cleaved, bingo, you have a knockout 4: separate out the mutants from the rest of the cells by centrifugation (they are denser from the build up of proteins within them) Recessive are often better because they are easier to test with complementation assays.

Question 2

Biochemistry to explore the secretory pathway?

Answer 2

1: Had a cell line which could not glycosylate proteins, place a viral protein within it. 2: Centrifuged out membranes of interest, which formed vesicles 3: Placed these vesicles with normal cell vesicles which had labelled glycosylation precursors, and proteins or inhibitors they thought would start or stop them from conjoining 4: if they conjoined, it would be shown by glycosylation and radio-labelled viral protein.

Question 3

Three coat proteins:

Answer 3

COP1: responsible for retrograde transport (from golgi to ER). First Cop got you on your way home. COP2: responsible for transport from ER to golgi. 2nd cop got you on your way from home (to the Hughes’). Clathrin: Transport from golgi to plasma membrane and from plasma membrane to Endosome to golgi.

Question 4

Rab proteins: Arf proteins:

Answer 4

For both: GTP is active (can bind to effectors) GDP is inactive. - Rab proteins: 1: lipid anchored 2: Slightly more flexible 3: activated/by GEF (GTP -> GDP exchange factor) 4: Inactivated by a GAP (GTPase-activating proteins, stimulate GTP -> GDP) - Arf proteins: 1: lipid anchored 2: Slightly less flexible 3: Activated by GEF (GTP, GDP exchange factor) 4: Inactivated by a GAP (GTPase-activating proteins, stimulate GTP -> GDP)

Question 5

Rab: - Signalling cascades:

Answer 5

• SIgnalling cascades:

Each Rab in a line of signalling will make a GEF which activates the next Rab in line.

The next Rab in line also activates the next Rab in line (by a gef) but also inactivates the previous Rab with a Gap

Allows one way, sequential flow of signals

Question 6

Structure of COP1, COP2, and clathrin

Answer 6

Question 7

Explain this flow chart

Answer 7

1: Rabs and Arfs occur in spatially isolated regions as do PIPs.
2: Rabs and Arfs are not always active, PIPs are (AKA Rabs and Arfs encode temporal specificity, they allow control of when I should be active)
3: Adaptors and cargo receptors recognize specific combintations of PIPs (possibly specific Rabs and arfs?)
4: PIPs can be interconverted when moved to a new compartment, into the spatially appropriate PIP for that compartment
5: These are in turn recognized by clathrin, cop1 or cop2, and transported!

Question 8

Explain this picture, why change the PIP?

Answer 8

Shows conversion of a PIP which signals for exocytosis (golgi to plasma membrane) for a PIP which signals for endocytosis (plasma membrane to golgi). There is only so much PIP, so if most of it is on one membrane, then most transport will be from that membrane. This balances exchange between the two membranes. Stopping one from shrinking and while the other expands

Question 9

Explain this picture:

Answer 9

This is COP2 transport (no PIPs involved in the ER)

Sar1 is phosphorylated, added to the membrane.

Sec24 (bound to cargo receptor) and Sec23 (bound to Sar1) can now associate

Adaptors and COP2 can now bind

Question 10

Explain this picture:

Answer 10

COP2 can form long tubular vesicle or small vesicle. This tubular vesicle allows transport of collagen and other large proteins out of the ER.

Question 11

COP1:

• Purpose:
• Two domain types:

Answer 11

• Purpose:

Bring back proteins to the ER that belonged there

• Two domain types:

KKXX at C terminus: resident membrane ER protein. Can bind directly to COP1

KDEL at C terminus: resident water soluble ER protein, binds to receptor. Receptor interacts with active (GTP) Arf1, which helps COP1 bind.

Question 12

Golgi trafficking:

• Which model is favored? Why?
• Glycosylation by position in golgi and in ER:

Answer 12

• Which model is favored? Why?

Cisternal maturation model. No evidence of vesicles moving from one golgi to the next. Moleculare are transported which are too large for vesicular transport

• Glycosylation by position in golgi:

ER: adds sugars, initial glycosylation occurs

CIS golgi: trims off sugars

Medial Golgi: trims off sugars

Medial/trans golgi: adds a lot of sugars back on (now complex sugars can exist)

Question 13

SNAREs:

• Once assembled how do they activate it:
• How do snares double check if fusion should occur?
• How is the SNARE complex disassembled after use so it can be used again?

Answer 13

• Once assembled how do they activate it:

Already very tightly bound, Calcium based activation (ATP used), water is cleared between membranes, so that they can fuse.

• How do snares double check if fusion should occur?

V-SNAREs (vessicle SNAREs) are specific and tSNARES (their docking sites) are only on certain organelles.

A specific Rab is also involved and is only phosphorylated to RAB-GTP by a membrane surface specific tethered protein.

• How is the SNARE complex disassembled after use so it can be used again?NSF uses ATP to pry them apart

Question 14

Explain the three different routes and explain insulin secretion.

Answer 14

• Constitutive Secretory Pathway:

Release of all proteins in Golgi which don’t have any further localization signals. AKA only had an ER localization signal.

• Signal mediated diversion to lysosomes:

As explained in previous lecture (Receptors recruit specific AP# proteins which recruit clathrin which localizes them)

• Signal mediated diversion for regulated secretion (insulin for example):

Cargo concentrated in zinc rich environment into a large crystal lattice which is subsequently released. See image.