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ER - Golgi Flashcards

1
Q

How are proteins concentrated into vesicles to go from the ER to the Golgi?

A

The ER membrane contains cargo receptors that select and concentrate luminal cargo. Receptors are TM proteins with a cargo binding domain on the ER side and a domain to recruit COAT proteins, often via di-acidic motifs (Asp-X-Glu) in their cytosolic protrusions.

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2
Q

What do COAT proteins do?

A

they bend the membrane into a vesicle.

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3
Q

What are COPII vesicles?

A

Vesicles which transport proteins from the ER to the Golgi.

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4
Q

What are the cargo receptors for luminal proteins?

A

p24 family proteins and ERGIC53 (a glycoprotein-binding receptor).
They have to be sufficiently promiscuous to bind to a number of soluble proteins that need to be packaged into vesicles.

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5
Q

How are proteins not going to the Golgi but to the cell surface put into vesicles?

A

They recruit their own COAT proteins instead of doing it via a receptor.

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6
Q

Where does cargo selection happen?

A

In the ER at ER exit sites, which are ribosome-free areas of the membrane. It is where proteins are concentrated into COPII coated buds.

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7
Q

How do proteins arrive at the Cis-Golgi Network?

A

As vesicular tubular clusters, which are vesicles which have fused together.
They are moved along by motor proteins on microtubules.

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8
Q

What are COPI vesicles?

A

Vesicles that take some material back to the ER to maintain an equilibrium otherwise there will be build up at the Golgi

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9
Q

Why should there be cycling of proteins between the ER and the Golgi?

A

To maintain membrane homeostasis.

To return proteins which belong in the ER like chaperones such as BiP, PDI and others which must function in the ER

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10
Q

How are resident ER proteins identified?

A

They have the same C terminus sequence; KDEL or HDEL - this has been shown to be a signal for ER residence (deletion of this sequence results in their secretion).
Membrane protein receptors for KDEL and HDEL have been identified - they operate by retrieval rather than by retention

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11
Q

ER resident proteins stay in the ER through retrieval rather than retention - how do we know this?

A

The binding of the KDEL to the receptor happens only at a pH less than 7.
The pH in the golgi is slightly lower (6.5) than in the ER (7). So its favoured in the golgi which is how the receptors manage to catch escapees. pH decreases the further into the Golgi you go so binding is increasingly more favoured/affinity for the receptor increases.
KDEL receptors only release their ligands at neutral pH which is back in the ER.

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12
Q

How do KDEL receptors return to the Golgi?

A

When the signals are empty, their cytosolic domain recruits COPII to return to the Golgi but recruits COPI again when going back to the ER.

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13
Q

How are ER MEMBRANE proteins retrieved from the Golgi?

A

It is not well characterised but it is postulated that some ER membrane proteins, like KDEL receptors, have KKXX motifs in their cytosolic domains that, under certain conditions, must permit the recruitment of COPI coats for the formation of transport vesicles that take them back to the ER.

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14
Q

Who devised the in vitro Golgi transport assay?

A

Jim Rothman

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15
Q

How would we look at the transport of proteins across the Golgi in the in vitro Golgi transport assay?

A

Golgi cisternae are isolated from two cell types that have different Golgi characteristics:
- one has a normal Golgi;
- the other has a Golgi with a defective N-acetylglucosamine transferase and a supply of radioactive GlucNacs.
If its activity is restored, there will be radioactive proteins.

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16
Q

What is N-acetylglucosamine transferase?

A

An N-glycan modifying enzyme that adds a GlucNac residue to proteins in the medial Golgi.

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17
Q

How was success of transport of proteins into the Golgi assessed in the in vitro Golgi transport assay?

A

Wildtype cells express VSVG, golgi is isolated and incubated with cytosol and radioactive rGlucNac. Immunoprecipitate VSVG and radiolabel WILL be detected in association with it.
In mutant cells, no radiolabel will be detected

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18
Q

How was the in vitro Golgi transport assay set up?

A

Golgi isolated from wildtype cells were incubated with Golgi from mutant cells that expressed VSVG. Cytosol and radioactive sugar was added. They looked for radioactive sugar in the VSVG immunoprecipitate.
It would only be radioactive if there were vesicle transport between cisternae as the enzyme needed to add radioactive sugar is only found in wildtype golgi.

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19
Q

What did Rothman observe in the in vitro Golgi transport assay?

A

He showed that he could successfully reconstitute Golgi transport in vitro, but only if cytosol was added.

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20
Q

Why could directionality of Golgi transport not be interpreted from Rothman’s study?

A

Because it is equally possible that VSVG was being brought to the WT golgi and that N-acetylglucosamine transferase was being brought to the mutant golgi.

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21
Q

Where is N-acetylglucosamine transferase found?

A

Embedded in the golgi membrane

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22
Q

Where is VSVG found?

A

Embedded in the golgi membrane

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23
Q

Who revealed all the steps of vesicle formation and fusion?

A

Rothman

24
Q

What do the enzymes in the Golgi do?

A
  • modify lysosomal enzymes (adding phosphates to their mannose sugars which is the signal to target proteins to the lysosome)
  • modify glycoproteins and glycolipids, (not glucose – this was for QC)
  • add sulphate to certain proteins, and to cleave other proteins
  • synthesise some lipids, and modify many others passing through it.
25
Q

What was observed about the Golgi structure?

A

Rothman noticed that each cisterna appears to disintegrate into tubules and vesicles at its rim.
He found that when he isolated golgi stacks, he saw cisternae but no vesicles.
But when he isolated golgi with added cytosol, he saw coated vesicles

26
Q

How was whether or not vesicle formation requires energy investigated?

A

Using a non-hydrolysable ATP/GTP so that any G/ATPases will be frozen when they bind their respective substrate.
GTPyS was used in this scenario.

27
Q

What is NEM?

A

n-ethylmalemide blocks thiol groups on cysteine residues to prevent sulphide bonds formation. Any process requiring these bonds are inhibited.

28
Q

What were the three conditions used to investigate energy requirements for vesicle formation and their outcomes?

A
  1. Golgi vesicles + cytosol and GTPyS = coated vesicles
  2. Golgi vesicles + NEM = uncoated vesicles
  3. Golgi vesicles + both = coated vesicles
    This shows that GTP must be needed for uncoating and NEM is needed for vesicle fusion
29
Q

How were the components which make up the golgi vesicle coats identified?

A

Golgi was incubated with cytosol and GTPyS. Vesicles were purified.

30
Q

What proteins were found and which was the most abundant protein in the golgi vesicle coat?

A

The most abundant was ARF, which is an ADP ribosylation factor that has GTPase activity.
They also found a complex of 7 proteins called COPs, named alpha, beta etc.
Both exist in the cytosol which show why vesicles only form when cytosol is added.

31
Q

How can ARF associate with the membrane?

A

it is myristoylated (i.e. has a covalent fatty acid – hydrophobic group).

32
Q

What are ARF’s two conformational states?

A

In its GDP bound state, myristate group is hidden. In its GTP bound state, it is exposed in an aqueous environment so becomes membrane bound.

33
Q

What is the role of ARF?

A

It is the first factor that recruits COAT proteins.

34
Q

What is the mechanism of COPI vesicles formation?

A
  1. A golgi-associated GEF activates soluble ARF-GDP to convert it from soluble to membrane bound ARF-GTP
  2. ARF-GTP recruits COAT proteins by offering binding sites on its cytosolic domain.
  3. Targeting sequences in the cargo proteins in the new vesicle make contact with COAT proteins (COP1).
  4. ARF-COP1 complex recruits GAP into the forming vesicle.
  5. Cargo is concentrated into vesicle while COAT scaffold formation causes vesicle budding
35
Q

What does GEF stand for and what is its role?

A

Guanine nucleotide exchange factor

It makes ARF specific to golgi vesicles by its presence only being near the golgi.

36
Q

What is GAP?

A

GTPase activating protein

37
Q

What are the roles of coat proteins?

A
  • concentrates cargo (via interacting cargo-receptors)
  • recruits the necessary machinery molecules
  • sculpts the membrane into a bud that can pinch off as a vesicle
38
Q

How are COPI and COPII different?

A

they recruit different proteins from the cytosol:
COPI = ARF1 and coatomer proteins
COPII= Sec complexes

they have different permanent membrane constituents:
COPI= cargo receptors
COPII = ERGIC53 and others (P24)

39
Q

How are COPI and COPII similar?

A

They both recruit GTP binding molecules from the cytosol (ARF1 and Sar1)
They both have membrane cargo as permanent membrane constituents

40
Q

What is the only difference between COPI and COPII vesicle formation?

A

COPII vesicle formation: instead of a lipid group, it has a hydrophobic N terminus which is hidden when GDP is bound but when GTP is bound by Sec12, it is exposed and inserted into the membrane. so Sar1 and ARF1 are similar

41
Q

What is the mechanism of COPII vesicle formation?

A
  1. A GEF on Sec12 activates Sar1 so it binds to the ER membrane.
  2. SarI recruits COPII proteins
  3. Vesicle forms and is pinched off.
42
Q

What transport is COPI vesicles involved in?

A

COPI vesicles are involved in intra-Golgi transport, in retrograde Golgi to ER transport (and in endosome-Golgi transport)

43
Q

What transport is COPII vesicles involved in?

A

COPII vesicles are exclusively involved in forward (anterograde) transport from the ER to the early Golgi

44
Q

Why is uncoating a necessary step?

A

It allows vesicular fusion, otherwise coat proteins block it.

45
Q

How does uncoating happen?

A

As ARF and Sar1 are weak GTPases, uncoating needs to be activated by a recruited GAP to hydrolyse GTP to GDP.
GTP hydrolysis leads to ARF diffusing from the membrane, which causes the release of coatomers and GAP, exposing the membrane for fusion.

46
Q

How does GAP know when to release the COAT proteins and when not to?

A

This is not yet clear. GAP may sense the curvature of the vesicle and gets rid of COAT when vesicle is formed/after it is pinched off.

47
Q

How did Rothman study vesicle fusion?

A

He prepared Golgi in presence of cytosol and NEM and vesicles did not fuse.
Wash off NEM and add fresh cytosol resumes fusing.
This means a cytosolic NEM sensitive protein must be involved.
Cytosol was fractionated and the fraction which recovered fusion was analysed one by one. A protein was found called NSF.

48
Q

What is NSF?

A

An ATPase required for vesicle fusion. But it needed factors called SNAPS (soluble NSF attachment proteins) which are cytosolic proteins that bind membrane receptors.

49
Q

What are SNAREs?

A

SNAP receptors

They mediate the fusion of vesicles.

50
Q

What are the two types of SNARES?

A

If they were on vesicles were originally termed v-SNAREs
If they were on target membranes were termed t-SNAREs
It was found that two t-SNARES were needed for every v-SNARE.
It forms a bundle of 4 intertwined parallel helices that have extraordinary stability. This requires 1 helix from the v-SNARE (blue), 2 helices from SNAP25-like t-SNARE (green) and 1 helix from the another t-SNARE (red):

When the four helices come together, there are lots of parallel interactions – mostly hydrophobic. V-SNARE always has an Arginine (R) for hydrophobic bonds. T-SNARES have Glutamine (Q) contributing to H bonds. They were renamed R-SNARES and Q-SNARES respectively

51
Q

What is TIRFM?

A

Total Internal Reflection Fluorescence Microscopy - it lets you image events that occur in very thin layers of matter, i.e good for membrane events.

52
Q

How does TIRFM work?

A

Shine light from below a cover slip at 90 degrees to the cells.
Light hits a critical angle of reflection where all of the light is reflected.
An evanescent wave comes from the point of incidence which had excited things in the membrane.
If you have a liposome containing fluorescent material, you can see it.

53
Q

How was TIRFM used to measure vesicle fusion?

A

Coverslip had an immobilised lipid bilayer with two t-SNARES; one provides one helix, the other provides two.
Docked vesicles containing fluorescent material were visualised. No fusion could happen without calcium, so adding Ca showed fusion, releasing fluorescent proteins.

54
Q

What do SNARES do to facilitate vesicle fusion?

A

They act to pull the membranes together. They squeeze out all the water molecules between the membranes so the lipids can fuse.
There are enough types of SNARES to provide specificity so that vesicles only fuse with where it needs to go.

55
Q

What is the role of NSF and SNAP adaptors in vesicular fusion?

A

NSF recycles the SNAREs after fusion. NSF is an ATPase that untwists the helical bundle and frees the SNAREs for interaction with another incoming v-SNAREs.

56
Q

What happens when NSF is inhibited?

A

NEM inhibits NSF which depletes the system of free SNARES so no vesicle fusion (as observed by Rothman)

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