MT4- COPII etc Flashcards

1
Q

sequence of events in ER if correct folding?

A
  1. glucosylase I and II trim two terminal glucoses.
  2. ER chaperones recognise eg. Calretriculum and ER57 to aid folding.
  3. If correct folding, terminal glucose removed from Mannose and packages into a vesicle for export at ER exit sites.
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2
Q

What is the Unfolded protein response?

A

This is a stress induced pathway which stops protein synthesis but increases synthesis of ER proteins and ER to respond to the overload of unfolded proteins. In response to too much protein for chaperones to deal with.

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3
Q

Yeast UPR?

A

Yeast upregulates over 300genes in response.

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4
Q

Overview of the UPR?

A
  1. After activating UPR, misfolded protein sensors are activated.
  2. These activate ER stress signals IRE1, PERK and ATF6. IRE1 causes mRNA splicing so initiates translation of genes. PERK reduces other protein translation by P translation initiator factors, but selects TF’s. ATF6 regulates proteolysis releases gene regulaory proteins. All upregulate TF.
  3. Downstream effect of all to upregulate genes to increase ER folding capacity.
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5
Q

ER stress signal proteins?

A

ATF6, IRE1, PERK. These are present on the ER membrane and assosicated with ER chaperone called BiP, held an inactive state. But when lots of unfolded protein BiP chaperone has a higher affinity for these so dissociates, and they can then go on to cause their downstream effects.

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6
Q

ER stress/oxygen deprival affect on cell survival?

A

Certain amount can cause cell survival mechanism by UPR, but prolonged too long or too strong can cause cell death signals. Both are activated but depends on the balance between.

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7
Q

Downside of the UPR?

A

Cancer cells can use to activate angiogenic factors in tumour to increase O2 and glucose to it.

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8
Q

How does ATF6 upregulate UPR genes?

A

Bip has a higher affinity for this so dissociates from ATF6. This enables ATF6s translocation to golgi. S1P and S2P proteases cleave AFT6 into cytosolic fragment which can then migrate to the nucleus and upregulate UPR genes.
This is done as a summative effect, so lots of proteins misfolded, lots of BiP dissociates, so lots of ATF8 translocation, lots of UPR genes activated.

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9
Q

Cholesterol homeostasis?

A

Regulate cell cholesterol synthesis, depending on diet intake. SREBP in SER regulates genes for cholesterol synthesis.

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10
Q

High cholesterol? Low?

A

High cholesterol: SCAP cholestol sensor (SREBP cleavage activating protein) ensures SREBP TF associates with an Insig ER chaperone and keeps it in the ER.
Low: SCAP allows dissociation from Chaperone, SREBP translocates to golgi, cleaved by proteases (same that cleave ATF6) so form can translocate to nucleus and act upon genes.

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11
Q

WHat is autophagy?

A

Autophagy is used by cells under stress to engulf their own cytoplasm and organelles. Forms an autophagosome around organelles to fuse with lysosomes to be degraded.

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12
Q

What can activate autophagy?

A

The ER often activates e,g can engulf extra ER made during UPR.
Cancer cells can use to get nutrients by lysing cells and releasing their nutrients.

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13
Q

Alzheimers cause?

A

Mutations can cause cleavage of the protein at different points e.g. 42AA instead of 40, and so create folding problems.

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14
Q

CRT? -/- impacts?

A

Calreticulin in ER binds to misfolded protein and prevents their export. Binds to sequester Ca.
Cardiac problems.

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15
Q

CNX -/- impacts?

A

Calnexin TM ER chaperone, quality control and assisting folding.
large myelinated fibres- dysmyelination

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16
Q

Glucosidases function?

A

Glucosidase I and II trims the first two glucose residues before folding, and then II trims the terminal glucose after chaperones ensure correct folding. UGGT adds the terminal glucose back on if incorrect folding so they are recognised by chaperones to give them another chance at folding.

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17
Q

Difference between CNX, CRT K/O and BiP?

A

All ER chaperones.
BiP fatal, suggests essential for all cultured cells, wheras CNX and CRT more tissue specific.
CNX- Dysmyelination
CRT- cardiac problems

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18
Q

3 examples of ER folding diseases?

A

CFTR.
Emphysema
Hypothyroidism

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19
Q

If downregulate which chaperone, helps CFTR DF508?

A

Hsp90 co-chaperone Aha1 downregulation by SiRNA.
This is an ATPase regulator.
Increase in not only band B but also band C (suggesting golgi translocation)
-with a 60% Aha1 K/D, 50-60% reduction in HSP90 bound to CFTR in immunoblots was seen. partial rescue in conductance also.

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20
Q

Emphysema cause?

A

Deficiency in secretion of alpha1-antitrypsin.
Trypsin is a protease thats secreted into the lungs and creates gaps in the tissue, altering permeability. Anti-trypsin is secreted to stop this here.
genetic predisposition but smoking increases risk.

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21
Q

Emphysema symptoms?

A

Lung tissue (alveoli) digested and gaps appear. Less surface area for gaseous exchange.

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22
Q

Hypothyroidism cause?

A

Production of mutant thyroglobulin, which is a precursor to the thyroid hormones. As compensation the Er undergoes massive proliferation, but too high UPR can act as a death signal to cells.

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23
Q

ALS/Motor neuron disease?

A

(either sporadic or familal) Death of motor neurons resulting in muscle wasting.
ERAD impairement, altered trafficking and protein aggregation which can result in autophagy.
ER stress signals associated with UPR.

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24
Q

ALS/Motor neuron disease and UPR?

A

PERK activation- gives protection (therapy shift towards?)

IREalpha- degeneration by autophagy or apoptosis.

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25
Q

How was COPII orginally found?

A

Orginally identified in Sec mutants, because without proteins can’t leave the ER to golgi.
Biochemically v different to ER- lipid and protein content v different so can separate.
Ultrastructurally v different- can see under electron microscope

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26
Q

COPII function?

A

Coat vesicles for cargo from ER to Golgi.

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27
Q

Function of Sar1?

A

small GTPase- initiates coat formation and budding following recruitment by sec12

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28
Q

Function of Sec12?

A

GEF-on ER membrane- recruits Sar1 to initiate coat formation.

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29
Q

What is a GTPase?

A

Enzyme that has GTP/GDP bound. In active state has GTP bound but then hydrolyses GTP to GDP as energy. as it activates another enzyme e.g. a GAP.
GEF removes the GDP to add GTP again.

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30
Q

WHat is a GEF?

A

Guanine exchange factor- removes GDP from a GTPase and add GTP to activate.

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31
Q

What is a GAP?

A

Guanine activating protein- Hydrolyses GTP to GDP, of a GTPases as energy.
(GAP bad, ‘please mind the gap,it will inactivate you’)

32
Q

What is Sec 23/24?

A

Adapter protein- Acts as a GAP (GTPase Activating Protein) and links cargo to the coat. Forms the pre-budding complex with cargo on ER membrane.

33
Q

Sar1 and sec 12 relationship?

A

Sec12 on the ER membrane recruits Sar1 and is a GEF so exchanges the GDP to GTP to activate Sar1.

34
Q

Sar1 and Sec23/24 relationship?

A

Sar1 recruites Sec23/24. Sec23/24 is a GAP, and hydrolyses the GTP of Sar1. This forms the pre-budding complex. This is an adapter protein which links cargo with the coat.

35
Q

What is Sec13/31?

A

The coat- Causes coat polymerisation and membrane curvature, and fission. Enhances GAP activity of Sec24/24.

36
Q

Briefly steps from ER cargo to coat formation? (4)

A
  1. Cargo without KDEL sequence.
  2. Sec12 recruits Sar1. Sec12 GEF- Sar1-GTP.
  3. Sar1 recruits Sec23/24. Adapter protein that binds to cargo=prebudding complex on ER.
  4. Sec13/31 coat is recruited. GAP enhancer. This polymerises the coat and causes membrane curvature for budding.
37
Q

After fission from the ER membrane what happens?

A

After vesicle budding, this stimulates Sec23 GAP activity- hydrolysing Sar1-GTP back to GDP which has reduced affinity for Sec23-24 complex so dissociates after vesicle budding.

38
Q

Two diseases associated with COPII assembly?

A

Fromme paper (2007)
CLSD- Cranio Lenticulo Sutural Dysplasia.
CMRD-Chylomicron retention disease.

39
Q

What is CLSD? symptoms

A

Cranio Lenticulo Sutural Dysplasia (CLSD) - categorised by facial abnormalities, late closing fontenelles and cataracts. Export of collagen disrupted.

40
Q

Fromme paper aim? focusing on what mutation?

A

The paper mainly focuses on the F382L mutation of SEC23A to establish the role of the prebudding complex for COPII vesicles. And why CLSD is a tissue specific disease.

41
Q

Fromme paper problem with mutation?

A

SEC23A F382L mutation poorly recruits Sec13/31, and so cargo (Collegen) budding disrupted.

42
Q

What paralogue could rescue budding in the Fromme paper? Experiment?

A

Permeabilized cell and cytoplasm removed so could add back specific componens. Western blot measured ERGIC-53 cargo secretion, hence COPII formation. Sar1A partially rescues the poor Sec23 mutated budding with Sar1B. (17%, vs 2% (control 20%)).

43
Q

What was the theory of why CLSD is tissue specific? Fromme (2007)

A

In other tissues there is a degree of redundancy e.g. the other paralogue SEC23B, but here only SEC23A (which is mutated).

44
Q

Experiment that investigated SEC23A mutant activity? Why? Result?

A

Use synthetic liposomes, one with SEC23A and another with Sar1 to test Decrease in fluorescence over time represents the hydrolysis of GTP on SAR1. This is testing the GTPase-activating protein (GAP) activity of F382L-SEC23A compared to wild-type SEC23A.

Why? Check still can function.
Result?Normal with Sar1A but not Sar1B.

45
Q

Experiment that showed F382L-SEC23A poorly recruits Sec13-31 in Fromme paper (2007)
Result?

A

liposome floatation assays to measure the amount of Sec13-Sec31 complex bound to membranes in the presence of F382L-SEC23A. One liposome contains each and if bind form complex pulled out together and float at a different density in an increasing sucrose concentration.
Also- western blot- addition of Sec13/31 in excess needed to pull out ERGIC-57 (therefore secrete) rescue budding.

Poorly recruits Sec13/31 with Sar1B

46
Q

Experiment that shows why CLSD may be tissue specific?

A

Western blot of different tissues, and calvarial osteoblasts have v little SEC23B to compensate. These are the cells that are responsible for suture closure.
compared to fibroblasts, kidney HEK cells etc.

47
Q

Morphology of the ER in CLSD patients different? (2 experiments)

A

EM images show there are invagination sites that are not budded off at ER exit sites, only 10% of buds are coated compared to 100% in WT.

Also anti-SEC31A antibody and gold-labeled secondary antibody to, quanitifies at ERES.
In mutant accumulation of Sar1 at ERES, but reduction in SEC31 (poor recruitment)

48
Q

Experiment into CLSD cargo sorting?

A

ERGIC-53 used as cargo and PDI as ER resident protein, segregation? Yes, so its the budding thats the problem. ERGIC-53 at ERES in pre-budding complex,

49
Q

Explain the higher lower affinities of sar and secs? (Fromme 2007)

A

Sar1a has highest affinity for Sec23/24A, higher than b to b etc.

50
Q

Summarise the key findings of the Fromme (2007) paper?

A
  • Mutated F238L Sec23A has lower affinity for Sec13/31, which is a GAP promoter, therefore it has lower GAP activity.
  • Sar1A can rescue the fault.
  • Tissue specific as Calvarial oseoblasts have v low Sec23B to compensate.
  • Not sorting problem for cargo but accumulation at ERES as v low Sec13/31 so not coated.
51
Q

Explain why Sec23A mutant will have lower GAP activity? (Fromme 2007)

A

Sec13-31 stimulates the GAP activity of Sec23-24 on Sar1. So if in the mutant less Sec13-31 is recruited/binding with SAR1B, the GAP activity will decrease.

52
Q

Why do the calvarial osteoblasts not just use SAR1A? (Fromme 2007)

A

They have suggested that the SEC13-31A has a higher affinity for SAR1A than SAR1B, and that SAR1A can rescue the Sec mutant- so why not use SAR1A?

Because SAR1B is thought to be required for large cargo such as collagen, which need larger cages. The mutant is not very efficient at binding to SAR1B.

53
Q

Mutant binding of Sec23/24 to SAR1B? (Fromme, 2007)

A

Poor, 6/8 of the AA differences in SAR1A and B binding sites are on the surface of the F382L mutation

54
Q

Main finding of the Fromme paper?

A

This paper provided the first evidence for distinct affinities between COPII subunit paralogs, suggesting that variation of Sar1 paralog expression is another means of regulation of cargo.

55
Q

Haemophilia is a defect at..?

A

COPII vesicle formation- Clotthing Factor V and VIII not secreted.

56
Q

COPII cargo examples?

A
  • Collagen (CLSD)
  • Factor V and VIII (Haemophilia)
  • TMP destined for ither organelles
  • Recycling proteins e.g. SNARES, cargo receptors
  • p23/24 family- chaperone for Hsp90
  • ERGIC-53 (cargo R for proteins between ER and Golgi)
  • Any proteins synthesised and ready to be processed at the golgi for secretion.
57
Q

What is classed as big cargo?

A

100nm normal COPII vesicle, Cylomicrons v big 600nm and collagen- about 500nm length

58
Q

2 models for large cargo coating?

A
  1. Simply make a larger COPII cage

2. Non vesicular intemediate, and COPII somehow facilitates.

59
Q

How has large cargo coating been researched?

A

Gorur (2017) Correlative Light electron microscopy,
Can see an area of fluorescing light and do cross-sectional slices through e.g. Tag pro-collagen to flouresce- could see it was packed into a large COPII vesicle.

60
Q

Evidence to suggest that UPR important for Pro-collagen?

A

In absence of UPR transducer, decrease in cargo proteins and notocord not lined up developmentally. e.g. AtF6 -/-.

61
Q

What has been found to be necessary for collagen secretion?

A

Tango1- binds to bulky cargos, similar protein also needed for chylomicrons in gut.

62
Q

COPI used for?

A

Transport vesicles retograde and anteriograde though secretory pathway.

63
Q

COPI structure?

A

Coatomer of 7 proteins:
Requires an adapter B, o, Y, Z (greek)
and COP coat A, B, E
One complex but those are different subcomplexes (F=adapters), B=coat

Also Arf1 GTPase, activtes a myristoyl switch

64
Q

What is Arf1?

A

Arf1 GTPase, activtes a myristoyl switchThe ARF1 protein is localized to the Golgi apparatus and has a central role in intra-Golgi transport.

65
Q

Cargo transported by COPI vesicles?

A

Retrograde: From golgi back to ER- cargo that has a KDEL sequence and di-lysine motif.
Anterograde: Cargo going through golgi

66
Q

TGN function?

A

Transgolgi network,

  1. sortingsecretory proteins between regulated and consitutive secretion.
  2. Sorting to apical/basolateral membranes.
  3. Sorting to establish polarity in the cell
  4. Sorting lysososomal proteins for degredation. e.g. lysosomal hydrolases
67
Q

How are proteins sorted at the TGN?

A

Using adapters to sort into vesicles and coating.]

e.g. AP3 to lysosome, AP1 to MVB.

68
Q

What is the sorting signal for lysosomal proteins?

Sorting of Lysosomal hydrolase?

A

M-6-P, Mannose-6-phosphate.
Mannose there from ER, Phosphate added at the cis golgi, at the trans bings to M6P Receptor, clathri added and vesicle buds off. At Late endosome, Acidic PH causes dissociation of receptor, then phosphate removed into mature form.
Receptor and coat recycled.

69
Q

What is the sorting signal for lysosomal proteins? How added?

A
  1. From the ER the lysosomal hydrolase has an N linked oligosaccaride with a terminal Mannose.
  2. It has a specific signal patch and this can bind to the GlcNAc phosophotransferase in the golgi.
  3. UDP- GlcNAc binds next to the enzyme on the phosphotransferase.
  4. GlcNAc added to the lysosomal hydrolase mannose. (UMP given off)
  5. Another enzyme cleaves off the GlcNAc to leave M-6-P.
  6. This is released and delivered to the endosome.
70
Q

Lysosomal hydrolase mutation disease?

A

I cell disease (i)
fibroblasts contain no lysosomal hydrolase, but found in the blood. Don’t get M-6-P added so are secreted.
Mutation in GlcNAc phosphotransferase in golgi.

71
Q

what is fluid phase endocytosis?

A

Fluid phase endocytosis is a low efficiency, non-specific process that involves the bulk uptake of solutes in exact proportion to their concentration in the extracellular fluid.

72
Q

Paper on muscular dystrophies?

A

Martin et al, 2009
Cats with muscular dystrophies. Sequence analysis of translated regions of the feline dystroglycan gene in affected cats were no different nor RT-PCR (showing levels), but reduction in glycosylated alpha-dystroglycan by immunoblot. And later studies found later that a loss of function of glycosyltransferase gene caused muscular dystrophies.

73
Q

WHat is CRD? Symptoms

A

Chylomicron retention disease. fat malaborption disease (abnormalities in absorbing fat across gut), where intestinal absorptive cells fail to secrete chylomicrons into blood stream to sequester the fats.

74
Q

CFTR paper figure 1:

A

Tested 3 different blockages of ER to golgi trafficking to force unconventional pathway and stops WT band C getting to PM but band B (ER core glycosylated does) but DF508 gets to surface as band B now! Biotin to AB to CFTR to visuallise on blot.
Mutant Sar1, mutant Arf1 (component of COPI for recycling) or Stx5 overexpression- sequests SNAREs.

If gets to surface- x10 less current but still rescue in 40% cells.

Immunocytochemistry evidenced insitu also e.g. accumulation in ER etc.

75
Q

CFTR paper what forces mutant to surface?

A

(figure 1 mutant sar1, overexpress stx 5, mutant arf1)
GRASPs- if inhibit with above, no expression, therefore necessary for the unconventional pathway. overexpression surface (insert gene plasmid)
Immyunoprecipitation:
AB to HA- HA tag CFTR-GRASP-myc- Myc AB - pull down with mutant Sar1.
Thapsigargin addition forces to surface also, and this causes the P of GRASPs. IREalpha siRNA stops surfacing after thapsigargin.

76
Q

What can rescue CFTR DF508 in vivo?

A

Overexpression of GRASP55.
Crossbred DF508 x GRASP mutant.
from under 10 week life span to 95% 12+weeks.