Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

BMS 109 > MT- secretory > Flashcards

MT- secretory Flashcards

1
Q

Why need membrane trafficking? (3)

A
  1. Compartmentalisation: targeting of proteins to specific locations.
  2. Retrieve proteins back to resident compartment e.g. a receptor to be internalised with antigen etc and need to go back to membrane.
  3. secretory pathway.
  4. Endocytic pathway. - for signalling or degradation.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What is constitutive secretion?

A

Constitutive secretion: Secretion without any signals needed.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What is regulated secretion?

A

Regulated secretion: Needs sorting signals to say where to go and signals for release.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

ER proteins have a ….?

A

KDEL sequence.
Signal sequence targets proteins to the ER.
e.g. V SNARES etc so can recycle back to the ER.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Organelles ER secretion?

A

ER- ER Golgi Intermediate compartment (ERGIC or VTC)-Cis Golgi Network- Cis Golgi- Medial Golgi- Trans Golgi- Trans Golgi Network (TGN)- Secretory vesicle to PM.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

Organelle order of endocytic pathway?

A

PM- Endocytic Vesicle-Early endosome- Late Endosome/MVB- intraluminal vesicles- Lysosome for degradation.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Glycosylations are? To where?

A

addition of a sugar residue to a protein. Either N (Asparagine) and O(found on serine and theonine) linked glycosylations in the ER or Golgi.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What is a common residue sequence sugars can be added to?

A

In the ER lumen, when theres an asparagine, something, then a serine/theonine (N-x-S/T) motif, a preformed sugar can be added by an oligosaccharidal transferase.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

Enzymes that can trim down glycosylations?

A

Glycosylase.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

Experimental evidence for glycosylation?

A

CFTR glycosylation- band C complex glycosylated in Golgi, or band B core glycosylated in ER.
Know? Delta F508 paper, if force the unconventional pathway (e.g. mutant Sar1, upregulate syntaxin 5 sequests SNARES) which skips the Golgi this can still get to surface but not complex glycosylated, only core. Can see this on western blots as band B has a lower MW.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Purpose of glycosylation?

A
  1. Asist and stabilises the folding of the protein.
  2. Form a site for adhesion to another protein or extracellular matrix. dystroglycan
  3. Interaction site for ligand. e.g Staphylococcus aureus toxin/
  4. Can affect trafficking to places on membrane.
  5. Give protein complexity, depending on enzymes present and levels can give different sugars- gives different properties on different cells etc. e.g. blood type.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Example of glycosylation giving specificity to a protein?

A

Blood types.

Different O-linked sugars.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Example of glycosylation affecting Interaction site?

A

Staphylococcus aureus toxin binds to specific sialic acid residue additions(monosaccaride often found at terminal end of oligosaccharides giving protein a negative charge) on certain human cell surface glycosylated proteins. Uses this to get its toxins into cells.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Example of glycosylation giving stability to a protein and interactions with other proteins?

A

Certain muscular dystrophies are caused by the absence of certain sugars on the protein dystroglycan. It isn’t a defect in a protein but in the sugar addition which normally makes muscles more robust.
Dystroglycan links dystrophin to the extracellular matrix around muscle cells. Its sugars are important for facilitating this interaction. Without dystroglycan people will get muscular dystrophy, but loss of sugars can cause different type.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

What might make a model suitable for studies on membrane trafficking?

A

Simplicity - trafficking occurs on a cellular scale so a single celled organism is likely to provide information.
Analysis of specific types of secretion e.g regulated secretion, would need a model system that is able to perform this function.
Often different systems reveal different information

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Positives and negatives of yeast model organisms for membrane trafficking?

A

Positives:cheap, no ethic problems, genetic studies easy(can grow haploid or diploid), genome sequenced, many conserved paths.

Negative: Limited gene diversity, limited cell-cell contact so unlikely to be informative about multicellularity, small (5µm), so high resolution imaging studies of intracellular compartments is difficult. Has a cell wall which can preclude some types of studies- e.g. microinjection studies.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

Study that investigated the secretory pathway in yeast?

A

Novick and Schekman 1980.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

What was the rationale behind the Novick and Schekamn 1980 experiment?

A

mutate certain genes and if vesicles couldn’t be secreted i.e cells were secretory deficient (sec-), the cell would increase its density as these vesicles accumulate. These cells would also accumulate proteins that are normally secreted (invertase, acid phosphatase).

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

Novick and Schekamn 1980 experimental procedure to separate sec mutants?

A
  1. yeast cells were mutagenized randomly and shifted to a restrictive growth temperature (37°C) and then fractionated in a gradient forming medium.
  2. A 5% increase in density of the sec mutants gave complete separation from a population of wild type cells.
  3. The densest 1-2% of cells were tested further (separated by centrifugation.) These were the mutated, and an increase in density was due to an accumulation of cargo that couldnt be secreted.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

What did Novick and Schekamn 1980 then do with the sec mutants identified?

A
  1. The densest 1-2% of cells were put under electron microscopy to see alterations in the normal ultra-structure of cells e.g accumulation of vesicles or Berkeley body formations inside cell.
  2. Certain proteins can be detected which are modified at different stages through the secretory pathway (e.g glycosylated or proteolytically cleaved) to see where the mutation was.
  3. Novick and Schekman analysed cells for their ability to secrete invertase and acid phosphatase at permissive and restrictive temperatures.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

How did Novick and Schekman 1980 define secretory mutants?

A

They defined secretory mutants as those strains which fail to export active invertase and acid phosphatase, but continued to synthesize protein under restrictive growth conditions.(so not synthesis problems) These assays looked for defects in secretion but not establishing the stage of the defect. Invertase- mutants plates did not go brown as they did not secrete this.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

How is the ultrastructure of Sec mutants different?

A

Vesicles budding off golgi or ER are seen in the cytoplasm unable to be secreted. Also formation of Berkleley bodies, double membraned strucures. Discovered in 1980 by Novick and Schekman, of the University of California, Berkeley.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

Example of well studied protein that’s modified before being secreted? How is it modified?

A

Alpha-factor. Made as a Pre-Pro form. Oligosaccharides added, then modified, e.g. mannose added. In late golgi, KEX 2 chops it up into 4 mature alpha-factor proteins. Oligosaccharides function to ensure the protease recognises the protein and folds correctly.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

Results of Novick and Schekman experiment?

A

23 sec genes were identified (genes required for secretion from ER to PM) in Yeast. These could be put into 5 categories Class A-E depending on the stage of the block in secretion.
A- accumulation in cytosol. B- RER, C-ER to Golgi vesicles, D-Golgi, E-secretory vesicles.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Q

Why weren’t all of the genes/proteins involved in the exocytic pathway identified by Novick and Schekman ?

A
  1. They only identified temperature sensitive mutants. Not all genes when mutated will cause this phenotype.
  2. They only considered secretion to the plasma membrane so defects in transport to endosome or vacuole would not be identified.
  3. Any ‘redundantly’ functioning genes would not be identified. (yeast normally low redundancy- so fewer gene compensation, however is some.)
  4. Some mutations would have simply killed the cell.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
26
Q

Temperature sensitive mutant vs restrictive vs permissive?

A

Temperature sensitive mutant- Genes that allow normal function at low temps but altered at high temperatures.
Permissive temp: temp at which a temperature sensitive mutant gene product takes on normal function/phenotype.
Restrictive temp: takes on mutated function i.e. here disturbing trafficking so not secreting.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
27
Q

A vacuole is what?

A

Bacterial version of a lysosome.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
28
Q

What is a lysosome/Vacuole?

A

Degradation of extracellular material taken up by endocytosis and autophagy (intracellular components) by degradative/proteolytic enzymes (break peptide bonds). Genetic studies in yeast found this.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
29
Q

How do proteins destined for the lysosome get here?

A

Active sorting after trans Golgi network to late endosome then to lysosome. Or endocytic sorting to early endo, then late.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
30
Q

Proteins used for Sec mutant studies, and VPS studies?

A

Sec: invertase and acid phosphatase.
VPS: CPY

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
31
Q

Use of CPY experimentally?

A

Vps (raymond 90’s)
Several labs have used, mutantised cells and see if CPY is secreted.
Carboxypeptidase Y is normally transported from Golgi→Lysosome/MVB, instead of being secreted (mutant).
If it is secreted know there is a mutation. A simple colour change assay could show secretion by using an antibody to CPY and a western blot/ ELISA.This cargo is well studied hence this pathway called the CPY pathway.
Over 60 vacuolar protein sorting (vps) genes have been identified in this way.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
32
Q

Pathway from Golgi to MVB?

A

CPY- carboxypeptidase Y.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
33
Q

How is CPY modified?

A

P1- pre-pro form in ER after being cleaved by signal peptidase.
Modified in Golgi=P2. - glycosylation and mannose additions.
Then trafficked to vacuole to be cleaved into the mature form.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
34
Q

Experimental evidence to test VPS?

A

E.g. Raymond 1992
CPY is normally trafficked from the golgi to vacuole/lysosome (via MVB) where it’s cleaved into the mature form. However, in vps mutant cells, a portion of the p2 (Golgi) form of CPY is secreted from the cell, where it isn’t normally. Depending on which stage the blockage was categories were made e.g.
Over 60 vacuolar protein sorting (vps) genes have been identified in this way.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
35
Q

What is VPS?

A

Vacuolar protein sorting proteins, involved in the intracellular sorting. (degradation/endocytic tests for sec mutants)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
36
Q

What did Norvick and Schekman 1980 show? (as well as 23 genes)

A

a secretory protein moves from the cytosol to the endoplasmic reticulum (ER), from the ER in vesicles to the Golgi, and then in secretory vesicles to the plasma membrane.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
37
Q

What were the phenotypes of VPS mutants?

A

They secreted CPY. Class A-F 6 complementation groups. Had different shapes/numbers of vacuoles etc. Different mutant’s depending on where the blockage was before the vacuole.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
38
Q

What were the classes of VPS mutants? (be aware of don’t need to know directly)

A

A Wild-type: 3–10 spherical vacuoles that cluster in one area of the cytoplasm. In dividing cells, the vacuole extends from mother to daughter cell along the cell axis.
B Fragmented vacuoles: more than 20 small, vacuole-like compartments.
C No identifiable vacuoles.
D Single, large vacuole that fails to extend into daughter cell buds.
E Vacuoles larger than wild-type, with a very large, aberrant late endosome/MVB (the class E compartment) adjacent to the vacuoles.
F One large vacuole surrounded by a number of fragmented vacuole structures.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
39
Q

4 destinations after the Late Golgi? Names of pathways?

A

Four possible destinations

  1. To plasma membrane- secretion
  2. To early endosome
  3. To late endosome/MVB- CPY
  4. To vacuole- ALP
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
40
Q

What receptor recognises CPY and targets it to the MVB?

A

sorting receptor Vps10 recognises CPY in the Golgi. Cycles between the late Golgi and MVB- where it dissociates from CPY.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
41
Q

How does CPY traffic from the Golgi to the MVB?

A

In Golgi CPY binds to Vps10 which helps sorting.The transport step requires cytoplasmic factors: clathrin and two adaptors called Gga1 and Gga2.

42
Q

How does Vps10 recycle?

A

By the retrograde pathway. Vps10 is retrieved to the late Golgi through a specific aromatic -based signal in its protein sequence (YSSL, FYVF). The retromer complex loads the receptors onto vesicles.

43
Q

Evidence for VPS10 as receptor of CPY?

A

Emr lab 1994- Yeast
Mutations in VPS10 result in the selective mis-sorting and secretion of CPY; all other vacuolar proteins tested are delivered to the vacuole in vps10 mutants.

Coimmunoprecipitated then Chemical cross-linking studies demonstrate that Vps10p and the Golgi-modified precursor form of CPY directly interact.

A single amino acid change in the CPY vacuolar sorting signal prevents this interaction.

Vps10p also interacts with a hybrid protein containing the CPY sorting signal fused to the normally secreted enzyme invertase.

44
Q

What signals are recognised by the retromer complex?

A

Kex2p and DPAP A- Golgi retention signals

VPS10- YSSL, FYVF aromatic sequences

45
Q

What is the retromer complex?

A

necessary for recycling of trafficking components e.g. endosomal to Golgi retrieval and endosome to PM sorting.

46
Q

Retromer and Vps10 disease link?

A

Dysregulation of retromer-mediated endosomal protein sorting leads to various pathologies, including neurodegenerative diseases such as Alzheimer disease and spastic paraplegia

genome-wide association studies (GWAS) showed certain members of Vps10 and retromer proteins are risk factors for Neurodegeneration.

47
Q

Evidence for VPS10 recycling?

A

Ungermann 2015

Method: GAL induced overexpressed Vps10–GFP from the inducible GAL1 promoter at the vacuole. cells were shifted from galactose-containing medium to glucose-containing medium to repress further expression. Localisation of Vps10 was monitored.

Result: Initially all vps10 shifted to the vacuole (cargo such as CPY from Golgi to here). After 7.5 hours after a synthesis inhibitor was added they relocated into punctae in the cytoplasm.

48
Q

Evidence for the retromer complex recycling VPS10

A

Ungermann 2015

Recycling from vacuoles depends on the retromer, Ypt7 and Vps1. In the absence of any retromer subunit, recycling from the vacuole was blocked. Absence of VPs1 also implicated recycling.

Visualisation by VPS10-GFP under GAL1 promoter seen by fluorescent microscope. ) Retromer subunits localize to Vps10 dots on the vacuole during retrograde transport from this organelle. Endogenous retromer subunits Vps17 and Vps35 were C-terminally tagged with mCherry.

49
Q

Retromer complex formed by?

A

yeast dynamin Vps1 and Rab protein YPT-7 and others/

50
Q

What is the ALP pathway?

A

golgi to vacuole. In the ALP pathway, the alkaline phosphatase ALP is packaged into vesicles through adaptor AP-3 interaction which in turn recruits clathrin.

51
Q

The ALP pathway recruits what adapter?

A

adapter Ap-3 and adapter protein complex, but does not need clathrin.

52
Q

Golgi to vacuole pathway?

A

ALP- bypasses endosomes.

53
Q

ALP and Vam3 have in common?

A

Both proteins Golgi-lysosomes.

Both have a di-leucine sorting signal required for sorting into the correct vesicles.

54
Q

What is KEX2?

A

Its a mating pheromone protease (processing enzyme). Golgi-Early endo-Late endo- recycle back to Golgi. Cleaves alpha factor for example in the late golgi.

55
Q

What does KEX2 need for movement from Golgi to early endosome?

A

Clathrin. Defects in clathrin blocks trafficking so vesicles of Kex2 stays in the golgi.

56
Q

Kex2p signal?

A

Kex2p has a late endosome retrieval signal and a Golgi retention signal= TLS2 (trans-Golgi network localisation sequence 2).

57
Q

What’s needed for Kex2 movement from early to late endosome?

A

phosphorylation

58
Q

Why do cells need endocytosis? (3)

A
  1. Retrieval of molecules that formed part of the secretory vesicle for recycling e.g. coating molecules/ SNARES
  2. Downregulation of signals (endocytose into a cell to be degraded)
  3. Remodelling cell surface lipid and protein composition.-
    Can internalise receptors to degrade to control content on the surface, and use AA’s to make others and secrete out.
59
Q

How do we know that there are multiple compartments and not just traffic straight from the surface to lysosomes?

A
  1. Genetic studies in yeast have generated mutants that are blocked at specific stages of these trafficking pathways. e.g. Sec mutants Novick and Schekman
  2. morphological differences in compartments under microscope,
  3. •Early and late endosomes can be separated based on their density.
  4. Biochemical analysis of the contents in each compartment shows distinct proteins/enzymes in each.
60
Q

fluid‐phase endocytosis mutants were found how?

A

A mutant yeast library generated by the European Functional Analysis Network (EUROFAN) 2001 was screened for strains defective in fluid‐phase endocytosis. Accumulation of Lucifer yellow in the vacuole was used as a marker for efficient endocytosis.

61
Q

Steps in the endocytosis process?

A
  1. There is a sequential assembly and disassembly of complexes involved in endocytosis. e.g. adapters and coating- AP1 and Clathrin lattice. As well as actin nucleation proteins.
  2. Actin network formation and polymerisation which extends the cargo inwards
  3. Scission machinery cuts the vesicles off.
    cytoskeleton plays a key role in the inward movement of vesicles in yeast and in mammalian cells
62
Q

Evidence supporting endocytosis mechanism?

A

Newpher (2005) Live imaging of Actin cytoskeleton can be seen in a time course to internalise at the same time as fluorescently tagged clathrin. Clathrin is recruited prior to a burst of intensity of the actin patch/endocytic marker, Abp1.

63
Q

End- are what?

A

Endocytosis mutants that could not internalise a fluid phase marker (lucifer yellow) or a bound pheromone alpha-factor.

64
Q

Enodcytic and exocytic cargo can both be transported to..?

A

Late endosome to then be degraded at the lysosome.

Either endocytosed into a lysosome, or secreted from late golgi.

65
Q

What is a MVB? How is it formed?

A

Late endosomes invaginate in their membranes to form internal vesicles, and this is why its called a multi-vesicular body. These then fuse with lysosomes so the cargo gets degraded.

66
Q

What are class E VPS mutants?

A 
Vacuoles larger than wild-type, with a very large, aberrant late endosome/MVB (the class E compartment) adjacent to the vacuoles.
mutants are defective in forming, and sorting proteins into, lumenal MVB vesicles.
67
Q

How do we know the endocytic and Exocytic pathway can intersect?

A 
At the MVB. 
Ste2 (endocytosed PM receptor)
CPS (golgi to MVB)
Vps10 (CPY receptor between LG and MVB)
In class E VPS mutants all accumulate in an aberrant late endosome, showing they are all together at one point.
68
Q

What is Vps10?

A

receptor for CPY. Recycled between the late golgi and Late endosome.

69
Q

What is carboxypeptidase S route?

A

Synthesised then travels from the Late golgi to MVB to mature in the lysosome.

70
Q

What is Ste2? Route?

A

pheromone receptor localises to the plasma membrane, is ubiquitinated and internalised by endocytosis. It is trafficked from EE to LE/MVB and to the vacuole.

71
Q

What are ESCRTs?

A

Endosomal sorting complexes required for transport. VPS class E mutantants tend to be one of the 4 complexes. ESCRTs recognise proteins on the LE and pull them into the MVB.

72
Q

Schekman found?
Rothman?
Sudhof?

A

Schekman: Key genes involved in trafficking. Sec mutants etc.
Rothman: SNARE complex
Sudhof: Brain synapses- found synaptotagmin Ca sensor.

73
Q

What mediates protein sorting into organelles?

A

Sorting Signals.

Can be linear or a patch that coes together during protein folding.

74
Q

As well as ensuring correct destination, sorting signals can also..? E.g..

A

Enable protein retrivel back to a compartment e.g. K-DEL sequence on ER resident proteins. K-DEL receptor recognises and delivers them back.

75
Q

ER signal sequence? structure?

A

K-DEL sequence for ER soluble proteins. KKXX for TM protein sequence. (di-lysine)
with at least 8 non-polar AA’s in the centre (so that it can cross the membrane).

76
Q

If a synthesised protein has no signal sequence where is it destined to?

A

PM or cytosol.

77
Q

How can RER and SER be separated experimentally?

A

Homogenise the cell.
RER and SER will reseal into microsomes.
Centrifuge and put into an increasing sucrose gradient.
RER is more dense (ribosomes add weight) so will sink and equilibriate at the higher sucrose concentration, whereas SER will float at lower concentrations.

78
Q

KDEL importance tests? (brief)

A

Necessary: remove- does the protein still go to ER?
Sufficient: Add to another protein- Does it go to ER?

79
Q

Methods to test whether proteins are in their organelle location? IN VITRO (3)
(Therefore can be used to test necessity of Signal sequences).
(Also what other factors are needed for protein translocation e.g. ATP if restrict etc)

A
  1. Radioactively label a protein that for example has a KDEL sequence. Homogenise and fractionate the RER and SER. See if protein found in either- with and without sequence.
  2. Signal sequences are usually removed after protein gets to desired location. If run the cell lysis with and without the organelle there will be two different MWs of the protein seen on gel electrophoresis. If the shorter length is present, suggests got to location and had signal removed.
  3. If proteases are added to the cell, those proteins not in organelles will be digested, but if in organelles will be protected. If detergent is then added the protein will escape and get digested. The need for detergent will evidence that the protein is in organelle.
80
Q

Method to test sufficiency of a signal sequence?

A

Genetically engineer a protein that normally is cytosolic to express the signal sequence of interest. Transfect this cDNA into cells and by immunostaining of cell fractionation then look at the location of the protein. if tis in the organelle it is sufficient.

81
Q

How has translocation machinery been used to study signal sequences experimentally?

A

Yeast with mutations in translocation machinery been useful, but these die so genetic engineering has had to be used.
Histidinol dehydrogenase is normally in the cytosol and converts histidinol to histadine. GM a ER sequence to enzyme means its targetted to ER. Then in a media free without added histadine these yeast die.
If these GM yeast then also contain a partial ER translocation mutation, they may survive as not all the enzyme is transported to the ER so some histadine can be made so can survive on histadine free media.
This shows that the signal was sufficient to target the enzyme to the ER.

82
Q

How can translocation mutations be identified in yeast?

A

A temp sensitive mutation in ER translocation will die at high temps as no ER proteins will get there, but if transfect the mutant cells with a plasmid that contains this mutated unmutated gene it will rescue the yeast and enable it to survive. Many different gene plasmids may have to be tested in order to work out the gene.

83
Q

Signal hypothesis experiment?

A

Blobel On microsomes. LOOK UP. Idea that newly synthesised proteins should end up at the ER lumen.

84
Q

What recognises signal sequences?

A

Signal Recognition particle- SRP, on newly made proteins (maybe during translation by ribsome hence RER formation.)

85
Q

SRP structure?

A 
1 RNA and 6protein complex. 
Has a GTPase and SRP receptor binding site.
SRP RNA. 
Signal sequence binding pocket.
Translational pause domain.
86
Q

Sequence of events of a protein translocating into the ER?

A
  1. Protein being translated by a ribosome.
  2. SRP binds to the signal sequence and pauses translation.
  3. SRP recognised by SRP Receptor on the RER membrane.
  4. SRPR located near to a protein translocator, and as the complex nears the translocator plug is removed.
  5. The SRP and SRPR are recycled as the protein associates with the translocator. Translation starts again.
  6. As the protein is translated the protein is fed through the translocator into the ER lumen.
  7. Signal peptidase cleaves the signal and either the protein is released into the ER or if there is a non-polar section may stay in the ER as transmembrane protein.
87
Q

Sec61 function?

A

Protein translocator. (orginally found in yeast)
In absence of a ribosome has a plug in, but opens after complex binds.
Opens only one way to let proteins into the ER

88
Q

Signal translocation for a transmembrane protein?

A

Has a start and stop translocation sequence. The Start sequence is the signal sequence which in these proteins is half way through the polypeptide so will have the N terminus out into the cytosol. Further along the protein is a stop transfer sequence which is hydrophobic. This will stop translocation and leave the C terminus into the cytosol. There may be more start stops also to create a multipass protein.
The signal sequence is not cut off here so stays in the membrane.

89
Q

Why are newly synthesised proteins translocated to the ER?

A

It has a key role in quality control. It is rich in chaperones and glycosylating enzymes.

90
Q

Why is ER quality control important?

A

If proteins are misfolded they can aggregate and even affect other proteins e.g. Prion B amyloid proteins in Alzheimers.

Retains precursor proteins in environment where they mature

Favours correct assembly by increasing subunit concentration

91
Q

Experimental example of ER quality control?

A

CFTR paper- DF508 mutated CFTR retained in the ER and not trafficked to the Golgi for complex glycosylation (Band C). But under ER stress under the unconventional pathway (GRASPS involved and other traditional ER-Golgi pathways blocked) the core glycosylated CFTR can get to the surface and 40% of the proteins can function, evidencing that it is held in the ER.

92
Q

The start and stop translocations are..

A

hydrophobic/ non-polar. Polar bears like water but it dissolves them.

93
Q

Whats UGGT?

A

glucosyl-transferase that acts as a folding sensor on ER. Transfers glucose back onto wrongly folded proteins so they are recognised by chaperones again so gives them another chance for refolding.

94
Q

Mannose trimming role?

A

Acts as a timer for degredation, like a lifespan.

95
Q

ER Chaperone examples?

A

After terminal glycosylation and sugar trimming, Protein interracts with ER chaperones. Ca dependent Calreticulum or ERp57- aid protein folding in the ER.

96
Q

If a protein keeps misfolding? (brief)

A

Deemed terminally misfolded, mannose trimmed (no glucose added) and so degraded. ERAD pathway.

97
Q

ERAD? (6 steps)

A

ER Associated Degredation.

  1. Unfolded protein recognition- e.g. exposed hydrophobic regions or immature glycans etc
  2. Protein sugars trimmed. (mannose)
  3. The protein is reverse translocated out of Sec61 out of the ER.
  4. N-Glycanases remove remaining sugars.
  5. Polyubiquitination of the protein.
  6. A proteosome recognises and degrades the protein.
98
Q

When also does the Unfolded protein response happen?

A

Developmentally- e.g. When B cells mature to T cells they upregulate their ER in order to be able to secrete many Antibodies.

99
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.
100
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.

BMS 109 (20 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions