Brainscape's Knowledge GenomeTM

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


See full index

y > ER Translocation > Flashcards

ER Translocation Flashcards

1
Q

Which protein would you stain for to visualise the ER?

A

Calreticulin which is an ER chaperone

You would use anti-calireticulin antibody

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

What is the entry point into the secretory pathway?

A

The endoplasmic reticulum

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

How has most research into the ER been carried out?

A

On microsomes

These are ER vesicles with ribosomes attached, which work as proxies for the entire ER.

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

How do you prepare microsomes?

A

They form when you homogenise a cell and separate the contents by density gradient fractionation

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

Describe the structure of signal peptides

A

Found on the N-terminal region of a protein
about 20 amino acids long
with basic/positively charged amino acids in the first few residues
hydrophobic stretches
alpha helical structure
small amino acids like alanine, serine or glycine are found at the signal peptide cleavage point

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

How wide is the lipid bilayer in amino acids?

A

17-18 amino acids

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

When does ER import happen for most mammalian proteins?

A

co-translation

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

If you add the ER after translation, what would you find?

A

protease sensitive protein with an intact signal sequence

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

If you add the ER before translation, what would you find?

A

protein is protected from protease and signal sequence is cleaved

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

When does ER import happen for most yeast proteins?

A

post-translation

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

Who proposed the Signal Hypothesis?

A

Blobel and Dobberstein (1975)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What is the Signal Hypothesis?

A

that information for targeting a protein to the ER lies at its N terminus. It emerges from the ribosome first, triggers the attachment of the free ribosome-nascent chain complex to the ER membrane for cotranslational importation through a channel.
This must start before the protein gets too long/folded.
Once in the lumen, the signal peptide is cleaved off and the protein can fold - it is a one way process.

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

What is the speed of translation?

A

100 amino acids per minute

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

How was the signal recognition particle discovered?

A

Walter and Blobel (1981) noticed that washing microsomes with salt to remove peripherally associated proteins, preprolactin were protease sensitive.
Fractions of proteins were added back and it was found that a large protein was what made preprolactin resistant.

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

What are the components of SRP?

A

P54 = recognises and binds to hydrophobic stretches on the ER signal sequence as protein leaves the ER
P68/P72 = sends ribosome to the ER
P9/P14 blocks translation while translocation happens by binding to aminoacyl site

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

What ensures that the ribosomes docks with the ER membrane and not other membranes?

A

Signal Recognition Particle (SRP)

The ER is targeted, not by the emerging signal peptide, but by the P68/72 proteins of the ribosome-nascent chain-SRP complex.
These SRP proteins interact with a duplex of receptors (SRP a and β receptors), unique to the ER membrane.

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

How can a ribosome-nascent chain complex move to the ER fast enough before chain extension and protein folding preclude import?

A

Signal Recognition Particle (SRP)

A ribosome-nascent chain complex does not have to move to the ER within milliseconds in order to ensure that the protein isn’t too extended or folded for import, because the binding of SRP P9/14 blocks the ribosome A site, preventing tRNA access and hence precluding continued translation.

This pause in translation gives time for the ribosome-nascent chain-SRP complex to find and dock with the ER membrane.

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

Describe the SRP cycle?

A

GTP binds to both SRP54 and the SRP receptor.
Then a conformational change “locks” the complex onto the membrane.
This leads to the transfer of the ribosome to other proteins.
SRP54 and SRP receptor are GTPases so GTP hydrolysis leads to SRP release (when BOTH are bound).
SRP dissociates from the signal peptide and elongation cycles can resume.

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

Is SRP found elsewhere?

A

SRP homologues have been found in fungi and bacteria, eg E. coli

However, in these systems, only a small number of proteins are targeted to the ER (or periplasm) in a ribosome-dependent, SRP-dependent manner.

Most targeting in lower eukaryotes and bacteria is SRP-independent

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

How was the existence of protein-conducting channels investigated in the ER

A

Simon and Blobel (1991) used electrochemical methods: used electrodes on either side of a membrane which separated two chambers. Allowed microsomes to fuse with the membrane. Measured currents across the membrane to measure ion movement across channels.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

How was the existence of protein-conducting channels in the ER membrane shown?

A

Measured membrane potential.
No potential at first bc while docking of ribosomes opens the channel, nascent protein blocks it.
Adding puromycin to mimic the 3’ end of charged tRNA caused the nascent protein to fall off quickly, resulting in a flux of chloride ion which could be measured.

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

If we get rid of ribosomes all together, do protein-conducting channels remain?

A

If you add salt to strip the ER membrane of ribosomes, the flux stops so we can conclude that the channels only work when translating ribosomes are physically attached

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

How was the environment of the mammalian protein translocation channel of the ER membrane investigated?

A

Crowley et al (1993) probed the signal peptide using fluorescence spectroscopy - A fluorescent probe (NBD) was incorporated into the signal peptide of nascent chains. Fluorescence of NBD is affected by the hydrophobicity of its environment. In a nonpolar/hydrophobic environment it fluoresces for 7-8ns whilst in an aqueous environment its fluorescence lifetime is just 1ns.

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

What is the environment of the mammalian protein translocation channel of the ER membrane? - is it aqueous or hydrophobic?

A

Crowley et al (1993) found that it was aqueous because When NBD-Lys was incorporated into the signal peptide of a truncated preprolactin and fluorescence lifetimes measured, the NBD had a lifetime of 1ns

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

What were the special requirements of Crowley et al’s experiment?

A
  • Truncated protein length to synchronise translation: so that signal peptides are in the ER channels during excitation of the fluorescent probe
  • Deleted lysines in preprolactin and added a single one at the 5’ end for NBD to bind to.
  • Lysyl tRNA needed to be purified, labelled with NBD and returned.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
26
Q

How would you test the contents of the protein conducting channels in the ER membrane?

A

collisional quenching of fluorescence
If iodide ions are able to collide with excited NBD molecules, the excited state will be lost and fewer photons will be emitted (i.e. fluorescence intensity drops).

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

How does collisional quenching of fluorescence work?

A

Quench (stop) fluorescence by adding quencher (iodine ions) and measure fluorescence intensity.
Put iodide ions in the cytosol vs in the ER lumen and see which condition results in no fluorescence

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

How did collisional quenching of fluorescence show whether channels are filled with cytosol?

A

Add iodide ions to the cytosol .
When translating ribosomes are membrane-bound, there is no change in fluorescence intensity.
This shows the cytosol is not what makes the channel aqueous and there must be some kind of seal

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

How did collisional quenching of fluorescence show whether channels are open to the ER lumen?

A

Streptolysin O is a gentle detergent which will punch holes in the membrane so iodide can go through.
Still no quenching so when the protein is short, the channel is still closed on both ends.
It was repeated when protein is longer and quenching occurred so channel is open from the luminal side

30
Q

How was the gate identified?

A

Hamman et al 1998:
Repeated experiment with ER lumen depleted of protein content.
Added them back slowly and found that BiP is the gate.

31
Q

How big is the aqueous pore in the ER lumen and how was it found?

A

Using different sized quenchers, it was found to be 1.5 nm while resting and 4-6nm when nascent chains are being translocated

32
Q

How was it shown that alpha helices begin folding in the ribosomal tunnel?

A

FRET - two fluorophores on an amino acid stretch destined to be an alpha helix. when it folds, energy can be transferred from one to the other so excite one with one wavelength of light and measure the expected.

33
Q

How was the composition of ER channels found?

A
  1. Cross linking
  2. Genetic studies
  3. Functional reconstitution of translocating membranes
34
Q

What are the two types of cross-linkers?

A
  1. Conventional cross-linkers = use standard chemistry. links proteins together that are close in proximity, but not very refined as they cross-link to membranes

2, Photoactivatable cross linkers = uses highly reactive chemicals activated by light, no need for suitable chemical groups to be present, can be incorporated into nascent chains so no need to ensure crosslinkers can diffuse into channel.

35
Q

How was cross linking used to find the composition of ER channels?

A

Photoactivatable cross linkers were incorporated into nascent chain. light was shone onto the area and everything was crosslinked.
Preprolactin was labelled with [35S] Met. Radioactive complexes were isolated. Mass of this minus preprolactin = size estimate of coupled protein
Antibodies can be used to confirm identity of components
Nowadays, mass spec would be used to sequence peptides and identify

36
Q

What is cross linked to nascent chains in the channels of the ER membrane?

A
  • SRP receptor
  • Signal peptidase
  • TRanslocating chain Associating Membrane protein (TRAM) – required for translocation of certain membrane proteins
  • Ribophorin (part of oligosaccharyl transferase) – enzyme complex that attaches glycan to asparagine residues
  • Sec61-a (alpha)
37
Q

Who confirmed the role of Sec61-a in the translocation of proteins across the ER membrane?

A 
Randy Schekman (1990) did a yeast screen and identified a yeast mutant where proteins accumulated in the cytosol, class A. Proteins couldn't translocate into the ER.
Complementation analysis of this mutant found that proteins with multiple hydrophobic stretches were needed to restore function.
38
Q

What proteins did Schekman identify?

A

Sec61, Sec62 and Sec63 genes.
Sec61 is particularly important as mutations in it completely blocked import into the ER.
It was equivalent to mammalian Sec61alpha.

39
Q

What is a limitations of cross-linking?

A

White it identifies neighbours, it doesn’t tell you whether they have a function in the process

40
Q

What did Gorlich and Rapoport conclude?

A

Sec61a is important for the membrane translocation of secretory proteins

41
Q

Who showed the minimal requirements for protein translocation across the ER membrane and how?

A

Gorlich and Rapoport 1993

Immunodepletion of particular proteins, membrane reconstitution, then translation of a secretory protein and a post-translational protease protection assay

42
Q

What did Gorlich and Rapoport do?

A

First, solubilise ER membrane proteins using a non ionic detergent which makes micelles around a protein.
Antibodies are added to bind to proteins which are used to remove proteins using Protein A Sepharose beads which bind the Ab.
Centrifuge the beads out.
Remove detergent to reconstitute the membrane, which are said to be immunodepleted.
Function can be tested in invitro cell free translation systems.

43
Q

What structural features of ER translocons were revealed by crystal structures?

A

Crystal structure of archael SecY by Wu et al (2019) showed 3 subunits; alpha, beta and gamma.
Alpha subunit has 10 TMDs which form the pore.
Pore is blocked by a plug which is kept in place by a pore ring; a cluster of hydrophobic residues

44
Q

How do WE THINK translocons work?

A
  1. Ribosome or a Sec62/63 complex binds to a closed channel
  2. Plug is displaced by the emerging signal peptide that then inserts in the channel through the pore ring as a loop
  3. The mature region of the translocating protein passes through a widened pore ring (and the SP is cleaved)
  4. Once through, the plug returns to its closed state and the pore ring narrows so the channel is self-sealing and the signal peptide triggers the opening of the pore.
45
Q

How does the proteinaceous channel form and behave in the absence of a ribosomal seal (e.g. during post-translational import)?

A

The channel itself has structural features (plug and pore ring) that might maintain the permeability barrier

46
Q

Do ribosomes seal the pore during co-translational import?

A

They may contribute to this, but from the X-ray structure of SecY, the channel itself appears to be self-sealing

47
Q

How do the hydrophobic domains of nascent membrane proteins leave the Sec61p channels and enter the lipid bilayer, and how do surface loops of multispanning membrane proteins become extruded to face the cytosol?

A 
The 'clam shell' arrangement means that the channel can change conformation to open in order to allow transmembrane domains to move laterally into the lipid bilayer.
Other proteins (hydrophilic ones) can translocate with the lateral gate closed and the central channel only rearranges slightly.
48
Q

What other components are needed for optimal translocation of proteins, other than translocons?

A

Mammals: TRAM and SRP receptor

Yeast: Sec62/63p, Bip and SRP receptor

49
Q

What does TRAM stand for?

A

TRanslocating chain Associating Membrane

50
Q

What other membrane proteins make contact with incoming protein chains?

A

oligosaccharyl transferase (adds glycan to protein)
signal peptidase
calnexin (an ER chaperone)

51
Q

How does post-translational translocation happen?

A

SRP does not recognise these proteins because the hydrophobicity of the peptide is a lot lower than SRP’s targets.
Cytosolic chaperones maintain the fully made preprotein in a translocation-competent state – prevents the protein from folding because once folded it cannot be translocated.
In yeast, the Sec62/63 complex is thought to recognise weaker signal peptides and deliver them to translocation pore.

52
Q

What did we learn from the first cryo-EM structure of the yeast post-translational translocon?

A
  • It consisted of the structure of Sec61, Sec 62, Sec 63 and other accessory proteins
  • Sec63 causes wide opening of the lateral gate of the Sec61 channel, priming it for the passage of low-hydrophobicity signal sequences into the lipid phase, without displacing the channel’s plug domain.
53
Q

What problems can arise with post-translational translocation?

A

Premature folding of the nascent protein would clog the translocation pore on the cytosolic side - usually happens when overexpressing a protein.

54
Q

What system has yeast evolved to overcome problems with post-translational translocation.

A

Ast et al 2016 discovered that a metalloprotease, Ste24, detects folded proteins in the translocation pore and frees up the pore by cleaving the protein.

55
Q

What is the driving force for translocation across the ER membrane?

A

For co-translational translocation, the force comes from elongation cycles, however GTP is needed for SRP docking.
The energy used for protein translation provides sufficient push force to drive the protein across the membrane.
But for post-translational translocation, two mechanisms have evolved. E. coli uses the push technique and yeast uses the pull technique.

56
Q

What is the push technique and where is it found?

A

E.coli uses it. SecA acts as a pump to push the protein into the translocon from the cytosolic side. It has ATPase activity, so upon hydrolysis, part of SecA bound to the peptide pokes into the translocon.

57
Q

What is the pull technique and where is it found?

A

Yeast use it.
BiP drives the protein in by waiting at the luminal side of the channel.
It has ATPase activity so binding ATP allows it to bind proteins and hydrolysis causes it to let go. It binds and unbinds in a cycle to pull the peptide in.

58
Q

What are the two ways that proteins enter the ER?

A

Type 1 = Head first

Type 2 = Tail first

59
Q

Explain the Type 1 way that membrane proteins enter the ER

A

Head first:
Signal peptide on the N terminus enters first in a loop, then drives the rest of protein into the channel, making the loop bigger.
When a TMD passes through the pore, SP is cleaved and it will diffuse laterally.
The N terminus will be in the ER lumen and the protein will be anchored to the membrane.

60
Q

Explain the Type 2 way that membrane proteins enter the ER

A

Tail first:
Type II proteins don’t normally carry a signal peptide but do contain a transmembrane domain. As the signal peptide is essentially a TM domain, a TM domain can act as a SP.
So TM domain is inserted in a loop but there is no cleavage as there is no SP. The TM domain diffuses out of the lateral gate.
Rest of the protein threads through. N terminus is in the cytosol while the C terminus is in the lumen

61
Q

How can you predict if a transmembrane protein is Type 2?

A

They will have positively charged amino acids near the cytosolic side of the membrane.
Charge distribution around the start-transfer sequence determines orientation: the ‘positive inside rule’ (inside = cytosol)

62
Q

How do big proteins with multiple TMDs get into the ER membrane?

A

Sec61 has 10 TMDs. Charge distribution in the first TMDs determines the topology of multipass membrane proteins - it will tell you which side goes into the lumen.

63
Q

What is Sec61?

A

A translocon

64
Q

What guides the insertion of the first TMD on ER membrane proteins?

A

ER Membrane protein Complex (EMC), discovered by Chitwood et al 2018 by knocking it out, which makes GPCRs enters the membrane incorrectly so they are degraded. Highlights importance of correct insertion of GPCRs.

65
Q

What are alternative routes for membrane proteins going into the ER membrane?

A

For proteins that are tail-anchored (e.g. cytochromes, SNAREs, next lectures), their TMDs cannot engage with SRP.
Instead, a dedicated Guide Entry of Tail-anchored (GET) protein complex inserts the protein into the membrane post-translationally.

66
Q

How were membrane proteins which use neither GET or SRP discovered?

A

Aviram et al 2016 fused model protein Gas1 to an FP. Visualised ER. Inserted fusion protein into library of yeast with different mutations. Looked at its localisation. Cytosol = failed translocation.
3 mutants showed this. Mutant proteins were named SND1-3 (SRP-independent targeting).

67
Q

What proteins are recognised by SND?

A

Ones which have TMDs far from the N or C terminus so SRP or GET cannot recognise them.

68
Q

If a TMD comes out of the ribosome first, what protein binds?

A

SRP is most likely to bind.

If not, then SND.

69
Q

If a TMD comes out of the ribosome last, what protein binds?

A

GET is most likely to bind.

If not then SND.

70
Q

What happens after translocation into the ER?

What are the events in the ER lumen?

A

SP is removed by Signal peptidase. Further cleavage can be carried out by signal peptide peptidase.
Chaperones prevent inappropriate interactions during folding.
Protein disulphide isomerase catalyse disulphide bond formation where appropriate.
Oligosaccharyl transferase (OST) adds sugars to Asn resiudes

y (6 decks)

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