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BMS376 Membrane trafficking > ER translocation > Flashcards

ER translocation Flashcards

1
Q

Who is Gunter Blobel, what did he discover and examples of it?

A

Nobel prize for the signal hypothesis
- Functions of cells depends on molecules in the right place at the right time
ExAmple- Insulin needs to be transported out of the cells the molecules are packaged into vesicles which deliver their cargo

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2
Q

Randy schekman- What did he do?

A

Discovered genes encoding proteins that are key regulators of vesicle traffic
looked at mutant cells
discovered genes that control transport

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3
Q

James rothman- what did he do?

A

found protein complex which enabled vesicles to fuse with their target membrane at the right location so cargo is delivered

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4
Q

Thomas C. suldhof- what did he do?

A

Found the role of proteins, signal transmitted and what senses calcium iron channels

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5
Q

What is the role of signals in ER translocation?

A
  1. mediate protein sorting
  2. sequence
  3. patching
    they can be linear and patch
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6
Q

Difference between patched and linear

A

in the unfolded proteins linear only has signal regions the the end of the protein whereas patch has them dispursed throughout

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7
Q

ER retrieval signals- 2 types

A

KDEL- resident ER soluble proteins found in the lumen of the ER
KKXX- resident ER membrane proteins

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8
Q

Resident ER protein t

A

Have to be targeted to many different organelles

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9
Q

What does the signal sequence contain ?

A

positively (Arg/lys) and negatively (glu/Asp) charged amino acids

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10
Q

What is the endoplasmic reticulum?

A

Rough- ribosomes closely associated with the membrane
Smooth- site of lipid synthesis
ER extends throughout the cell

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11
Q

Isolation of ER

A

Homogenization- RER more dense so stops sedimenting and floats at high sucrose conc (bottom)
SER- less dense so floats higher at low sucrose level

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12
Q

Isolation of signal sequence

A
  1. transfection approach for defining signal sequence

2. biochemical approach

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13
Q

transfection approach for signal sequence

A

Plasmid used to transfect cells

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14
Q

3 different biochemical approaches

A
  1. labelling protein co-fractionates with the organelle during centrifugation
  2. the signal sequence is removed by a specific protease that is present inside the organelle-
  3. protein is protected from digestion when proteases are added to the incubation medium but its susceptible if a detergent is first added to disrupt the organelle membrane
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15
Q

What is the genetic approach of studying protein translocation?

A
  • grow yeast on histamine
    WT yeast- enzyme in cytosol, cell lives without histadine as a nutrient
    Engineered yeast- enzyme targeted to ER, cell dies without histamine as nutrient
    Engineered mutant- not all enzymes taken up into ER, cells live without histamine as nutrient
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16
Q

What is the nurture of the ER signal sequence

A

variable

At least 8 non polar aa at the centre

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17
Q

What are the early experiments of lysomes establish

A

Signal hypothesis- newly made protein should end up in the lumen of the ER

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18
Q

How is the signal sequence interpreted

A

Contains

  • translational pause domain
  • GTPase and SRP receptor binding site
  • signal sequence binding pocket
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19
Q

What does binding of SRP ensure

A

Coupling of translation to translocation
1- SRP binds to signal sequence in polypeptide
2- causes pause in translation
3- SRP ribosome attaches to SRP receptor in ER membrane
4- Translation continues
5- SRP and receptor displaced and recycled
6-translocation continues

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20
Q

Pore- what is led through and how is it opened

A

Non polar aaa (sec61) led through

the complex sits in the pore when the ribosome is absent the pore has plug in it

21
Q

What is the purpose of Sec61?

A

facilitates transfer of nascent chain across the ER membrane
released into ER and folds up

22
Q

Translocation of soluble proteins

A
  • inactive protein
  • active translator
  • mature protein in the lumen
23
Q

Integration of double pass transmembrane protein

A

2 sequences- start and stop transfer

  • the mature transmembrane proteins gets into the ER membrane but not fully through into the lumen as signal sequence not cleaved off arrest anchor
  • stops in membrane
24
Q

What is the ER lumen rich in?

A
  • chaperones and gylcosylating enzymes
  • Glycosylation- membrane asymmetry and quality control
  • chaperones-secreted and membrane proteins
25
Q

What is the quality control within the ER

A
  • prevents the transport of aberrant proteins
  • retains precursor proteins in environment where they mature
  • favours correct assembly by increasing subunit conc
  • reduces risk of toxicity by inhibiting aggregation and degrading terminally misfiled proteins
26
Q

steps to show terminal glycosylation diagram

A
  • glycosylated
  • trimmed
  • proteins interact with chaperones
  • CNX cycle
27
Q

What is terminal glycosylation dependent on?

A

Ca2+

caelrecticulus- synthesising protein packaged

28
Q

What happens if the protein is miss folded?

A

Get 2nd attempt
recycled back- transfer glucose back on sugars so another go
if terminally unfolded= degradation

29
Q

UDP-glucose

A

Acts as folding sensor in glyscoylation

Glycoprotein glucose transferase (UGGT) acts as a folding sensor

30
Q

Mannose trimming

A

acts as a trimer for degradation

31
Q

What is ERAD?

A

ER associated protein degradation

  • regulates mechanism to remove unassembled or misfiled proteins
  • reverse transcribed
32
Q

How does ERAD work?

A

Chaperone + misfiled protein

  • ER protein translocator with accessory proteins
  • N-glycanase
  • UBIQUITIN ADDED
  • degraded
33
Q

Different types of ubiquitination ?

A
  • mono
  • homotytic polyubiquitination
  • heterotypic polyubiquitination = mixed, branched, UBI-modified, modified by chemical (phosphorylated acetylation)
34
Q

What is UPR?

A

Unfolded protein response

  • stress induced signalling pathway
  • increased synthesis of ER and ER chaperones
  • inhibition of translation
35
Q

What is a chaperone

A

Proteins that assist folding or unfolding of structures

36
Q

What are the sensors for misfolded proteins?

A

IRE1
PERK
ATF6

37
Q

IRE1 sensor

A

regulated mRNA splicing indicates translation of gene regulatory proteins

38
Q

Perk sensor

A

Phosphorylation inactivates translation initiation factor

selective translation of gene regulatory protein 2

39
Q

AFT6 sensor

A

regulated proteolysis releases gene regulatory protein 3

40
Q

What does activation of all these sensors result in?

A

Activation of genes to increase protein folding capacity of ER

41
Q

What does ER stress determine?

A

cellular outcome

42
Q

DIAGRAM OF ER stress from stress stimulus

A

ER stress response in high or low magnitude

  • low= pro survival, inhibit death
  • high= pro death, inhibit survival
43
Q

Graphs of ER stress

a) physiological conditions
b) early ER stress
c) late ER stress
d) E2F1 expression and time after stimulus graph

A

a- E2F1/PBR- E2F1 causes G1/2 cell cycle transition and inhibits puma and noxA
b- cel survival- increase PUMA, NOXA, MLC,BLC
c- cell death indiction- decrease MCL,E2F1 still increase puma and nova
d- shows point of no return from survival to death dependent on E2F1 expression and time after stimulus
- long time after stimulus the E2F1 expression is still high= death

44
Q

AFT6 involvement in UPR with stress

A
  1. membrane-bound AFT6 transits from ER to golgi
  2. S1p and s2p proteases cleave AFT6 yielding a cytosolic fragment
  3. AFT6 fragment migrates to nucleus to activate transcription of UPR gene
45
Q

What is the control for UPR

A

Bip

UPR dissociates from BIP cleaved in golgi releasing TF

46
Q

What is selective exit from ER?

A

Controlled by cholesterol homeostasis regulated by SREBP

47
Q

What happens to exit from ER in high and low cholesterol conc?

A

High- SCAP retains SREBP in ER, associates with insig

Low- SCAP dissociates from inside and SREBP transmits golgi, cleaved by proteases

48
Q

Autophagic signals to autophagy

A

Autophagic signals- Pas- PM/golgi/mitochondria- phagophore- autophagasome- lysosome- autophagolysosome

BMS376 Membrane trafficking (5 decks)

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