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z:Cell 300 > Section 2: The Endoplasmic reticulum > Flashcards

Section 2: The Endoplasmic reticulum Flashcards

1
Q

newly created proteins follows secreted pathway that originates _____

A

at the ER

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2
Q

First step of protein trafficking _____ earned ____ Nobel prize

A

-(entry into the ER)
-Blobel

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3
Q

Blobel cell-free reconstitution of protein targeting

A

-isolate rough microsomes (RM)
-analyze product by SDS-PAGE + autoradiography (AR)

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4
Q

radioactive a.a –> ____ proteins will be radioactive

A

-only newly made

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5
Q

labelling with radioactive a.a is an _____

A

easy identification of product of in-vitro translation system

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6
Q

autoradiography

A

-gel with radioactive + invisible proteins + film to identify position of bands

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7
Q

size of secreted protein is ____ than nascent protein

A

less

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8
Q

all ribosomes, free or membrane-bound, are ____

A

the same

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9
Q

translocation: mRNA signal sends ____ to the ____

A

ribosomes to ER

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10
Q

translocation: peptide signal is ___

A

removed from protein before secrection

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11
Q

How do ribosomes attach to ER membrane?

A

ionic interaction
hydrophobic interactions

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12
Q

Ribosomes can be removed for ER membrane by ____

A

washing with a high-salt conc.

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13
Q

protein translocation (def.)

A

the way proteins move between organelles + compartments in cell

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14
Q

vesicle interaction protects protein from ____

A

degradation

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15
Q

what happens to translocation when microsomes are added after translation?

A

no translocation occurs

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16
Q

photo crosslinking

A

linking of proteins with UV light

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17
Q

NAC stands for

A

Nascent polypeptide associated complex

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18
Q

NAC binds to ____

A

ribosome near where polypeptide chains emerge

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19
Q

SRP stands for

A

signal recognition particle

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20
Q

SRP made of _____

A

6 proteins + RNA molecule (need for function)

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21
Q

SRP dissociated with___

A

EDTA

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22
Q

SRP has two domains:

A

ribosome binding + signal sequence binding (SRP54)

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23
Q

SRP binds to signal sequence through ____

A

hydrophobic cleft in SRP54 and ribosomes

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24
Q

adding SRP to sequences cause pausing of _____

A

translation after 70 residues

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25
Q

adding rough microsomes to blocked ribosomes-SRP causes ____ indicating that ___

A

translation + translocation to continue indicating that something of surface of microsomes binds to SRP + releases block

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26
Q

SR stands for

A

signal recognition particle receptor

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27
Q

SR is ____ of a + B subunits, where B acts as ____, and a binds to ___

A

-heterodimer
-membrane anchor
-srp on cytosol side

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28
Q

overview of ER protein targeting

A
  1. NAC binds
    2.SRP binds + pause in translation
  2. SRP binds to SR so SRP54 = empty state
    4.GTP binding to SRP54 + SR = release signal sequence from block
  3. GTP hydrolysis on SRP54 and SR = dissociation of complex
  4. export of peptide into ER lumen though pore
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29
Q

SR is on the ___ membrane

A

ER

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30
Q

SRP54 and SR subunits are both ____

A

GTP-binding proteins

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31
Q

GTP bound= active or inactive?

A

active (on)

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32
Q

GDP bound= active or inactive?

A

inactive (off)

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33
Q

puromycin does what?

A

releases nascent protein chains and terminates translation, unclogging translocation pore

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34
Q

Sec61p

A

-heterotrimer that an form translocation pore

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35
Q

crosslinking (def.)

A

chemical linking of peptide chains

36
Q

3 protein components of the translocation pore

A

-SRP receptor (recruits ribosome)
-Sec61p trimer (docks ribosome/pore)
-TRAM (chaperone)

37
Q

Translocon interacts with ___ and ____ chaperones

A

-cytosolic
-ER-luminal

38
Q

other possible protein components of translocation pore

A
  1. EMC -ER membrane complex = transmembrane proteins
  2. TRAP = insertion of signal peptides
  3. OST = N-Glycosylation
39
Q

cytosolic chaperones associated with translocon

A

Hsp40/70

40
Q

ER-luminal chaperones associated with translocon

A

-Bip

41
Q

what does signal peptidase do?

A

recognizes sequence in signal peptide and cleaves signal off the nascent protein

42
Q

_____ residues after _____ core indicates cleavage site for signal peptidase

A

-polar
-hydrophobic

43
Q

4 signal sequence characteristics

A

-at N-terminus
-removed from final product
-has 1 or more + residues after 6-12 hydrophobic ones
-little sequence conservation

44
Q

_____ properties indicates signal sequence

A

-physical

45
Q

Type 1 transmembrane protein

A

N-terminus in the ER lumen

46
Q

Creating Type 1 transmembrane protein

A
  1. cleave n-terminus signal sequence by signal peptidase
  2. insert stop transfer anchor sequence
47
Q

types of membrane topology

A

-transmembrane, single/multi-spanning
-peripheral membrane association

48
Q

Type 2 transmembrane protein

A

N-terminus in cytosol

49
Q

Type 3 transmembrane protein

A

N-terminus in ER lumen (without signal sequence)

50
Q

positive inside rule

A

-positive residues found around cytoplasmic side

51
Q

Creating Type 2 transmembrane protein

A
  1. uncleaved internal signal anchor targets nascent protein to membrane
  2. acts a membrane anchor because cluster of basic (+) residues at N-terminus of transmembrane domain = positive inside rule
  3. N-terminus directed into cytosol
52
Q

Mutation can transform Type II into Type I protein:

A

inverting charged residues = altered orientation

53
Q

Type II membrane protein : cluster of acidic (-) residues on _____ of transmembrane domain

A

luminal

54
Q

Type III membrane protein : cluster of basic (+) residues on _____ of transmembrane domain

A

cytoplasmic

55
Q

topology of multipass transmembrane proteins is due to combinations of ___

A

start and stop signal sequences

55
Q

topology of multipass transmembrane proteins is due to combinations of ___

A

start and stop signal sequences

56
Q

4 types of topogenic sequences

A
  • N-terminal, cleaved Signal Sequence (canonical)
    -Stop/Signal Anchor Sequence
    -reverse Stop/Signal Anchor Sequence
    -C-terminal Signal Anchor Sequence
57
Q

C-terminal signal-anchor

A

only exposed after completion of protein synthesis
recognized by specialized factor (Get3), not SRP
targeted to ER membrane receptors (Get1/Get2)
inserted in membrane without channel; coupled to ATP hydrolysis

58
Q

membrane protein= single-spanning, long N-terminus in lumen

A

Type I

58
Q

membrane protein= single-spanning, C-terminus in lumen

A

Type II

59
Q

membrane protein = single-spanning, short N-terminus in lumen

A

Type III

60
Q

O-linked glycosylation

A

sugar attached to OH group (serine)
single sugar added one at a time

61
Q

O-linked glycosylation occurs in ___ after ____

A

-ER lumen (rarely in cytosol)
-translocation

62
Q

N-linked glycosylation

A

suggar attached to N on side chain of asparagine (N-X-S/T)
-core oligosaccharide added en bloc

63
Q

N-linked glycosylation occurs only in ___ ____

A

-ER lumen co translationally (OST associated with pore

64
Q

N-glycosylation occurs upon conjugation of a pre-formed _____to the Asn residue within the N-X-S/T motif

A

14 unit core that is attached to lipid anchor (dolichol)

65
Q

Oxidoreductive enzymes: ____ and _____ depend on ER redox and create disulfide bonds

A

-PDI (protein disulfide isomerase)
-PPI (peptidyl prolyl isomerase)

66
Q

Chaperones: ___, ___ and ____ depend on ER energy levels and fold _____surfaces into the interior of proteins

A

-BiP, calnexin and calreticulin
-hydrophobic

67
Q

Gels with DTT/B-MER (non-reducing gels) allow us to ____

A

distinguish disulfide bond folding intermediates

68
Q

PDI promotes ___ and ___ of disulfide bonds while forming

A

-formation
-shuffling
-transient covalent intermediate

69
Q

oxidized PDI promotes ____

A

disulfide bond formation (PDI becomes reduced)

70
Q

reduced PDI promotes ____

A

reformation of disulfide bonds (PDI remains reduced)

71
Q

Function of chaperons

A

minimize aggregation and prevent interactions between unfolded chains

72
Q

Folding of hydrophobic protein segments

A

-BiP-ADP binds hydrophobic segments normally found in protein interior
-ADP release followed by ATP binding causes release of BiP from protein
-Sec63 complex promotes ATP hydrolysis

73
Q

BiP-ADP binding to nascent chain prevents ______

A

-backsliding
-aggregation

74
Q

Calnexin (CNX) and calreticulin (CRT)

A

Critical for the folding of glycosylated proteins.
Substrates undergo glycosylation/deglycosylation reaction until fully folded
works on monoglycosylated substrates
Require Ca2+ for their folding activities

75
Q

UGGT action

A

Adds glucose from UDP-glucose to generate monoglucosylated protein
-works only on unfolded protein

76
Q

difference between calnexin and calreticulin

A

-calreticulin: soluble
-calnexin: membrane bound

77
Q

ERp57 protein similar to ____

A

PDI

78
Q

Failure of ER Quality Control leads to ____

A

the accumulation of unfolded proteins

79
Q

Proteasome

A

an ER-Associated Degradation Pathway

80
Q

20S of proteasome

A

4 heptameric rings that form a narrow inner channel with multiple protease activities

81
Q

19S of proteasome

A

Recognition of substrates that acts as gate and unfolds proteins in ATP-dependent manner

82
Q

proteasome: ER associated degradation pathway

A
  1. Ubiquitin (Ub) is a small 8 kDa protein.
    2.Transferred to target protein by group of three enzymes (E1, E2 and E3) to a specific Lys. Forms isopeptide bond.
    3.. Targeted protein then becomes poly-ubiquitinated by repeated transfer to Lysine 48 of ubiquitin
  2. Poly-ubiquitinated (>4) proteins are recognized by 19 S complex.
  3. Protein is unfolded and fed through the inner channel of proteasome.
  4. Ub removed by isopeptidase during unfolding for recycling.
83
Q

proteasome in ER?

A

No

84
Q

E1/E2/E3 in the ER?

A

No

z:Cell 300 (17 decks)

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