Section 2: The Endoplasmic reticulum Flashcards
(86 cards)
1
Q
newly created proteins follows secreted pathway that originates _____
A
at the ER
2
Q
First step of protein trafficking _____ earned ____ Nobel prize
A
-(entry into the ER)
-Blobel
3
Q
Blobel cell-free reconstitution of protein targeting
A
-isolate rough microsomes (RM)
-analyze product by SDS-PAGE + autoradiography (AR)
4
Q
radioactive a.a –> ____ proteins will be radioactive
A
-only newly made
5
Q
labelling with radioactive a.a is an _____
A
easy identification of product of in-vitro translation system
6
Q
autoradiography
A
-gel with radioactive + invisible proteins + film to identify position of bands
7
Q
size of secreted protein is ____ than nascent protein
A
less
8
Q
all ribosomes, free or membrane-bound, are ____
A
the same
9
Q
translocation: mRNA signal sends ____ to the ____
A
ribosomes to ER
10
Q
translocation: peptide signal is ___
A
removed from protein before secrection
11
Q
How do ribosomes attach to ER membrane?
A
ionic interaction
hydrophobic interactions
12
Q
Ribosomes can be removed for ER membrane by ____
A
washing with a high-salt conc.
13
Q
protein translocation (def.)
A
the way proteins move between organelles + compartments in cell
14
Q
vesicle interaction protects protein from ____
A
degradation
15
Q
what happens to translocation when microsomes are added after translation?
A
no translocation occurs
16
Q
photo crosslinking
A
linking of proteins with UV light
17
Q
NAC stands for
A
Nascent polypeptide associated complex
18
Q
NAC binds to ____
A
ribosome near where polypeptide chains emerge
19
Q
SRP stands for
A
signal recognition particle
20
Q
SRP made of _____
A
6 proteins + RNA molecule (need for function)
21
Q
SRP dissociated with___
A
EDTA
22
Q
SRP has two domains:
A
ribosome binding + signal sequence binding (SRP54)
23
Q
SRP binds to signal sequence through ____
A
hydrophobic cleft in SRP54 and ribosomes
24
Q
adding SRP to sequences cause pausing of _____
A
translation after 70 residues
25
adding rough microsomes to blocked ribosomes-SRP causes ____ indicating that ___
translation + translocation to continue indicating that something of surface of microsomes binds to SRP + releases block
26
SR stands for
signal recognition particle receptor
27
SR is ____ of a + B subunits, where B acts as ____, and a binds to ___
-heterodimer
-membrane anchor
-srp on cytosol side
28
overview of ER protein targeting
1. NAC binds
2.SRP binds + pause in translation
3. SRP binds to SR so SRP54 = empty state
4.GTP binding to SRP54 + SR = release signal sequence from block
5. GTP hydrolysis on SRP54 and SR = dissociation of complex
6. export of peptide into ER lumen though pore
29
SR is on the ___ membrane
ER
30
SRP54 and SR subunits are both ____
GTP-binding proteins
31
GTP bound= active or inactive?
active (on)
32
GDP bound= active or inactive?
inactive (off)
33
puromycin does what?
releases nascent protein chains and terminates translation, unclogging translocation pore
34
Sec61p
-heterotrimer that an form translocation pore
35
crosslinking (def.)
chemical linking of peptide chains
36
3 protein components of the translocation pore
-SRP receptor (recruits ribosome)
-Sec61p trimer (docks ribosome/pore)
-TRAM (chaperone)
37
Translocon interacts with ___ and ____ chaperones
-cytosolic
-ER-luminal
38
other possible protein components of translocation pore
1. EMC -ER membrane complex = transmembrane proteins
2. TRAP = insertion of signal peptides
3. OST = N-Glycosylation
39
cytosolic chaperones associated with translocon
Hsp40/70
40
ER-luminal chaperones associated with translocon
-Bip
41
what does signal peptidase do?
recognizes sequence in signal peptide and cleaves signal off the nascent protein
42
_____ residues after _____ core indicates cleavage site for signal peptidase
-polar
-hydrophobic
43
4 signal sequence characteristics
-at N-terminus
-removed from final product
-has 1 or more + residues after 6-12 hydrophobic ones
-little sequence conservation
44
_____ properties indicates signal sequence
-physical
45
Type 1 transmembrane protein
N-terminus in the ER lumen
46
Creating Type 1 transmembrane protein
1. cleave n-terminus signal sequence by signal peptidase
2. insert stop transfer anchor sequence
47
types of membrane topology
-transmembrane, single/multi-spanning
-peripheral membrane association
48
Type 2 transmembrane protein
N-terminus in cytosol
49
Type 3 transmembrane protein
N-terminus in ER lumen (without signal sequence)
50
positive inside rule
-positive residues found around cytoplasmic side
51
Creating Type 2 transmembrane protein
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
Mutation can transform Type II into Type I protein:
inverting charged residues = altered orientation
53
Type II membrane protein : cluster of acidic (-) residues on _____ of transmembrane domain
luminal
54
Type III membrane protein : cluster of basic (+) residues on _____ of transmembrane domain
cytoplasmic
55
topology of multipass transmembrane proteins is due to combinations of ___
start and stop signal sequences
55
topology of multipass transmembrane proteins is due to combinations of ___
start and stop signal sequences
56
4 types of topogenic sequences
- N-terminal, cleaved Signal Sequence (canonical)
-Stop/Signal Anchor Sequence
-reverse Stop/Signal Anchor Sequence
-C-terminal Signal Anchor Sequence
57
C-terminal signal-anchor
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
membrane protein= single-spanning, long N-terminus in lumen
Type I
58
membrane protein= single-spanning, C-terminus in lumen
Type II
59
membrane protein = single-spanning, short N-terminus in lumen
Type III
60
O-linked glycosylation
sugar attached to OH group (serine)
single sugar added one at a time
61
O-linked glycosylation occurs in ___ after ____
-ER lumen (rarely in cytosol)
-translocation
62
N-linked glycosylation
suggar attached to N on side chain of asparagine (N-X-S/T)
-core oligosaccharide added en bloc
63
N-linked glycosylation occurs only in ___ ____
-ER lumen co translationally (OST associated with pore
64
N-glycosylation occurs upon conjugation of a pre-formed _____to the Asn residue within the N-X-S/T motif
14 unit core that is attached to lipid anchor (dolichol)
65
Oxidoreductive enzymes: ____ and _____ depend on ER redox and create disulfide bonds
-PDI (protein disulfide isomerase)
-PPI (peptidyl prolyl isomerase)
66
Chaperones: ___, ___ and ____ depend on ER energy levels and fold _____surfaces into the interior of proteins
-BiP, calnexin and calreticulin
-hydrophobic
67
Gels with DTT/B-MER (non-reducing gels) allow us to ____
distinguish disulfide bond folding intermediates
68
PDI promotes ___ and ___ of disulfide bonds while forming
-formation
-shuffling
-transient covalent intermediate
69
oxidized PDI promotes ____
disulfide bond formation (PDI becomes reduced)
70
reduced PDI promotes ____
reformation of disulfide bonds (PDI remains reduced)
71
Function of chaperons
minimize aggregation and prevent interactions between unfolded chains
72
Folding of hydrophobic protein segments
-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
BiP-ADP binding to nascent chain prevents ______
-backsliding
-aggregation
74
Calnexin (CNX) and calreticulin (CRT)
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
UGGT action
Adds glucose from UDP-glucose to generate monoglucosylated protein
-works only on unfolded protein
76
difference between calnexin and calreticulin
-calreticulin: soluble
-calnexin: membrane bound
77
ERp57 protein similar to ____
PDI
78
Failure of ER Quality Control leads to ____
the accumulation of unfolded proteins
79
Proteasome
an ER-Associated Degradation Pathway
80
20S of proteasome
4 heptameric rings that form a narrow inner channel with multiple protease activities
81
19S of proteasome
Recognition of substrates that acts as gate and unfolds proteins in ATP-dependent manner
82
proteasome: ER associated degradation pathway
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
4. Poly-ubiquitinated (>4) proteins are recognized by 19 S complex.
5. Protein is unfolded and fed through the inner channel of proteasome.
6. Ub removed by isopeptidase during unfolding for recycling.
83
proteasome in ER?
No
84
E1/E2/E3 in the ER?
No
