Unit 6: Protein Sorting to Organelles - 1 Flashcards
1
Q
A typical mammalian cell contains up to __ different proteins
A
10 000
2
Q
A typical mammalian cell contains up to 10,000 different proteins, and each must be __
A
localized to the correct organelle.
3
Q
protein: Na+/K+ ATPase
location: _
A
plasma membrane
4
Q
Protein : RNA polymerase
location: ?
A
nucleus
5
Q
protein: proteases
location:?
A
lysosome
6
Q
protein: catalase
location? _
A
peroxisome
7
Q
protein: ATP synthase
location: ?
A
mitochondria
8
Q
protein: hormones
location : ?
A
extracellular space
9
Q
most of the proteins (“nuclear DNA proteins”) synthesized in eukaryotic cells are (3):
A
(1) Encoded by nuclear DNA
(2) Synthesized on ribosomes in the cytosol
(3) Are delivered to the organelle of destination from the cytosol
10
Q
most of the proteins synthesized in eukaryotic cells are encoded by:
A
nuclear DNA
11
Q
most of the proteins synthesized in eukaryotic cells are synthesized on:
A
ribosomes in the cytosol
12
Q
most of the proteins synthesized in eukaryotic cells are delivered to the organelle of destination from
A
the cytosol
13
Q
a few proteins “organelle-specific proteins” synthesized in eukaryotic cells are (3):
A
(1) Are encoded by the DNA present
in MITOCHONDIRA and CHLOROPLASTS
(2) Are synthesized on ribosomes inside
mitochondria and chloroplasts
(3) Are incorporated directly into compartments within mitochondria and chloroplasts
14
Q
A few proteins (“Organelle-specific proteins”) synthesized in eukaryotic cells are encoded by:
A
the DNA present
in mitochondria and chloroplasts
15
Q
A few proteins synthesized in eukaryotic cells (“Organelle-specific proteins”) are synthesized on:
A
ribosomes inside
mitochondria and chloroplasts
16
Q
A few proteins synthesized in eukaryotic cells (“Organelle-specific proteins”) are incorporated:
A
directly into compartments within mitochondria and chloroplasts
17
Q
Define “Protein sorting” :
A
the process by which newly-made proteins are directed to the correct location
ex; proteins A and B are sorted to different organelels
18
Q
Each protein has a __ that can range from 3-60 continuous amino acids.
A
sorting signal (signal sequence)
19
Q
Each protein has a sorting signal (signal sequence) that can range from
A
3-60 continuous amino acids
20
Q
Each protein has a sorting signal (signal sequence) that can range from 3-60 continuous amino acids. The signal sequence is often, but not always ___
A
removed once the protein arrives at its destination.
21
Q
an element that is necessary and sufficient for protein sorting
A
Signal sequences
22
Q
Signal sequences can be (4):
A
(1) Hydrophobic
(2) positively charged
(3) negatively charged
(4) polar
23
Q
(signal sequence): signal for import into ER is (2) __ (either: hydrophobic/
/positively charged
/negatively charged
/polar) ?
A
Hydrophobic
Negatively charged
24
Q
Signal sequence for import into mitochondria is (1) : (either: hydrophobic/
/positively charged
/negatively charged
/polar) ?
A
positively charged
25
Signal sequence for import into nucleus is (1) : (either: hydrophobic/
/positively charged
/negatively charged
/polar) ?
positively charged
26
Signal sequence for export from nucleus is (1) : (either: hydrophobic/
/positively charged
/negatively charged
/polar) ?
hydrophobic
27
3 steps in protein sorting
1. Recognition of the signal sequence by a shuttling cytosolic receptor
2. Targeting to the outer surface of the organelle membrane
3. Import of the targeted protein into the membrane or transport of the protein across the membrane
28
what is step 1 in protein sorting?
Recognition of the signal sequence by a shuttling cytosolic receptor
29
What is step 2 in protein sorting?
Targeting to the outer surface of the organelle membrane
30
What is step 3 in protein sorting?
Import of the targeted protein into the membrane or transport of the protein across the membrane
31
what is a general problem for protein import into organelles?
How to transport the protein across membranes that are normally impermeable to hydrophilic molecules
32
What are the three main mechanisms to import proteins into a membrane-enclosed organelle:
(1) Transport through nuclear pores
(2) Transport across membranes
(3) Transport by vesicles
33
Three main mechanisms to import proteins into a membrane-enclosed organelle: Method 1 Transport through nuclear pores --> transport __ and proteins __
(1) transports specific proteins
(2) proteins remain folded during transport
34
do proteins remain folded during transport through nuclear pores?
yes proteins remain folded during transport through nuclear pores
35
There are three main mechanisms to import proteins into a membrane-enclosed organelle: Method 2: transport across membranes: includes transport across the membranes of which organelles (4)?
(1) ER
(2) mitochondria
(3) chloroplasts
(4) peroxisomes
36
There are three main mechanisms to import proteins into a membrane-enclosed organelle: Method 2: transport across membranes: Are proteins folded or unfolded in order to cross the membrane?
Proteins are unfolded in order to cross the membrane
37
There are three main mechanisms to import proteins into a membrane-enclosed organelle: Method 2: transport across membranes: Requires
protein
translocators
38
There are three main mechanisms to import proteins into a membrane-enclosed organelle: Method 3: Transport by vesicles used from __ and through
From ER onward and through endomembrane system
39
Transport by vesicles: collect __ and __ from the membrane
Transport vesicles collect CARGO PROTEIN and PINCH OFF from membrane
40
Transport by vesicles: deliver cargo by:
fusing with another compartment
41
Proteins remain folded / unfolded during transport by vesicles?
Proteins remain folded during transport
42
Nuclear import: Entry into the nucleus proceeds through
a protein structure called the nuclear pore complex (NPC)
43
Nuclear import : nuclear pore complex (NPC) composed of
~30 proteins
44
Nuclear import: Entry into the nucleus proceeds through a protein structure called the nuclear pore complex (NPC). Composed of ~30 proteins, each with ( such that the NPC contains __ proteins when assembled
Composed of ~30 proteins, each with multiple copies such that the NPC contains 500-1000 proteins when assembled.
45
Nuclear import: nuclear pore complex (NPC) can transport __ molecules/sec and in which direction?
1000 molecules/sec, both directions simultaneously
46
nuclear import:NPC transports molecules up to what size?
40 kDa
47
_ can move through the nuclear pore complex (NPC) by passive diffusion.
Small, water-soluble molecules and proteins up to ~40 kDa
48
Nuclear Localization Signal (NLS):
Targets proteins to nucleus
49
Nuclear import components: cytosolic fibrils :
project outwards and helps channel cargo to the npc
50
Nuclear import components: scaffold nucleoporins
membrane-bending, stabilize membrane curvature
51
nuclear import channel components: channel nucleoporins:
line the central pore, many unstructured regions containing FG repeats, makes up the meshlike nature of the NPC
52
nuclear import channel components: nuclear basket:
Fibrils inside the nucleus converge at their distal ends to form a basket, function is not well understood
53
nuclear import channel components: membrane ring proteins
anchor the NPC to the nuclear envelope
54
Proteins synthesized in the cytoplasm are targeted for the nucleus by
a nuclear localization signal (NLS),having basic residues.
55
What is the NLS receptor?
importin a/B heterodimer
56
The Ran “gradient” ensures __ to nuclear transport.
directionality
57
nuclear import: proteins with an NLS bind to
an NLS receptor ; aka ; importin a/B heterodimer
58
nuclear import: what happens after proteins with an NLS bind to an NLS receptor ?
the protein/importin complex associates with cytoplasmic filaments
59
Nuclear import: What happens after the protein/importin complex associates with cytoplasmic filaments?
the protein/ importin complex passes through the NPC
60
What happens after the protein/importin complex passes through the NPC?
it associates with a GTPase called Ran
61
Nuclear import: How is importin B transported back to cytoplasm?
The Ran●GTP-importin b complex is transported back to the cytoplasm where Ran is converted to Ran●GDP and brought back in to the nucleus
62
Nuclear import: How is importin a returned to the cytoplasm?
importin a is returned to the cytoplasm via a protein called exportin
63
The Ran “gradient” ensures directionality to nuclear transport. The GTP-bound form only exists in
the nucleus
64
The Ran “gradient” ensures directionality to nuclear transport:GDP-bound form only exists in
the cytosol.
65
Describe the 5 steps involved in nuclear import:
1. Proteins with an NLS bind to an NLS receptor (importin a/b heterodimer).
2. Theprotein/importincomplexassociateswithcytoplasmicfilaments.
3. Theprotein/importincomplexpassesthroughtheNPC.....
4. .....andassociateswithaGTPasecalledRan.
5. The Ran●GTP-importin b complex is transported back to the cytoplasm where Ran is converted to Ran●GDP and brought back in to the nucleus. Importin a is returned to the cytoplasm via a protein called exportin.
66
Mitochondrial import:for import into the matrix, a (usually) __ is required.
N-terminal sorting sequence
67
For import into the matrix, a (usually) N-terminal sorting sequence is required. If the protein localizes to the intermembrane space:
a second sorting sequence is needed
68
Matrix-targeting sequences are rich in hydrophobic, positively-charged and hydroxylated (Ser, Thr) residues, but lack
acidic residues
69
Matrix-targeting sequences tend to form
amphipathic helix.
70
mitochondrial import: targeting sequence characteristics (4):
(1) Usually N-terminal
(2) Rich in hydrophobic, positively-charged and hydroxylated residues (Ser, Thr)
(3) Lack acidic residues
(4) Tends to form amphipathic helix
71
Import into the mitochondria only occurs at points where
the inner and outer membranes are in close contact.
72
mitochondrial import:in the cytosol, Precursor proteins are kept in an unfolded state by the action of
the cytosolic chaperone Hsc70.
73
Precursor proteins are kept in an unfolded state by the action of the cytosolic chaperone Hsc70. This requires
energy in the form of ATP hydrolysis.
74
mitochondrial import: receptor in the outer mitochondrial membrane is called:
TOM 20 or TOM 22
75
mitochondrial import: The matrix-targeting sequence interacts with a receptor in the outer mitochondrial membrane called TOM20 or TOM22, what does this receptor do?
The receptor transfers the protein to the general import pore of the outer membrane composed of the protein TOM40.
76
mitochondrial import:the general import pore of the outer membrane composed of the protein:
TOM 40
77
Mitochondiral import: At contact sites between the inner and outer membranes, the protein passes through
the import pore of the inner membrane
78
mitochondrial import: At contact sites between the inner and outer membranes, the protein passes through the import pore of the inner membrane composed of the proteins (2):
(1) TIM 23
(2) TIM17
79
mitochondrial import:Matrix Hsc70 binds to
TIM44.
80
mitochondrial import: Matrix Hsc70 binds to TIM44. __ by this complex helps power translocation of the protein into the matrix.
ATP hydrolysis
81
mitochondrial import: As the matrix-targeting sequence emerges in the matrix, it is
cleaved by a matrix protease.
82
mitochondrial import: what happens before the protein can fold into its final conformation in the mitochondrial matrix?
As the matrix-targeting sequence emerges in the matrix, it is cleaved by a matrix protease.
83
mitochondrial import:As the matrix-targeting sequence emerges in the matrix, it is cleaved by a matrix protease.The protein can then fold into its final conformation, often (but not always), it is :
assisted by matrix chaperonins.
84
what is required for protein transport into the mitochondria?
The H+ electrochemical gradient generated by oxidative phosphorylation
85
The H+ electrochemical gradient generated by oxidative phosphorylation is also required for protein import into mitochondria. This ensures that:
only mitochondria that are actively respiring can import proteins. Hence, uncouplers block import.
86
mitochondrial import: Targeting of proteins to the intermembrane space requires a
second, hydrophobic targeting sequence
87
mitochondrial impoty: Targeting of proteins to the intermembrane space requires a second, hydrophobic targeting sequence that
does not allow the protein to completely pass through the TIM23/17 import pore.
88
mitochondrial import: Targeting of proteins to the intermembrane space requires a second, hydrophobic targeting sequence that does not allow the protein to completely pass through the TIM23/17 import pore.
The stalled protein is then
released from the pore into the membrane where a membrane- anchored protease cuts the protein, releasing it into the intermembrane space.
89
Unlike the proteins that enter the nucleus, mitochondria, chloroplasts and peroxisomes, most of the proteins that enter the endoplasmic reticulum begin to be translocated (transported) across the ER membrane ___
before the protein is completely synthesized.
90
Endoplasmic Reticulum (ER) Import
Unique Characteristic:
Protein begins translocation before complete synthesis
91
Unlike the proteins that enter the nucleus, mitochondria, chloroplasts and peroxisomes, most of the proteins that enter the endoplasmic reticulum begin to be translocated (transported) across the ER membrane before the protein is completely synthesized.
This requires that
the ribosome that is synthesizing the protein be attached to the ER membrane, giving it a rough appearance (and the name rough ER).
92
There are two separate populations of ribosomes in the cytosol:
1. membrane-bound ribosomes (attached to the ER)
2. free ribosomes
93
membrane-bound ribosomes are
attached to the cytosolic surface of the ER membrane and are synthesizing proteins that are translocated into the ER.
94
free ribosomes are
unattached to any membrane and are synthesizing all of the other proteins
95
ER Import: Steps 1 + 2 : 3
The emerging polypeptide with its ER signal sequence exposed is engaged by:
A complex of six proteins and an associated RNA molecule called the signal recognition particle (SRP)
96
ER Import: Steps 1 + 2 :
The emerging polypeptide with its ER signal sequence exposed is engaged by a complex of six proteins and an associated RNA molecule called the signal recognition particle (SRP). This binding:
halts translation and delivers the ribosome/polypeptide to the ER.
97
ER import: SRP delivers the ribosome/polypeptide to the
SRP receptor
98
ER import: SRP delivers the ribosome/polypeptide to the SRP receptor. This interaction is enhanced by
the binding of GTP to both SRP and its receptor.
99
ER import: what happens after SRP delivers the ribosome/polypeptide to the SRP receptor?
The ribosome/polypeptide is then transferred to the translocon, inducing it to open and receive the polypeptide which enters as a loop
100
in ER import, what enables another round of import?
Hydrolysis of GTP by SRP and its receptor free these factors for another round of import.
101
ER import; what happens after 2 rounds of import;
Translation resumes and the signal sequence is cleaved by a membrane-bound protease called signal peptidase
102
ER import: what happens. after the digestion by signal peptidase?
the rest of the protein is synthesized and enters the lumen of the ER.
103
ER import: steps 7 + 8: Following completion of translation
the ribosome is released causing the translocon to close. The newly-synthesized protein then folds.
104
Membrane proteins of the plasma membrane, Golgi, lysosome and endosomes are all inserted into
the ER membrane.
105
Membrane proteins of the plasma membrane, Golgi, lysosome and endosomes are all inserted into the ER membrane. From there, they are
transported to their correct location using amino acid and carbohydrate sorting signals.
106
membrane-anchored proteins:Type I:
- single pass,
-cleaved signal sequence at the N-terminus,
-uses SRP-SRP receptor to get to ER membrane,
-Nout-Cin
107
There are _ main types of membrane-anchored proteins:
6
108
membrane-anchored proteins:Type II:
-single pass
-no cleavable signal sequence
-uses SRP-SRP
receptor to get to ER membrane
-Nin-Cout
109
membrane-anchored proteins: Type III:
same as type II but Nout-Cin
-single pass
-no cleavable signal sequence
-uses SRP-SRP
receptor to get to ER membrane
110
membrane-anchored proteins:Tail-anchored:
-single pass,
-no cleavable signal sequence,
-hydrophobic
membrane-spanning sequence at C-terminus,
-does not use SRP-SRP receptor but the GET1/2/3 system to get to ER, posttranslational insertion, -Nin-Cout
111
membrane-anchored proteins:Type IV:
-multispanning
-no cleavable signal sequence
-uses SRP-SRP receptor for insertion of the first membrane-spanning domain but not subsequent ones,
IV-A are Nin-Cin,
IV-B are Nout-Cin
112
Difference IVa vs IVb
IV-A are Nin-Cin, IV-B are Nout-Cin
(in=cytosol, out=lumen or extracellular space)
113
membrane-anchored proteins:. GPI-anchored
entire protein is lumenal (out), cleaved signal sequence at the N-terminus, uses SRP-SRP receptor to get to ER membrane, anchored at C-terminus to membrane and then transferred to GPI anchor
114
memorize slide 26
115
116
26-38 missing
117
The roles of glycosylation include:
1. Promote folding of proteins (e.g. protein secretion is blocked for certain proteins when tunicamycin is used or if Asn is mutated)
2. Provide stability to proteins (e.g. some non-glycosylated proteins (fibronectin) are transported from the ER but are degraded faster)
3. Promote cell-cell adhesion on plasma membrane proteins (e.g. leukocyte-endothelial cell attachment during inflammatory response)
4. Act as a transport signal (e.g. mannose-6-phosphate directs proteins to the lysosome).
118
molecular chaperones assist in protein folding by preventing
aggregation of hydrophobic stretches of amino acids
119
Ultimately, if mannose residues are removed, the protein is targeted for
dislocation (transport out of the ER) and degradation in the cytosol.
120
Two types of ER chaperones:
1. Classical chaperones 2. Carbohydrate-binding chaperones
121
Classical chaperones examples
Hsp70 (BiP), Hsp90, GRP94)
122
Carbohydrate-binding chaperones examples:
calnexin, calreticulin
123
Carbohydrate-binding chaperones bind to
polypeptides that are monoglucosylated. Terminal glucose is removed
and if folded the protein can exit the ER. If not, a glucosyltransferase adds one glucose back and the cycle repeats.
124
type 1 membrane anchored protein: signal sequence:
yes
125
type 1 membrane anchored protein: SRP/SRP receptor:
yes
126
Type 2 membrane anchored protein: signal sequence:
no
127
type 3 membrane anchored protein: signal sequence:
no
128
tail anchored membrane anchored protein: signal sequence:
no
129
type 4 membrane anchored protein: signal sequence:
no
130
GPI anchored membrane anchored protein: signal sequence:
yes
131
type 2 membrane anchored protein: SRP/SRP receptor:
yes
132
type 3 membrane anchored protein: SRP/SRP receptor:
yes
133
tail anchored membrane anchored protein: SRP/SRP receptor:
no
134
type 4 membrane anchored protein: SRP/SRP receptor:
yes
135
GPI anchored membrane anchored protein: SRP/SRP receptor:
yes
136
type 1 membrane proteins use a
cleavable signal sequence and a stop-transfer anchor (STA) sequence that acts as the membrane spanning domain. The translocon opens to release this hydrophobic stretch into the membrane.
137
Type II and III membrane proteins use a
signal-anchor (SA) sequence
138
Type II and III membrane proteins use a signal-anchor (SA) sequence that acts as a
a dual signal sequence (directing the protein to the ER by the SRP) and the anchor or membrane-spanning sequence.
139
Type II and III membrane proteins use a signal-anchor (SA) sequence that acts as a dual signal sequence (directing the protein to the ER by the SRP) and the anchor or membrane-spanning sequence.
The orientation is determined by
the positioning of the SA sequence within the translocon
140
Type II and III membrane proteins use a signal-anchor (SA) sequence that acts as a dual signal sequence (directing the protein to the ER by the SRP) and the anchor or membrane-spanning sequence.
The orientation is determined by the positioning of the SA sequence within the translocon.
This is in turn determined by the positioning of
positively-charged residues relative to the SA sequence:
141
Type II and III membrane proteins use a signal-anchor (SA) sequence that acts as a dual signal sequence (directing the protein to the ER by the SRP) and the anchor or membrane-spanning sequence.
The orientation is determined by the positioning of the SA sequence within the translocon.
This is in turn determined by the positioning of positively-charged residues relative to the SA sequence: if they are between the N- terminus and the SA, then it will be
Type II
142
Type II and III membrane proteins use a signal-anchor (SA) sequence that acts as a dual signal sequence (directing the protein to the ER by the SRP) and the anchor or membrane-spanning sequence.
The orientation is determined by the positioning of the SA sequence within the translocon.
This is in turn determined by the positioning of positively-charged residues relative to the SA sequence:if between SA and C- terminus, then it will be
Type III.
143
Type II and III membrane proteins use a signal-anchor (SA) sequence that acts as a dual signal sequence (directing the protein to the ER by the SRP) and the anchor or membrane-spanning sequence.
The orientation is determined by the positioning of the SA sequence within the translocon.
This is in turn determined by the positioning of positively-charged residues relative to the SA sequence: These positive residues remain
cytosolic.
144
Tail-anchored proteins are inserted into the ER __
after translation is completed
145
Tail-anchored proteins are inserted into the ER after translation is completed since
the hydrophobic stretch that is inserted into the bilayer needs to fully emerge from the ribosome.
146
slide 29 missing diagram slides 1-4
147
GPI (glycosylphosphatidylinositol)-anchored proteins insert into the ER like
Type I membrane protein
148
GPI (glycosylphosphatidylinositol)-anchored proteins insert into the ER like Type I membrane protein using a
stop-transfer anchor (STA) sequence
149
GPI (glycosylphosphatidylinositol)-anchored proteins insert into the ER like Type I membrane protein using a stop-transfer anchor (STA) sequence. An enzyme then (i) cleaves the protein within the lumen of the ER and (ii) transfers it to the assembled GPI anchor. -- what is this enzyme?
transamidase
150
GPI (glycosylphosphatidylinositol)-anchored proteins insert into the ER like Type I membrane protein using a stop-transfer anchor (STA) sequence. An enzyme (transamidase) then (2):
(i) cleaves the protein within the lumen of the ER and (ii) transfers it to the assembled GPI anchor.
151
GPI (glycosylphosphatidylinositol)-anchored proteins:The purpose of transferring one lipid anchor for another (2) :
1. The GPI anchor more readily diffuses in the membrane
2. GPI can act as a targeting signal (e.g. apical localization versus
basolateral).
152
Type IV membrane proteins use combinaitions of:
stop-transfer anchor (STA) and signal-anchor (SA) sequences.
153
Type IV membrane proteins use combinations of stop-transfer anchor (STA) and signal-anchor (SA) sequences.
If the first SA sequence is a Type II SA (i.e. N-term+++++SA), then the protein will be
Nin (like a Type II membrane protein).
154
Type IV membrane proteins use combinations of stop-transfer anchor (STA) and signal-anchor (SA) sequences.If the first SA sequence is a Type III SA, then the protein will be
Nout (like a Type III membrane protein).
155
Hydropathic plots can help:
determine the type of membrane protein.
156
Hydropathic plots can help determine the type of membrane protein.
* The more hydrophobic an amino acid is, the more ___ the hydropathic
index.
positive
157
Hydropathic plots can help determine the type of membrane protein,The more hydrophilic the amino acid, the more __ the hydropathic index
negative
158
Besides proteins that reside in the ER, this organelle is the starting point for (3):
1. Soluble proteins that will be secreted from the cell (e.g.
hormones)
2. Soluble proteins that are destined for the Golgi, lysosome or
endosomes (e.g. acid hydrolases)
3. Membrane proteins that will embed in the Golgi, lysosome,
endosomes or plasma membrane (e.g. Na+/K+-ATPase).
159
Besides proteins that reside in the ER, this organelle is the starting point for 3 other categories of proteins with other destinations, in order to export these proteins, the ER ensures that they are properly modified, folded and assembled by a process known as
quality control.
160
Four principle modifications that occur in the ER:
1. Disulfide bond formation
2. Glycosylation (the addition and processing of carbohydrates) 3. Folding of polypeptides chains and assembly of multisubunit
complexes
4. Proteolytic cleavage of amino-terminal signal sequences
161
what is glycosylation
the addition and processing of carbohydrates
162
ER modification: describe Disulfide bond formation
covalent bond formation between thiol groups of cysteine residues either on the same protein (intramolecular) or on two different proteins (intermolecular)
163
Disulfide bond formation is dependent upon
ER resident enzyme protein disulfide isomerase (PDI).
164
Disulfide bond formation is dependent upon the ER resident enzyme protein disulfide isomerase (PDI). Thus, only __ undergo this modification.
(i) secreted proteins or (ii) lumenal or extracellular domains of membrane proteins
165
Disulfide bonds __ protein structure
stabilize
166
Disulfide bonds stabilize protein structure – important for proteins that
will be subjected to either extremes in pH or environments with high levels of proteases.
167
2. Glycosylation – begins with
the addition of a common oligosaccharide addition to asparagine residues in the consensus sequence Asn-X-Ser/Thr.
168
2. Glycosylation – begins with the addition of a common oligosaccharide addition to asparagine residues in the consensus sequence Asn-X-Ser/Thr. Referred to as
N-linked glycosylation
169
2. Glycosylation – begins with the addition of a common oligosaccharide addition to asparagine residues in the consensus sequence Asn-X-Ser/Thr. Referred to as N-linked glycosylation since
the oligosaccharide is added to the amine group of asparagine.
170
Glycosylation:The precursor oligosaccharide is transferred to the protein as the consensus sequence emerges from the translocon. Requires an ER membrane – bound enzyme complex called
oligosaccharyl transferase.
171
Glycosylation; The precursor oligosaccharide is assembled in a step-wise fashion on a lipid molecule called __
dolichol
172
glycosylation: dolichol contains __ carbons
75-95 carbons
173
glycosylation: Assembly of the 2 N-acetylglucosamine (GlcNAc) residues and the first 5 mannose residues takes place on
the cytosolic surface of the ER.
174
glycosylation: Assembly of the 2 N-acetylglucosamine (GlcNAc) residues and the first 5 mannose residues takes place on the cytosolic surface of the ER. The dolichol containing the seven sugar residues then
flips (using a transporter) to display the oligosaccharide in the lumen of the ER. The remaining mannose residues and glucose residues are added one at a time until the complete precursor is made.
175
glycosylation: Attachment of sugars to dolichol is mediated by
nucleotides (UDP- GlcNAc, UDP-glucose, GDP-mannose).
176
glycosylation: Tunicamycin is
a molecule that blocks the attachment of the first GlcNAc residue to dolichol
177
glycosyaltion: Tunicamycin is a molecule that blocks the attachment of the first GlcNAc residue to dolichol. This results in
non-glycosylated proteins
178
Since glycosylation is used as a sign of protein folding, and folding is required for export from the ER,tunicamycin increases
he level of unfolded proteins in the ER inducing the unfolded protein response (UPR).
179
molecular chaperones assist in protein folding by
preventing aggregation of hydrophobic stretches of amino acids.
180
Two types of ER chaperones:
1. Classical chaperones
2. Carbohydrate-binding chaperones
181
ER classical chaperones include:
Hsp70 (BiP), Hsp90, GRP94
182
ER carbohydrate binding chaperones (2):
calnexin, calreticulin
183
ER chaperones: arbohydrate-binding chaperones (calnexin, calreticulin) – bind to
polypeptides that are monoglucosylated. Terminal glucose is removed
and if folded the protein can exit the ER. If not, a glucosyltransferase adds one glucose back and the cycle repeats.
184
if mannose residues are removed, the protein is targeted for
dislocation (transport out of the ER) and degradation in the cytosol.
