Chapter 7 Flashcards
1
Q
Why do cells depend on signals on proteins?
A
to ensure they arrive at their proper subcellular destination
2
Q
Proteins with NO targeting signal will be translated entirely on and remain in?
A
free ribosomes and remain in cytosol
3
Q
Which organelles receive proteins from the cytosol after their translation is complete?
A
mitochondria, nucleus, chloroplasts, and peroxisomes
4
Q
What are translated by ribosomes attached to the ER and are translocated at the same time?
A
ER, Golgi, lysosomes, plasma membrane, and secreted proteins
5
Q
ER, Golgi, lysosomes, and plasma membranes, and secreted proteins arrive at the other locations via
A
vesicle budding and fusing
6
Q
Secretory pathway
A
transport of proteins through nucleus, ER, Golgi, and to the membrane
7
Q
Lumen of organelles of the secretory pathway are topologically equivalent to
A
each other and to the exterior of the cell
8
Q
Proteins targeting selectively recognizes
A
nascent proteins with signal sequences
9
Q
The signal sequence binds to
A
signal recognition particle
10
Q
Binding of signal sequence to the signal recognition particle causes
A
translation to temporarily halt
11
Q
Which 3 arrive at the ER
A
ribosome, nascent polypeptide, SRP
12
Q
What happens when the ribosome, nascent polypeptide, SRP interact with a receptor at the ER?
A
a channel (part of the translocon) opens and the nascent proteins begins to be co-translationally translocated
13
Q
When was the signal hypothesis proposed?
A
1970
14
Q
Why was the signal hypothesis proposed?
A
to explain how proteins got targeted to the RER
15
Q
When is the ER targeting signal cut off?
A
after the protein begins its translcoation
16
Q
Pre-protein
A
nascent protein prior to its signal sequence removal
17
Q
Normally ER bound protein synthesized in an in vitro cell-free system for gel
A
larger, migrates more slowly
18
Q
When translated in vitro in the presence of purified ER (microsomes) for gel
A
protein smaller, migrates faster, located in ER lumen (microsome lumen)
19
Q
What particle is SRP?
A
ribonucleoprotein
20
Q
SRP made up of
A
6 polypeptides and small (7S) RNA molecule
21
Q
What of SRP has what activity?
A
GTPase activity
22
Q
The part of SRP that binds to the nascent polypetide’s signal sequence has a
A
large number of Met residues (hydrophobic)
23
Q
function of part of SRP that binds to the ribosome
A
slows translation until docking at the ER
24
Q
Part of SRP bins to what? (2)
A
a. nascent polypeptide’s signal sequence
b. ribosome
25
SRP receptor structure
Dimer of two subunits: SRalpha and SRbeta
26
Where are SRalpha and SRbeta?
SRalpha - on cystol face
SRbeta - transmembrane
27
SRalpha and SRbeta are what proteins?
GTP-binding porteins (GTPases)
28
Coordinated GTP binding and hydrolysis by SRP and SR are required for
a. proper targeting of nascent chains to the ER
b. for their transfer to the translocation channel
c. for the recycling of SRP to the cytosol
29
What happens when SRP releases the ribosome?
Ribosome engages the translocon and the nascent polypeptide begins translocation
30
Transport of the polypeptide into the ER lumen through
an aqueous channel
31
Translocon comprised of
channel and other proteins closely associated with it
32
Ions cannot cross the membrane while
polypeptide is being translocated through a channel
33
After polypeptide is released and the ribosome is still attached, ions
permeate through the channel
34
Sec61 function
forms the channel through which the translocating protein passes
35
Sec61 structure
heterotrimeric complex, shaped like an hourglass in cross-section
36
What commits the chain to translocation?
Recognition and insertion of the signal sequence
37
What is displaced as translocation begins?
channel plug
38
During translocation, some proteins are able to
transiently slip out of the gap between ribosome and translocon, positioning a loop of the protein in the cytosol
39
Some organisms (especially unicellular eukaryotes) are able to translate and translocate proteins in where?
translation in cytosol, keep them unfolded, and translocate them into ER
40
Protein is no longer associated with ribosome when
it is translocated
41
Chaperonins of the hsp70 family associate with
nascent polypeptides
42
Chaperonin of the hsp70 family function
prevent holding
43
What is most important for recognition by the channel?
hydrophobicity of the signal sequence
44
What is the main energy source driving posttranslational translocation and co-translational translocation?
ATP hydrolysis by the ER-lumenal hsp70 BiP protein
45
What does the active pulling model propose?
ATP hydrolysis causes a conformational change in BiP which causes the polypeptide to be actively pulled (or pushed with SecAp for prokaryotes) through the channel
46
Steps of polypeptide translocation using BiP
1. BiP interacts with Sec63 and binds the polypeptide
2. polypeptide can diffuse inwards (ER lumen), but BiP prevents backward diffusion
3. when enough polypeptide is exposed, a second BiP binds
4. diffusion occurs again
5. process repeats, advancing the polypeptide incrementally
47
What side is the N-terminus on?
on the cytosol face or on the non cytosol face
48
What side is the C-terminus on?
on the opposite face from the N-terminus or may be on the same face
49
Steps of protein translocation (N-terminus)
1. protein translocation begins with signal sequence at the N-terminus
2. channel recognizes transmembrane domain
3. transmembrane domain enters the lipid bilayer through side of the channel
4. translation continues until termination
5. integrated polypeptide
50
Signal anchor proteins target by using an
internal transmembrane domain (signal anchor)
51
Simplest situation of transmembrane orientation
proteins spans the membrane 1x, the N-terminus faces ER lumen, and C-terminus faces the cytosol
52
Two possible orientation?
N-terminus region translocated into the ER-lumen or the C-terminus translocated into the ER-lumen
53
Polytopic proteins
span the membrane multiples times; may have transmembrane regions integrate one-at-a-time or they may integrate in pairs
54
If the ER-signal peptide is ________, it is almost always..?
N-terminal; cleaved after it has served its purpose
55
Signal peptidase complex structure
5 subunits: 2 of which have proteolytic activity
56
Exact cleavage site is?
variable
57
Exact cleavage site is influenced by the
amino acid residues in the immediate vicinity of the cleavage sites
58
Following its removal, signal peptide is often processed by
signal peptide peptidase
59
GPI anchoring
lipid GPI added to some translocated proteins
60
GPI anchoring to proteins renders them attached to the membrane as
IMPs
61
IMPs
integral membrane proteins, non-removable from the membrane by salt extraction procedure
62
GPI anchoring always tethers a protein to the
non-c-face of a membrane (ER-lumenal face first)
63
GPI
glycosylphophatidylinositol
64
Where does GPI begin at and move to?
begins on the c-face of the ER membrane, then a flippase (translocase) moves it across to the lumenal side
65
What happens to GPI at the lumenal side
more sugars are added along with 3 phosphoethanolamines
66
Signal for GPI-anchoring
small C-terminal hydrophobic domain of variable length
67
What recognizes the signal for GPI anchoring?
integral membrane complex
68
Integral membrane complex function
cuts the signal off from the protein and transiently attached to new C-terminus (omega site) and attaches omega site to the terminal phosphoethanolamine residue of GPI and the enxyme complex liberates itself
69
3 reasons why GPI-anchoring occurs
1. way of targeting proteins in polarized cells, lipid rafts...
2. give an IMP more lateral mobility compared to transmembrane IMPs
3. way to free a protein from membrane association by enzymatic cleavage of its attachment, thus passing along a message into the interior of a cell
70
More than half of secretory and membrane proteins in a cell are
glycosylated
71
Glycosylation in the ER is termed ____. Why?
N-linked because sugars are attached to asparagine residues
72
OST
oligosaccharyltransferase
73
Function of OST
transfers an oligosaccharide en bloc onto the translocating protein
74
Oligosaccharide presynthesized beginning in the
cytosol
75
Oligosaccharide presynthesized with the minor membrane ____.
phospholipid dolichol-phosphate
76
Sugars are added to ____.
dolichol-P
77
dolichol-P moved to where and by what?
flippase moves it to the lumen-face of the membrane
78
After oligosaccharide is added, what happens and where?
removal of some sugar residues and addition of others within the ER and Golgi
79
Most abundant protein in the ER lumen
BiP (binding protein)
80
BiP
Chaperonin
81
BiP most abundant in
ER lumen
82
BiP is a member of the
hsp70 family
83
BiP binds to
exposed hydrophobic patches (buried within a globular protein, interacting with each other to drive protein folding)
84
Why does BiP bind to exposed hydrophobic patches?
To help them get folded properly
85
ER-lumenal chaperonins no longer associate when?
once the protein has achieved its proper folding?
86
Cytosol has a __________ environment
reducing
87
In the reducing environment of a cytosol, what is not formed?
disulfide bonds
88
ER lumen and ECM have _________ environment
oxidizing
89
In the oxidizing environment of the ER lumen and ECM, what is formed?
disulfide bonds
90
The formation of the disulfide bonds in the oxidizing environment is catalyzed by
protein disulfide isomerases
91
Where in the cell has oxidizing environment and reducing environment?
Reducing environment - cytosol
Oxidizing environment - ER lumen and ECM
92
PDI
protein disulfide isomerase
93
PDI catalyzes
disulfide bond rearrangement and formation
94
PDI reduced when it
oxidizes the cysteines in a translocating protein
95
Reduction of PDI helps
folding
96
What oxidizes PDI by being itself reduced?
ER protein EroP1
97
PDI is oxidized by
an ER protein EroP1
98
EroP1 oxidized by
FAD
99
What is calnexin and where is it?
IMP in the ER memebrane
100
What is calreticulum and where is it?
protein in the ER lumen
101
Calnexin and calreticulin binds to
sugar residues
102
Binding of calnexin and calreticulin to the sugar residues indicate that they are
lectins
103
The purpose of calnexin or calreticulin activity is in
protein quality control (QC)
104
The calnexin or calreticulin activity is to
make certain that proteins are properly folded before they are allowed to leave the ER
105
What is the ticket to enter the calnexin cycle?
To have been N-glycosylated
106
After entering the calnexin cycle, the 2 distal glucose residues are removed by
glucosidase I and II
107
Association of calnexin helps the
protein contact ERp57
108
ERp57
a chaperonin and PDI family member
109
ERp57 function
catalyzes the disulfide bond formation and rearrangement and allows folding/refolding
110
What protein lets go of calnexin
unglycosylated protein
111
If the protein is not folded properly, a glucose is re-added by
UDP-glucose-glycoprotein glucosyl transferase (UGGT)
112
How does UGGT know that a protein is not folded properly?
it would be exposed clusters of hydrophobic residues
113
Proteins in the secretory pathway are retained in
ER by chaperone-association until the assembly of its subunits are completed
114
3 Retention mechanisms
a. associating a subunit with BiP
b. exposed cysteine binding to a PDI
c. exposed ER retention signal that will be masked later
115
BCRs or secreted Ig depend upon the
association of 4 polypeptides, 2 H chains (transmembrane) and 2 L chains (free in ER-lumen)
116
Retrograde translocation is used to
export misfolded proteins back into cytosol
117
What happens to the misfolded proteins in the cytosol?
multi-ubiquitinated and degraded by a proteasome
118
Ubiquitin
ubiquitous small protein used as a tag for protein degradation
119
Proteasome
cytoplasmic recycling barrel, a large protease complex
120
Degradative pathway (ubiquitinated protein degraded by proteasome) is known as
ER-associated degradation (ERAD)
121
ER-associated degradation (ERAD)
Misfolded proteins that cannot get refolded properly moves into the cytosol, and they are tagged with ubiquitin, marking them for degradation by proteasome
122
One of the retrograde translocation channels
Sec61
123
Unfolded protein response (UPR)
signaling pathway from the ER lumen to the nucleus
124
UPR function
a. allows the cell to monitor folding conditions
b. increase the expression of ER chaperonins when necessary
125
Protein that senses folding conditions in the ER and transmits the info
ER-membrane protein Ire1p
126
Ire1p has the ability to
dimerize by self-association of its lumenal domain
127
Under normal conditions, _______ bound to the lumenal domain of _______ resulting in?
BiP; Ire1p; inhibition of dimerization
128
Under stress, BiP is
too busy with misfolded proteins, so it leaves Ire1p to dimerize and send a signal
129
Cytosolic domain of Ire1p contains
a serine-threonine kinase
130
Serine-threonine kinase
it phosphorylates proteins on serine and threonine residues
131
When Ire1p dimerizes, it does what two things?
autophosphorylates and activates a second cytosolic domain
132
Dimerization of Ire1p catalyzes what?
the removal of an intron from the mRNA of a specific gene, HAC1
133
After Ire1p has removed HAC1, tRNA ligase does what and resulting in?
tRNA ligase joins the axon, allowing Hac1p (mRNA) to be translated
134
What happens if HAC1 (intron) is left in?
causes ribosomes to stall on the mRNA, causing no Hac1p to be translated
135
What is Hac1p?
transcription factor
136
What does Hac1p bind to?
a regulatory sequence called the unfolded protein response element (UPRE)
137
What does binding of Hac1p to UPRE stimulate?
transcription of several chaperonin genes
ex) BiP
138
Making the additional chaperonins help
QC with unfolded proteins during stressful conditions
139
Unfolded protein response (UPR) influences
transcription of a much broader collection of genes beyond those for chaperonins
140
What process is altered as a consequence of the cell's UPR?
increased lipid synthesis for ER expansion
141
Primary site of sythesis of the cell's phospholipids
ER
142
Where is the cell's phospholipid made in?
cytosolic leaflet of the ER membrane
143
Kennedy pathway
two fatty acyl CoAs are attached to glycerol 3-P to form diacylglycerol (DAG), which is hydrophobic enough to insert into the c-face of the ER membrane
144
From the membrane-associated DAG, what group can be added to form a complete phospholipid?
polar head groups
145
How is the polar group added? (STEPS)
1. head group phosphorylated
2. attached to CDP
3. Head group + a P is transferred to DAG
4. Releases CMP
146
What is the most abundant membrane phospholipid?
Phosphatidylcholine
147
What 4 membrane phospholipid is made by Kennedy pathway?
Phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PL), phosphatidylinositol (PI)
148
The 4 membrane phospholipid (PS,PE, PC, PL) is made by the Kennedy pathway in the ?
cytosol-face of the ER
149
Where can PE be made other than ER?
MItochondria by modifying PS
150
A specialized ER region that is responsible for transport of PS from ER
mitochondrial-associated membrane
151
What is mitochondrial-associated membrane (MAM)?
specialized ER that is very close to a mitochondria, making very close contact with its membrane
152
What synthesis also occur in the ER?
Sterol (most cholesterol)
153
Following their synthesis in the ER, some phospholipids must be transferred to where?
all the other membranes in the cell and NOT randomly
154
Transferring phospholipid to other membranes may occur in which regions?
where ER makes direct contact with other organelles of the cell
155
Phospholipid transfer proteins thout to move lipids from one bilayer to another but they cannot?
account for membrane growing because after they bring a lipid to membrane, they leave with a different one
156
Flippases neceessary to?
flip half of the newly-synthesized lipids from ER c-face to the non-c-face
157
Flipping of the lipid occurs randomly, but?
other phospholipid translocators must be more selective because memrbanes in cells are varied in their lipid composition from each other and within any membrane the two faces are varied
158
Where are PC, PS, PE, and PI enriched?
PC in non-c-face of the plasma membrane
PS and PE in the c-face
PI in c-face (EXCEPT GPI-anchored proteins)
159
ER form morphologically and in other regions
Morphologically, ER forms large, flat sheets (cisternae)
In other regions, it is in form of long, curving tubules
160
The flat sheets are generally found next to the
nuclear envelope
161
Tubules extend as a
network throughout the cell, contracting other organelles and the plasma membrane
162
RER is abundant in cells which secrete ?
proteins like Ig (plasma B cell), hormones,,
163
SER responsible for
lipid metabolism steroid synthesis, glycogen metabolism, and drug detoxification
164
SER is abundant in cells which secrete
steroid hormones (testes' leydig cells, ovary follicular cells) or liver hepatocytes
165
SER amount goes up with an?
increase in drug use
166
SR in skeletal muscle cells, cntain
calsequestrin
167
What is calsequestrin?
protein that has several Ca2+ binding sites
168
SR stores
intracellular Ca2+
169
SR in other cell types enables?
rapid Ca2+ regulation
170
What is calreticulin?
glycoprotein chaperone and a Ca2+ binding protein
171
When does BIP, calnexin, and calreticulin functio less efficiently?
when ER Ca2+ stores are depleted
172
Prolonged calcium depletion can induce?
UPR
173
Ca2+ release from the ER is implicated in?
triggering apoptosis
174
Tubular elements of the ER are in
continual flux, aligned to the cytoskeleton
175
Cytoskeleton is not required for the
formation of tubules or networks in vitro
176
Role of cytoskeleton ensures that
after the ER network forms, it is properly distributed throughout the cell
177
In response to increase in what drug expands SER?
phenobarbital
178
Removal of phenobarbital causes
rapid return of the SER to its normal size
179
The plasma B cells proliferate their RER in order to
meet the need for the cell to secrete large amounts of Ig proteins
180
Nucleus uses
internal signal patches
181
Internal signal patches are comprised of
non-contiguous aa residues that are brought together by tertiary and quaternary structure
182
What is not removed after import into the nucleus through the nuclear pore complexes?
nuclear localization signals (NLSs)
183
Why are NLSs not removed after import into the nucleus through the NPCs?
because many of the proteins might need their signal if they are outside after telophase and it is difficult to remove internal signals
184
Nucleus is able to import proteins in what form dues to what?
folded form due to size of the NPCs
185
Mitochondria and chloroplasts must import proteins in what form?
unfolded form
186
Peroxisome import proteins in what form?
folded form
187
Organelle targeting signals are
N-terminal or C-terminal
188
When are organelle targeting signals removed?
post-translocation
189
What spans the outer mitochondrial memebrane?
Translocase of the outer membrane (TOM)
190
Each mitochondrial membrane is spanned by a
different multi-protein complex that includes translocation channel to import proteins
191
What spans the inner mitochondrial membrane?
Translocase of the inner membrane (TIM)
192
What is the mitochondria targeting signal?
An N-terminal amphipathic alpha-helical stretch of 20-55 aa residues with hydrophobic sequences on 'side' of the helix and basic aa residues on the other
193
Why is chaperone cytosolic hsp70 family necessary?
To prevent folding of the protein prior to translocation
194
Portions of what are in close proximity?
TIM and TOM
195
Why are TIM and TOM in close proximity?
to ensure that most proteins are passed between complexes without dissociating into the intermembrane space
196
What binds protein as it emerges from the channel
mtHsp70
197
mtHsp70 act as what 2?
ratchet or motor or both
198
mtHSp70 binding to protein to act as a ratchet or motor requires
ATP energy
199
Membrane potential across the inner membrane (ETS) is important in
translocation
200
Most mitochondrial signal sequences are cleaved by
soluble mitochondrial processing protease (MPP)
201
One way that proteins arrive at final location of transmembrane IMPs in the mitochondrial inner membrane is via
Oxa1p pathway
202
What happens in the Ox1p pathway?
Proteins from cytosol get into the mitochondrial matrix, have their mitochondrial signal removed, exposing a second signal with directs them to the membrane for insertion
203
Proteins translated within the mitochondrial matrix could arrive at that destination with a
single targeting sequence
204
Proteins destined for chloroplast are kept unfolded in the cytoplasm by
cytoplasmic hsp70s
205
Channel that preproteins destined to chloroplast first meet?
Translocon of the outer envelope of chloroplasts (TOC)
206
Channel that proteins destined to chloroplasts go through and is attached to the TOC
translocon of the inner envelope of chloroplasts (TIC)
207
What requires energy for the proteins to go to the chloroplasts?
chloroplast stroma hsp70-type of chaperone
208
What regions of the chloroplasts must receive their own subset of specific proteins?
Thylakoid space and thylakoid membranes
209
What translocation system to thylakoid proteins use?
Twin-arginine-translocation system (tat)
210
TAT system uses what signal sequence?
second signal sequence since used after the chloroplast-targeting signal is cleaved
211
Translocation across the thylakoid membrane requires what 2?
energy and pH gradient produced during photosynthesis
212
What signal sequences can be used to import proteins into peroxisomes?
C-temrinal (PTS1) or N-terminal (PTS2)
213
Proteins are imported into the peroxisomal matrix by
posttranslation and post folding
214
Receptor that is carried into the peroxisome and then is exported alone to be used again
Peroxisomal targeting signal receptor
215
Why do peroxisomal translocation channel has to be tightly regulated?
the internal environment of peroxisomes would be toxic to the cell if the peroxisomal membrane were as permeable to protein and small molecule movement as is the nuclear envelope
216
What are the two main transport routes?
exocytic pathway (secretory pathway) and endocytic pathway
217
New proteins destined for any locations within the endocytic/exocytic pathway must be first targeted to where?
ER
218
What are the two ways to exit the ER?
a. to fail to fold properly, retrograde transport out to be ubiquitinated and proteasomed
b. to exit via budding into a transport vesicle
219
What compartments form during endocytosis?
Endosomes
220
Transport has to be ___________ lest the donor compartments cease to exist
bi-directional
221
What mechanisms return some vesicle components to the donor compartments?
Recycling mechanisms
222
What mechanism returns resident proteins which escaped from their donor compartments?
Salvage mechanisms
223
When is pulse-chase experiment used?
To experimentally show the pathway that proteins take as they move through the secretory pathway
224
What is regulated selection?
when cells accumulate proteins to be secreted in vesicles near the plasma membrane releasing them upon stimulation
225
Regulated secretion is aka
inducible secretion
226
What are 3 examples of regulated secretion?
a. digestive enzymes (pepsinogen, trypsinogen)
b. hormones (insulin, ADH)
c. histamine
227
What is constitutive secretion?
when cells continuously secrete a protein
228
What 4 examples of constitutive secretion?
a. immunoglobulins
b. yolk protein
c. bacterial infection-promoting proteins
d. insulin
229
What compartment is the most abundant membrane in most eukaryotic cells?
ER
230
What is Golgi apparatus made of?
Golgi stack made of cis, medial, and trans cisternae
231
What part of Golgi is near the entry face and what part is nearest the exit face?
cis-Golgi near the entry face
trans-Golgi near the exit face
232
Within the Golgi stack, what are modified sequentially to highly sialylated structures?
high mannose oligosaccharides (added to proteins in the ER)
233
What is the function of cis-Golgi network (CGN)?
a. receive proteins from the ER export sites
b. in QC by allowing ER resident proteins that escaped to be returned
234
Trans-Golgi network sorts for distribution to what 3 places?
a. lysosomes
b. plasma membrane/constitutively secreted proteins
c. regulated secretion
235
What are the 4 endocytosis functions?
a. internalizing of nutrients
b. regulating the cell surface expression of receptors, transporters
c. uptake, recycling of EC debris
d. recovery of membrane from the plasma membrane
236
What is the range from least to most degradative?
early endosome, late endosome, lysosome
237
What are V-ATPases?
H+ ATPases that transport protons from cytosol into the organelle lumen
238
Why is the acidification of early endosomes important?
for the dissociation of internalized ligand receptor complexes and recycling of cell membrane receptors to the plasma membrane
239
Why is the acidification of late endosomes important?
for the delivery of lysosomal enzymes from the trans-Golgi network (TGN)
240
What 2 cells are professional phagocytes?
macrophages and dendritic cells
241
What do phagocytes do?
ingest pathogens and clear senescent or apoptotic cells via phagocytosis
242
RME
Receptor-mediated endocytosis
243
Where does transcytosis occur?
in epithelial cells lining the intestine and other body cavities
244
What does transcytosis allow? (2)
a. nutrient uptake via the formation of a clathrin-coated vesicle
b. allow infants to take Ig from mother's milk by binding the Ig to gut apical receptors, transferring Ig through to the other side, and releasing the Ig into the blood plasma
