Module 5 Flashcards
(110 cards)
1
Q
what is the RER a site for?
A
I) co-translational transport
II) protein mods
III) formation of vesicles to transport proteins from ER to Golgi
2
Q
what is the SER a site for?
A
I) fatty acid and phospholipid synthesis
II) carbohydrate metabolism
III) where Ca2+ is sequestered to regulate Ca2+ concentrations in the cytosol
3
Q
what is glycosylation?
why is it important?
A
covalent addition of polysaccharides (add carbohydrate groups)
important for proteins that mediate cell interaction with the extracellular matrix
4
Q
what are the post translational modifications that happen in the ER?
A
I) glycosylation (N-linked)
2) protein folding (Lectins)
3) proteolytic cleavage
4) disulphide bond formation
5) mods to proteins targeted in the ER lumen (will only occur in the lumen of the protein)
5
Q
what is the most common form of glycosylation?
A
N-linked glycosylation, the addition of an amino group to the R-group of asparagine
6
Q
how do enzymes know what proteins to fold in the Er?
A
by recognizing amino acids with N-linked polysaccharides
7
Q
where is calnexin located?
A
throughout the cytosol
8
Q
what are lectins?
A
recognized modified proteins and assists in folding them, examples include calnexins and carlereticulin
9
Q
what is BiP?
A
a HSP70 chaperone of the ER
10
Q
what are BiP’s co-chaperones?
A
HSP40 and NEF
11
Q
what is a NEF?
A
nucleotide exchange factor and a co-chaperone to HSP70 BiP
12
Q
when does BiP bind to a protein and what does it do?
A
binds as soon as the protein appears on the luminal side of the membrane during co-translational transport, this occurs through the ER translocon
13
Q
true or false: BiP plays a role in the unfolding protein response in the ER
A
true
14
Q
in terms of disulphide bond formation, what are the reducing and oxidizing environments?
A
reducing: cytoplasm
oxidizing: ER
15
Q
disulphide bridges form between what amino acids?
A
two cysteines
16
Q
what are disulphide bridges?
A
covalent bonds between the sulphydryl groups of two cysteine residues that are essential in higher (3,4) structures of some proteins
17
Q
true or false: oxidative reactions can occur anywhere in the cell in eukaryotes?
A
false, oxidative reactions occur uniquely in the ER lumen
18
Q
why are disulphide bonds useful for inside the cell?
A
because the proteins may be exposed to harsh and denaturing conditions and the bonds help maintain the shape and fold of the protein
19
Q
what is PDI and what does it do?
A
this is a protein from the ER that can promote oxidation
it can also correct inappropriate disulphide bridge formation within or between proteins
20
Q
name resident ER proteins
A
I) PDI
II) BiP
21
Q
how does PDI accelerate the rate of reaction of disulphide bridges?
A
by creating an intermediate structure
22
Q
what is the difference between intramolecular and intermolecular bonds?
A
intra = within a molecule inter = between molecules
23
Q
true or false: RNase has 5 disulphide bridges
A
false, it has 4
24
Q
what does RNase do?
A
it helps with intestinal digestion processes by cleaving RNA
25
why does RNase have so many disulphide bridges?
because the acidic conditions of the intestine would cause the protein to unfold, but the bridges help keep the structure
26
true or false: the insulin protein is the target of 2 other peptidases
false, it is the target of 3 peptidases during transport in secretory vesicles
27
true or false: a cell can have too many unfolded proteins and this is not dangerous
true and false. Yes a protein can have too many unfolded protein but FALSE, it is dangerous for the cell
28
how can a cell have too many unfolded proteins?
1. too much protein production
2. delays in the processing steps
3. exposure to toxins
4. exposure to heat or other denaturing stresses
29
true or false: unfolded proteins are moved out of the ER
false, unfolded proteins cannot leave the ER ecause this causes an increased risk of protein aggregation and consequential cell death
30
how does the cell INITIALLY react when there are too many unfolded proteins?
through the UPR response (unfolded protein response), which works by trying to slow down protein translation OR removing unfolded proteins from the ER for degradation through ubiquitinylation
THEN increases the production of chaperones to help with protein folding
31
what proteins are essential in the UPR process?
BiP and Ire1
32
why is BiP important in the UPR process?
because its chaperone function prevents aggregation of proteins and promote their folding
will also lead to the formation of more protein folding regulators
33
true or false: Ire1 and BiP are associated proteins
true
34
what are the steps of the UPR response (Ire1 + BiP)
1. BiP is sequestered while Ire1 is inactive
2. an increase in unfolded proteins causes the dissociation of BiP from Ire1
3. Ire1 will now form homodimers in the ER membrane
4. these dimers can now act as active endonucleases
35
why does BiP dissociated from Ire1 when there are many unfolded proteins?
because BiP has a higher affinity for the hydrophobic patches on the unfolded proteins than it does for Ire1
36
True or false: Ire1 homodimers can act as actie kinases once dissociated from BiP
false, Ire1 homodimers act as active endonucleases
37
what can endonucleases do?
they are able to make internal cuts within nucleic acids (mRNAs)
38
Talk about Hac1
the mRNA for the Hac1 gene is the specific target of the Ire1 endonuclease
unsliced Hac1 mRNA contains an untranslated sequence that inhibits ribosome translation
39
true or false: Hac 1 is spliced by the Ire1 endonuclease?
true and this allows the synthesis of the Jac1 protein by removing the translation inhibition 'intron' sequence
40
Hac1 expression activates transcription of genes that code for _____ ?
I) PDI
II) BiP
III) Lectins
IV) Signal peptidases
41
true or false: misfolded proteins induced their own folding through a cascade of events?
true, this is what occurs in the UPR
42
what is the difference between anterograde and retrograde transport?
from ER --> Golgi --> cytosol = anterograde
| from cytosol --> Golgi --> ER = retrograde
43
what are the techniques for studying vesicular transport?
1. pulse-chase labelling
2. fluorescent microscopy of GFP-labelled proteins in mammals
3. genetic mutations that disrupt transport in yeast
44
what is the pulse and the chase in pulse chase labelling?
pulse: brief incubation in medium with radioactive a.a.
chase: the unlabelled medium
45
what is the basis of pulse-chase labelling?
you tag proteins for a brief amount of time so that only a subset of proteins are labelled and you are able to follow their journey
the pulse is a set time (of tagging)
and the chase observations can vary in time so that the cells can be seen at different stages of their journey
46
in GFP-tagged secreted proteins, what is VSV?
this is the virus that carries the gene that codes for protein enveloppe breakdown --> the G-protein
47
where is the G-protein synthesized?
in the host cell ER where it is glycosylated before being transported to the membrane
48
what is the permissive and restrictive temperature of the VSV-G protein?
restrictive: 40C --> misfolded protein, retained in ER
permissive: 32C --> protein folds we see fluorescent
49
true or false: when inserted in mammalian cells VSV-G virus is not disabled
false, it is disabled all we want to utilize is the fluorescence to track the progression of the cell thorughout its journey on the cell membrane
50
when does VSV-G stay in the ER?
at the restrictive temperature: 40C (no protein folding)
51
when does VSV-G make its way out of the ER?
at the permissive temperature: 32C (protein folding)
52
how does yeast metabolize sucrose?
through hydrolysis and converting it to glucose and fructose, this performed by the invertase protein secreted by the yeast cells
the glucose and fructose are then imported into the cell
53
true or false: if there are enough cells the community of cells in the yeast can help feed one another
true
54
what happens if invertase is not secreted properly in yeast cells?
an accumulation of vesicles at the restrictive temperature
55
what are sec mutants? how many classes are there?
different classes of mutations made to the yeast genome that prevent transport into the ER
there are 5 classes of sec mutants
56
what are Class A sec mutants?
invertase will accumulate in the cutosol
| and prevent transport into the ER
57
what are Class B sec mutants?
invertase will accumulate in the rough ER
| and they will bud off into vesicles coming from the rough ER
58
what are Class C sec mutants?
invertase will accumulate in the ER-to-golgi transport vesicles
they will prevent fusion of transport vesicles with the Golgi
59
What are Class D mutants?
invertase Will accumulate in the golgi
| will have defective transportation from the golgi to the secretory vesicles
60
what are class E mutants?
invertase will accumulate in secretory vesicles
| and have defective transport from the secretory vesicles to the cell surface
61
how did researchers determine the steps of the progression of protein transport?
by looking at double mutants of the sec mutant classes
| EX. a cell with Class A and Class B mutations will show ONLY a Class A phenotype
62
true or false: downstream mutations in either pathway mask the appearance of upstream phenotypes that are early in the pathway
FALSE, upstream mutations mask the phenotypes of downstream mutations that occur later in the pathway
EX. A double (BC mutant) will only show B phenotype as it masks the C mutant's appearance (B is required before C in the pathways)
63
what are cisternae and where are they located?
they are elongated flat sacs on the Golgi complex
64
what are endosomes?
these are vesicles that are formed at the cell membrane that capture and transport macromolecules outside of the cell
65
what do we call secretory vesicles held in the cell
secretory granules
66
true or false: GFP is used to recognize the Golgi complex
FALSE, what germ agglutinin is used to recognize the Golgi
67
what is wheat germ aglutinin?
this is a lectin that reognizes N-linked poly saccharaiades found in the Golgi cisternae
68
what is the cis-golgi network?
a collection of coalescing vesicles coming out from the ER to form the cis-cisternae
the medial-cisternae are in the center and the trans-cisternaw are the FURTHERST from the ER
69
true or false: the trans-cisternae are the closest to the ER
FALSE, they are in fact the furthest from the ER, the cis-cisternae are the closest to the ER
70
describe and compare model A and model B of anterograde transport.
anterograde transport is the movement from the ER to the Golgi.
Model A: the idea that vesicles move from cis- to medial- to trans-cisternae (Vesicular Transport Model)
Model B: the idea that the cisternae themselves rotate from cis- to medial- to trans- locations relative to the ER (Cisternal Maturation Model)
71
true or false: Model A is generally accepted concerning cisternal transport
false, model B is accepted
72
what are the implications of the Cisternal Maturation Model?
1. that new cis-golgi cisternae are formed by vesicles of the ER
2. trans-golgi cisternae dissipate into transport vesicles
3. Golgi-resident proteins must be escorted in the ANTEROGRADE direction
essentially, cis-golgi will become medial-golgi, and medial-golgi will become trans-golgi and the trans-golgi will be escorted by vesicles
73
true or false the bulk of protein cargo is being transported in the anterograde direction
true
74
in what direction fo the golgi cisternae cargo move?
anterograde
75
name the steps of vesicle formation
1. vesicles bud, by arising from the membranes donor compartment
2. cargo proteins are loaded into the buds by cargo signals
3. vesicle is formed and released
4. the vesicle docks and fuses to the membrane of the recipient compartment
76
what are coated proteins?
small GTP-binding proteins with GTPase activity
77
what are 3 types of coated vesicles? and why are they needed?
1. clathrin vesicles
- needed for transport from the trans-golgi network towards endosomes of the membrane + used in endocytosis
2. COP I
- used for retrograde transport from the Golgi to the ER
3. COP II
- required for anterograde transport from the RER to the cis-Golgi network
78
what do G-proteins do?
they bind to GTP and GDP
79
when is the G-protein inactive?
when it is bound to GDP
80
true or false, G-proteins can hydrolyze GTP to GDP?
true
81
what is GEF and what does it do?
this is a guanine exchange factor and it helps with the hydrolysis of GDP to GTP
82
true or false: COP II mediates retrograde transport and COP I mediates anterograde transport
FALSE,
COP II --> anterograde (ER to Golgi)
COP I --> retrograde (Golgi to ER)
83
describe step 1 of vesicle formation
step 1 : Vesicle Budding
required: GTPase + Sar1
Sec12 is a transmembrane protein foudn on the membrane of the donor compartment (ER), it is a GEF, that will render Sar1 active (Sar1-GTP)
Sar1-GTP binds with HIGH affnity to COP II proteins
this activation changes Sar1 conformation and reveals hydrophobic N-termius that will acnhor Sar1 in the ER membrane
84
what COP II coat proteins associated with Sar1-GTP
```
Sec 23
Sec 24
Sec 13
Sec 31
Sec 14 (binds indirectly)
```
85
tell me about the COP II complex
it has an inherent curvature that causes the membrane to curve, this is due to the dimerized sec 23 and sec 24 proteins binding to the anchored Sar1 protein
Sec 13 and sec 31 accumulate within the complex
86
what protein is functionally similar to Sar1 in the formation of COP I and clathrin-coated vesicles?
ARF protein (ARF-G protein)
87
describe step 2 of vesicle formation
step 2: loading cargo
1) cargo receotirs accumulate in the COP II bud and pick up soluble proteins
2) this is accomplished by the interaction of the cytosolic domains of the transmembrane cargo receptors with the coat proteins
3) the coat proteins act to collect vesicle-specific cargo
88
true or false: sometimes cargo is accidentally loaded, such as ER resident proteins
true, but never in high concentrations
89
describe step 3 of vesicle formation
step 3: vesicle completion and release
1) Sar1 is no longer anchored and the loaded vesicle is ready for transport
2) in CLATHRIN vesicles: the clathrin coat forma a polyhedral lattice using different clathrin proteins
90
what is the clathrin coat made of?
clathrin heavy light chains
clathrin light chains
adaptor proteins (AP)
- these make the triscallion that can interact with one another to make the clathrin coat
91
what is dynamin?
a G-protein required for the clathrin-coated vesicle release
| it can associated with the neck of the budding vesicle
92
tell me about dynamin structure
dynamin is able to assemble into spirals shaped like corkscrews
93
name and describe both of the dynamin models
1. Pinchase
- suggests that dynamin helices constrict and squeeze the membrane for vesicle release
2. Poppase
- suggest that dynamin helices elongate and pish the vesicle away from the donor compartment
94
what is shibbire?
a gene that codes for dynamin proteins in drosophila
it is temperature sensitive mutant
permissive T: 35
Restrictive T: 30
95
what is potential evidence for the poppase model?
when adding non-hydrolysable GTP we can see how the helical polymer was elongated
- suggest a combination of models
96
true or false: folding of the dynamin protein in drosophila is reversible?
true, the paralyzed phenotype of the flies at the restrictive temperature can be reversed, this allows us to manipulate the phenotype and learn more about the structure and function of dynamin
97
describe step 4 of vesicle formation
step 4: vesicle docking and fusion
1) Rab GTPase controls vesicle docking
Rab-GDP is free in the cytosol and Rab-GTP is bound to vesicles by a hydrophobic anchor
2) Rab-GTP interacts and binds to the Rab effector to facilitate docking
3) vesicle fusion is mediated by SNARE proteins
98
is docking sufficient to deposit the cargo?
no, the membranes MUST fuse to deposit the cargo
99
what are SNARE proteins?
membrane-anchored protein helices that mediate vesicle fusion
they are anchored onto the vesicle membrane
100
true or false:you can find V-SNARES on the target membrane
FALSE, v-SNARES are on the vesicle membrane, the target membrane will have t-SNARES
101
examples of V-SNARES
VAMP
102
examples of t-SNARES
Syntaxin
| SNAP25
103
explain membran fusion
interactions between v-SNARES and t-SNAREs cause membrane fusion
I) 4 helix bundle forms: 1 from VAMP, 1 from syntaxin, 2 from SNAP 25
II) this complex pulls the membranes together and allows fusion
104
what is required to dissociate the SNARE complexes?
Cytosolic SNARE proteins, NSF and alpha-SNAP
105
what proteins need to be returned to the ER?
I) memebrane V-SNAREs
II) COP II vesicle cargo receptor
III) unfolded proteins
IV) incorrectly sorted ER-resident proteins
106
what sequence is on ER-resident proteins?
KDEL sequence, this allows them to be identified when they are mislocated (Lys-Asp-Glu-Leu)
107
what is the KKXX sequence
this is on ER resident membrane proteins
| Lys-Lys-X-X
108
what is the cargo acceptor sequence from COP II vesicles?
Asp-X-Glu
109
what type of vesicles bring ER-resident proteins back to the ER?
this is retrograde transport therefore COP I vesicles accomplish this task
110
what is the endomembrane system?
this is the system of membranes suspended in the cytosol of a eukaryotic cell (nuclear membrane, RER, SER, Golgi...etc)
