F: Week 6 + Module 5 Flashcards
(112 cards)
1
Q
Is the RER dynamic or stationary?
A
dynamic (shape, fission/fusion, migration)
2
Q
what is the function of the RER?
A
co-translational transport, protein modification, formation of vesicles that will transport proteins from ER to Golgi
3
Q
what is the function of the SER?
A
fatty acid and phospholipid synthesis, carbohydrate metabolism, regulate Ca2+ conc in the cytosol
4
Q
what are the post translational modifications in the ER?
A
- glycosylation
- protein folding
- disulphide bond formation
- proteolytic cleavage
5
Q
Where do modifications occur in ER proteins
A
Proteins targeted to ER lumen
Can have modifications along the entire length of the protein
Proteins targeted to the ER membrane
Modifications will only occur in the luminal portion
6
Q
this protein modification is important for…
proteins that mediate cell interactions with the extracellular matrix and for receptor-ligand recognition
A
glycosylation
7
Q
this protein modification is important for…
proteins that are secreted from the cell and proteins embedded in the cell membrane
A
glycosylation
8
Q
Glycosylation
A
Addition of a polysaccharide or sugar group
9
Q
what is the most common form of glycosylation?
A
N-linked
10
Q
N-linked glycosylation adds a polysaccharide to which group of which amino acid?
A
NH2 group of the R-group of asparagine
11
Q
Where is the glycosylated portion found during transport and once embedded
A
Modified portion on luminal side during transport
Appears on exterior surface of a protein embedded in the ER membrane
Further explanation
Luminal side of ER → Inside of vesicle → Extracellular side of the plasma membrane.
Cytosolic side of ER → Outside of vesicle → Cytosolic side of the plasma membrane.
Doesn’t flip, reintegrated backwards after vesicle transport
12
Q
Disulphide bonds
A
Covalent bonds between sulfhydryl (-SH) groups of two cysteines
13
Q
does disulphide bond formation occur in reducing or oxidizing environment?
A
oxidizing
14
Q
is cytoplasm a reducing or an oxidizing environment?
A
reducing
15
Q
is ER (lumen) a reducing or an oxidizing environment?
A
oxidizing
disulphide bond formation only occurs here
16
Q
this protein…
is one of many proteins secreted into the intestine where it aids in the digestion of RNA by cleaving it into small pieces
A
Pancreatic RNAse A
4 disulphide bridges allow it to keep its structure in the acidic environment and do cleavage
17
Q
what is the protein that…
resides in the ER that promotes oxidation?
A
Protein disulphide isomerase = PDI
oxidation –> disulphide bridge formation
18
Q
these proteins recognize modified proteins and assist in protein folding in a similar way as chaperones
A
lectins
19
Q
what are the two types of lectins?
A
calnexin and calreticulin
20
Q
where is calnexin found?
A
ER membrane
21
Q
what is BiP? (3 functions)
A
ER-resident HSP70 chaperone
- transfers proteins from ER through the translocon by binding to proteins as soon as they appear on the luminal side of the membrane during co-translational transport
- initiate unfolded protein response in the ER
22
Q
what are the co-chaperones of BiP?
A
Hsp40 and NEF (nucleotide exchange factor)
23
Q
proteolytic cleavage
A
Cleavage of the peptide backbone of a protein
24
Q
does proteolytic cleavage in the ER happen in the lumen or the cytosol?
A
lumen
25
what is the N-terminal signal sequence of type I integral proteins cleaved by?
= proteolytic cleavage example
signal peptidase
26
what are the two responses of the unfolded protein response (UPR)?
1. restore normal cell function by slowing down new protein translation or removing unfolded proteins from the ER for degradation through ubiquitylation
2. increase production of chaperones BiP and Ire1)
27
what are the protein chaperones essential to UPR?
BiP and Ire1
28
this protein...
is involved in the UPR and functions as a chaperone to assist in proper folding and prevent aggregation of misfolded proteins
BiP
29
this protein...
is a transmembrane protein that is involved in the UPR
forms homodimers that work as activated endonucleases
Ire1
30
Endonucleases
make internal cuts in nucleic acids like mRNAs
31
Ire1 endonuclease specifically targets which gene's mRNA?
Hac1
32
what does unspliced Hac1 do?
inhibit translation
Association with inactive Ire1 inhibits BiP
When Hac1 spliced, it can be synthesized and translation can occur
33
this protein...
is a transcription factor that activates transcription of several genes including the genes that code for BiP, lectins, PDI, and signal peptidases
Hac1
34
where is the Hac1 protein transported to?
nucleus
35
moving from the ER towards the cell membrane
anterograde
towards the anterior of the cell
36
moving from the cell membrane towards the ER
retrograde
37
what is pulse-chase labeling?
why might it be done?
tags proteins with radioactive aminos for only a brief period of time, so that only some proteins are labeled
used to see vesicle transport from ER out of cell
0 mins chase @ ER
17 mins chase @ golgi
117 mins chase @ secretory vesicles
38
what are the 3 types of techniques used for studying vesicular transport?
1. pulse-chase
2. fluorescent microscopy of GFP-labeled proteins
3. genetic mutations that disrupt transport
39
Tracking GFP-tagged secreted proteins
- can track a viral protein encoded in the viral genome, but synthesized and embedded in the host cell membrane
- protein becomes part of the viral envelope that surrounds the virus
ex. use the GFP tagged VSV - vestibular stomatitis virus glycoprotein that gets secreted when in the host cells sec. sys.
= allows visualisation
40
GFP tagged VSV temperature sensitivity
32 deg permissive temp = proper folding and transport
40 deg restrictive temp
41
what is the protein identified for protein secretion in yeast?
Saccharomyces cerevisiae
invertase (does metabolism)
42
yeast metabolism benefit
product can feed other cells
43
yeast mutation experiment
- Researchers generated random mutations to figure out steps in protein transport
- They looked for temperature-sensitive mutations that, when shifted to the restrictive temperature, failed to secrete invertase out of the yeast cells
44
where is invertase seen in class A mutation and what is the defective function?
cytosol; transport into the ER
45
where is invertase seen in class B mutation and what is the defective function?
rough ER; budding of vesicles from the rough ER
46
where is invertase seen in class C mutation and what is the defective function?
ER-to-Golgi transport vesicles; fusion of transport vesicles with golgi
47
where is invertase seen in class D mutation and what is the defective function?
Golgi; transport from golgi to secretory vesicles
48
where is invertase seen in class E mutation and what is the defective function?
Secretory vesicles; transport from secretory vesicles to cell surface
49
effect of upstream mutations
Upstream mutations mask the appearance of downstream mutations
- If there’s a defect in multiple, only the earlier one will be detected
- It will show only the phenotype of the earlier mutation because it won’t make it to the second location
50
Constitutive vs Regulated Secretory Pathway differences and direction
Con
- Used by proteins that are released immediately after protein synthesis and transport
Reg
- Used by proteins that are kept in the cell (in secretory granules) until a signal triggers release
rER → Golgi [cis-cisternae → trans-cisternae] → cell membrane
51
this organelle is comprised of a series of elongated, flat sacs called cisternae
Golgi complex
52
ER to golgi: cis-cisternae or trans-cisternae?
Golgi to membrane: cis-cisternae or trans-cisternae?
Vesicles transport to cis-cisternae
Vesicles transport from trans-cisternae
53
Function of the trans-golgi network
Sorts proteins into vesicles targeted for different destinations
54
Trafficking of soluble lysosomal enzymes from the TGN and cell surface to lysosomes
- M6P receptors are embedded in the plasma membrane and sticking out
- they recognize M6P (phosphorylated at M6 sugar) residues on lysosomal enzymes from outside the cell
- clathrin-coated vesicles transport them to lysosomes
55
Human I cell disease
- due to a lack of N-acetylglucosamine phosphotransferase
= lysosomal enzymes lack M6P targeting signal
= constitutively secreted
56
this is a collection of coalescing vesicles emanating from the ER to form the cis-cisternae
cis-golgi network (CGN)
57
Visualizing the Golgi
Rather than an antibody…
→ Use a fluorescently-labeled wheat germ agglutinin to recognize Golgi complex
This is a lectin that recognizes N-linked polysaccharides found in Golgi cisternae
58
Two models of movement through the golgi
Model A: Vesicular Transport Model
Vesicles carrying proteins cargo move from cis- to medial-cisternae and from medial- to trans-cisternae
- anterograde
Model B: Cisternal Maturation/Progression Model
Proteins stay in cisternae, but the cisternae themselves are moving forward through the golgi. This still requires vesicles that move backwards
- retrograde
59
Immuno-TEM testing golgi transport model
Antibody to cell membrane protein
- Cell membrane protein only found in cisternal sacs, not in vesicles
- Moves in anterograde transport
Immuno-TEM images:
- Cell membrane protein moved = MODEL B is correct
Antibody to Resident Medial Golgi protein:
- Protein only found in medial-Golgi
- If cisternae are moving together through the Golgi, then the medial cisterna should move into the trans-cisterna position
- Proteins are mis-localized/resorted in retrograde direction in vesicles
- Therefore, medial-cisternae go in both directions
= MODEL B is correct
60
Steps of vesicle transport
1. Vesicles are formed by budding: buds arise from the membrane of a donor compartment
2. Cargo proteins are loaded into bids via cargo signal sequences and receptors
3. Vesicle formation and release
4. Vesicle docking and fusion to membranes of the recipient compartment
61
what are the three types of coated vesicles?
clathrin, COPI, COPII
62
what kind of protein are the coated vesicle proteins?
small GTP-binding proteins with GTPase activity
63
what protein is needed to convert GTP (active) to GDP (inactive)?
GAP = GTPase accelerating protein
64
what protein is needed to convert GDP (inactive) to GTP (active)?
GEF = Guanine exchange factor
65
coated vesicles needed for...
transport from trans golgi to endosomes and to cell membrane
clathrin
66
coated vesicles needed for...
retrograde transport (golgi → ER)
COPI
(lower number is going down)
67
coated vesicles needed for...
anterograde transport (rER → Cis golgi)
COPII
68
Sar1
a cytosolic GTPase protein that is active when bound to GTP and inactive when bound to GDP
69
Sec 12
transmembrane protein found on the membrane of donor compartments (ER) and is a GEF that facilitates the exchange of GDP for GTP on Sar1, making it active
70
what are the four COPII proteins?
sec 23, 24, 13, 31
71
which COPII proteins bind to Sar1?
directly = sec 23
indirectly = sec 24
72
which COPII proteins accumulate after sec 23 and sec 24?
sec 13, 31
73
curving of the membrane
The COP II complex has an inherent curvature, causing the membrane to curve, causing the budding of new vesicles
74
ARF-G
functionally similar protein to Sar1, used in the formation of COPI and clathrin-coated vesicles
75
Cargo receptors
accumulate in the curved bud and pick up soluble proteins
76
Accumulation of cargo
accomplished by the interaction of cytosolic domains of the receptors of transmembrane cargo with coat proteins - some cargo can be accidentally loaded, but will not accumulate to a high concentration
77
Uncoating
Once loaded, GTP-hydrolysis by Sar1 causes it to no longer be membrane anchored, releasing Sar1 and the coat proteins, the uncoated vesicle is then recognized by a motor protein and carried along microtubules from the donor to the recipient membrane.
78
Studying the steps in vesicle transport
disrupting the process and accumulating transition states
79
Preventing uncoating
adding a non-hydrolyzable form of GTP to maintain Sar1-GTP, or mutations in the Sar1 G-protein that inhibits GTPase activity
80
what are the 3 clathrin proteins? what do they do together?
clathrin heavy chains, light chains, adaptor proteins
form a polyhedral lattice
Tri-Scallion:
Composed of 3 light and 3 heavy chains
Interact with each other on the surface of the budding membrane to form the clathrin coat
81
what is the protein required for clathrin vesicle release?
dynamin, a G-protein that accumulates around the neck of the budding membrane when active GTP-bound
when inactive releases vesicle due to structural change
82
Creating intermediates in vesicle release
stopping GTP hydrolysis, causing dynamin to accumulate around the neck of the budding membrane, creating a long neck that winds around in rings - dynamin forms polymers that assemble into spirals and wrap in a helical pattern
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83
Poppase model
dynamin helices elongate and push the vesicle away from the donor membrane to induce vesicle release
84
Dynamin-mediated pinching off
- dynamin hydrolyzes GTP
= conformational change
= clathrin membrane fusion
= vesicle release
85
Pinchase model
dynamin helices constrict and squeeze the membrane to initiate release
86
Experiment 1 to test dynamin mechanism
lipid tubes were used as models for vesicle necks, addition of GTP allows a conformational shift that narrows the internal diameter of the dynamin spiral, and there is a constriction of the lipid tubules, consistent with PINCHASE
87
Experiment 2 to test dynamin mechanism
EM images of lipid tubes in different states were examined:
Undecorated tubes carry no dynamin
dynamin-GTP stage using non-hydrolyzable GTP (GTP gamma-S)
dynamin-GDP after hydrolyzation
After hydrolysis, the polymer has elongated indicated by increased space between ridges of dynamin rings, consistent with POPPASE
88
Dynamin in the model system of flies
Neurotransmitters are loaded from the cytosol into vesicles, and vesicle fusion with the cell membrane will release them through exocytosis. In flies, the shibire gene codes for dynamin. A temperature sensitive mutation in shibire disrupts the dynamin proteins at the restrictive temperature, and no vesicle release occurs, causing flies to be paralyzed. Folding of dynamin is reversible, so going back down to the permissive temperature ends paralysis
89
Release of cargo requires
vesicle docking and release
90
what is the protein required for vesicle docking?
Rab GTPase
91
what is the membrane-anchored protein required for vesicle fusion?
SNARE complex
92
what is the SNARE protein on the vesicle membrane?
v-SNARE; VAMP
93
what is the SNARE protein on the target membrane?
t-SNARE; syntaxin, SNAP-25
94
VAMP, SNAP25 and syntaxin
4 helices, 2 from SNAP25, 1 from Syntaxin and 1 from VAMP spiral together to form a 4-heix bundle, which pulls the two membranes close together to allow fusion
95
which proteins associate with the end of the SNARE complex and unwind the 4 helices?
NSF and alpha-SNAP
96
what occurs when snares spiral together
transmembrane domains of SNARES are pulled apart as the cytosolic domains spiral together, then a hole forms in the two simultaneously, and the vesicle and target membranes become continuous
97
Disassembling SNARES
NSF and alpha-SNAP protein associate with the end of the SNARE complex and unwind the helicies so the SNAREs can diffuse in the membrane for recycling
98
what proteins need to be returned to the ER?
v-SNARES (recycle)
COPII vesicle cargo receptor
unfolded proteins
incorrectly sorted ER-resident proteins
99
returning signal
signal is specific to ER resident proteins that are required for loading into COP I vesicles
100
ER [membrane] resident proteins return signal
LysLysXX (KKXX)
- lysine rich
101
ER resident proteins return signal
Lys-Asp-Glue-Leu (KDEL): on soluble ER resident proteins
102
COP II cargo receptor return signal
Asp-X-Glu
103
KDEL sequence
recognized by the KDEL receptor in the golgi complex, and is loaded into the COP I vesicles where it can accumulate proteins to bring back to the ER (retrograde)
k like keep in the dl
104
Secretory and endocytic pathways
1. protein synth in rough ER
2. transport vesicle packaging
3. retrograde retrieval
4. cis-golgi cisterna move up
5. retrograde transport vesicles
6. constitutive secretion
7. regulated secretion
8. sorting to lysosomes
9. endocytosis
105
The transferrin cycle
- in all growing cells
Transferrin contains iron = Ferrotransferrin/Transferrin-Fe(III)
- binds to transferrin receptors on Reticulocytes = high levels of transferrin receptor-1
- brought into cell
- in endosome, iron released while carrier protein+receptor stay together
- iron exported to the cytosol to make heme
- heme synthesized and incorporated into globin chains
= hemoglobin
106
Endocytic pathway for internalizing LDL
- endocytic pathway delivers ligands (like LDL) to lysosomes for degradation
- LDL comes from outside of the cell
- LATE endosome acidic environment dissociates most receptor-ligand complexes for receptor recycling to the plasma membrane and ligand degradation in lysosomes
107
Precursor-product relations for internalizing LDL - graph + experiment
- LDL labelled with radioactive iodine
- cultured human skin fibroblasts with iodine-LDL
@ 4 degrees
- LDL binds to surface LDL receptors = not endocytosed
- unbound LDL washed away
@37 degrees
- receptor mediated endocytosis / chase activated
Results
- I-LDL binding (on surface) decreases fast bc endocytosed
- I-LDL internalized (in vesicles) increases then decreases
- After 15 mins I-LDL degradation (in lysosomes) increases
108
Good vs. bad (?) cholesterol
Low Density Lipoprotein = bad?
- one type of apolipoprotein (ApoB)
High Density Lipoprotein = good?
- one type of apolipoprotein (ApoA1)
109
Cholesterol cycle
1. VLDL (LDL precursor) comes from liver
2. Turns into LDL
3. Cells have LDL receptors and use it
4. Excess LDL is taken up by the peripheral tissues
5. Excess cholesterol gets sent to HDL
6. Goes back to liver and disposed of
110
What makes LDL "bad"
- accumulation of LDL is a problem as they have nowhere to go
- get modified = oxidized
- can now ignore receptors on cells
- go through the "back door"
- fill the cells with fat
- forms pimple -> plaque -> thrombotic event
- uncontrolled uptake of ox-LDL can cause cardiovascular disease
111
Formation of atherosclerotic lesions
- LDL moves into the sub-endothelium and is oxidized by macrophages and SMCs
- release of growth factors and cytokines attracts additional monocytes
- foam cell accumulation and SMC proliferation result in growth of the plaque
112
Lowering cholesterol
- diet
- meds
... continue this ig
