Midterm No. 3, Opus 1 Flashcards
(96 cards)
1
Q
How are cellular compartments linked?
A
By vesicular transport
2
Q
Can protein transfer between compartments (such as between the cis-golgi and medial-golgi compartments) occur in a cell free system?
A
Yes
3
Q
When doing experiemtns with vesicles using non-hydrolyzable GTP, an accumulation of distinct classes of coated vesicles was observed. That does this suggest?
A
Suggests that monomeric GTPases are involved in the uncoating step of the vesicles
4
Q
COPII – transport step mediated
A
ER –> cis golgi
5
Q
COPII – coat proteins
A
Sec23+Sec24, Sec13+Sec31, Sec16
6
Q
COPII – associate GTPase
A
Sar1
7
Q
COPI – transport step mediated
A
Cis golgi → ER
Cis golgi → golgi cisternae
8
Q
COPI – coat proteins
A
Coatamers with 7 different COP subunits
9
Q
COPI – associate GTPase
A
ARF
10
Q
Clathrin + AP – transport step mediated
A
Trans golgi → endosome
11
Q
Clathrin + AP – coat proteins
A
Clathrin +AP1 complexes
12
Q
Clathrin + AP – associate GTPase
A
ARF
13
Q
Describe the formation of COPII vesicles
A
Adapters Sec23/24 are recruited to the ER membrane by Sar1, a monomeric GTPase, and the help of a localized GEF
Amphiphilic helix is hidden, then after the localized GEF phosphorylates GDP → GTP it’s revealed
Sec23/24 associate with Sar1-GTP and the cargo receptor’s cytosolic tail
Coat proteins Sec23/24 and later Sec13/31 bend the membrane. The coat sculpts the membrane
14
Q
How are a vesicle’s cargo proteins selected?
A
Sorting sequences
15
Q
How are adaptors Sec23/24 recruited to the ER membrane?
A
They’re recruited by Sar1 (a monomeric GTPase) and a localized GEF
16
Q
What’s the usual max size for a COPII vesicle?
A
~50 nm
17
Q
What is the second layer in COPII vesicle coats?
A
Self-assembled Sec13/31
18
Q
Why do COPII vesicles need to shed their coats?
A
Because the coat hides the vesicle’s v-SNARE, which has to be revealed so it can fuse with the target membrane’s t-SNAREs
19
Q
How does COPII coat shedding occur?
A
Because the vesicle has budded and is moving away from the donor site, Sar1 is leaving its GEF (Sec12) behind. In response Sar1 hydrolyzes GTP, which retract’s Sar1’s tail. The tail retraction causes the shedding
Sec23/24 ultimately serves as a GAP for Sar1
20
Q
What is Sar1’s state dependent on?
A
Relative activity levels of Sec12 (a Sar-GEF) and Sec23 (a GAP)
21
Q
What is Sec12?
A
A GEF for Sar1
22
Q
What serves as a GAP for Sar1?
A
Sec23
23
Q
What dictates a vesicle’s donor membrane specificity?
A
Rab interacting with a protein on the target membrane
V-SNARE and t-SNAREs twisting and fusing
24
Q
What maintains a cytosolic pool of Rabs?
A
GDI
25
GDI
Guanine dissociation inhibitors
They prevent the GDP-bound form of Ras from immediately going to the GTP-bound form
26
Describe the SNARE hypothesis
Every vesicle has its own v-SNARE (usually 1 per vesicle)
Every target membrane has its own t-SNAREs (usually 3)
The interaction between SNAREs is a recognition step, confers specificity
27
Can vesicle fusion occur in the absence of Rab?
Yes, but it's slow and inefficient
28
Why is Rab hypothesized to accelerate the fusion process between vesicle and donor membranes?
Because in the absence of Rab (which we've done experimentally), fusion still occurs via the SNARE proteins, however it's slower and less efficient
29
What pulls vesicle and donor membranes together?
The coiling of intertwined SNAREs
Their alpha helices coil together, aligning so that any hydrophobic strips are next to each other
30
What forms the final bridge between the vesicle and target membranes?
A hydrophobic surface on the SNARE proteins, located close to the protein's respective membranes on the cytosolic side
31
Is the fusion process between a vesicle membrane and its target membrane fast or slow?
Fast
32
Does fusion between a vesicle membrane and its target membrane require energy?
No
Coiling of SNARE proteins is energetically favorable, doesn't use energy
At 1.5 nm (distance between two membranes) fusion is energetically favorable, happens spontaneously with no energy needed
33
How do the v-SNAREs and t-SNAREs unwind after fusion?
NSF (a protein complex) uses ATP to unwrap them
34
Is SNARE unwinding energy independent or energy dependent?
Energy dependent
Requires ATP hydrolysis
35
Anterograde
Forwards
36
Retrograde
Backwards
37
What direction do COPII vesicles go in?
Anterograde (forwards)
38
What direction do COPI vesicles go in?
Retrograde (backwards)
39
What are retrograde vesicle paths used for?
Returning v-SNAREs, bringing back sneaky or accidental stowaway proteins, etc
40
How are proteins targeted to remain in the ER?
They have a KDEL sequence on their C-terminus
41
How are stowaway proteins brought back to the ER?
Their C-terminal KDEL sequence binds to a receptor in the cis-golgi's lumen, binding is favorable in the golgi's acidic lumen
The cis-golgi buds into a COPI vesicle for retrograde direction back to the ER
Once fused into the ER, the less acidic ER lumen favors release of the KDEL sequence from the cis-golgi's receptor
42
What environments favor KDEL binding to the cis-golgi's receptor?
Acidic
43
What environment's favor KDEL dissociating from the cis-golgi's receptor?
Less acidic / basic
44
In an acidic environment, will a protein's C-terminal KDEL sequence bind to the cis-golgi's receptor?
Yes
45
In an basic environment, will a protein's C-terminal KDEL sequence bind to the cis-golgi's receptor?
No
46
Briefly describe the golgi apparatus's compartments
3 compartments: cis, medial, and trans
Compartments are usually stacked
Compartments are associated with distinct activities
47
Does the golgi apparatus contain N-acetylglucosamine transferase enzymes?
No, but it does contain enzymes to further modify N-linked glycosylations
48
What kind of glycosylation occurs in the golgi apparatus?
O-linked glycosylation
N-linked glycosylation is modified further (but is only ever originally added in the ER!)
49
What is O-linked glycosylation?
Amino acids with -OH as part of their R groups are glycosylated by transferase enzymes in the golgi
50
Will a protein in human epithelial cell be glycosylated in the same way as that same protein in a fly's neuron?
Probably not
Glycosylation enzymes can vary depending on cell specialization and between organisms
51
Why is it important to know that a protein may be glycosylated differently depending on the cell specialization and on the organism?
Because when making protein-based medicines, the protein may be glycosylated differently than expected, and the immune system might not recognize the abnormally glycosylated protein, or the abnormally glycosylated protein may not have the same effect as intended
52
How does the golgi apparatus move through vesicles?
Still not quite clear... currently two schools of thought
The "Stable Compartment" school (less favored rn)
The "Cisternal Maturation" school (more favored rn)
53
Stable Compartment school of golgi-vesicle motion
Post-translational modifiers stay in their golgi compartments
The cargo moves sequentially forwards through the compartments
Anterograde movement of cargo, no movement of modifiers
54
Cisternal Maturation school of golgi-vesicle motion
Each new cohort of cargo stays in the compartment
After the post-translational modifications of one cargo cohort are complete, the modifiers move backwards into a new golgi compartment behind them
Retrograde movement of modifiers, no movement of cargo
55
Evidence for the "Cisternal Maturation" hypothesis
There are some cases where the cargo is too large to fit into the 40-80 nm COPI vesicles
COPI vesicles are enriched in ER and golgi resident proteins
The modifier/enzyme content of a particular cisterna changes over time. If tagged with different colored fluorescent tags, it's observed that cisterna are originally filled with cis golgi enzymes, then over time the cis golgi enzymes empty out and trans golgi enzymes fill in their place
56
Where are clathrin coated vesicles seen?
Cargo leaving the trans-golgi
Endocytosis
57
What is adaptin?
An adaptor protein
Binds to the cargo receptor, then to the second coat layer
They bind to the cytosolic side of the receptor, the on the adaptin protein's other side they bind to clathrin
58
What is dynamin?
A GTPase
It self-assembles around the vesicle's base
It's hydrolysis induces a shape change, it's coil becomes tighter and pinches off the vesicle from the donor membrane
59
What enzyme is involved in clathrin+adaptin vesicle uncoating?
Hsp70 (an ATPase!)
Hsp70 binds to clathrin. It's ATP hydrolysis allows the clathrin to pop off the vesicle, uncoating it
60
Does clathrin self-assemble?
Yes, it self-assembles into triskelion structures
The triskelions then self-assemble to form the clathrin-coated vesicle
61
What sculps the clathrin-coated vesicle?
Intrinsic curvature
62
What sculpts the membrane of COPII vesicles?
Coat assembly
63
What happens when dynamin can't hydrolyze GTP?
It keeps extending it's spiraled stalk, but never finishes pinching off
64
What causes a clathrin-coated vesicle to pinch and bud off from its donor membrane?
Dynamin's hydrolysis of GTP --> GDP + Pi
65
How does dynamin function in the presence of non-hydrolyzable GTP?
Dynamin keeps making a longer and longer neck/stalk, but never actually pinches off the vesicle
66
What happens to clathrin-coated vesicles in the presence of non-hydrolyzable ATP?
Hsp70 can't hydrolyze ATP, and the vesicle never uncoats, causing an accumulation of clathrin-coated vesicles in the cytosol
67
What is the function of the trans-golgi network?
To sort proteins into vesicles targeted for various destinations
68
What signal tells the trans-golgi network to deliver the lysosomal enzyme to a lysosome?
An M6P residue
69
How are lysosomal enzymes delivered to the lysosomes?
They bear M6P residues that are recognized by M6P receptors, signal for the proteins' trafficking via a clathrin-coated vesicle to a lysosome
70
When/how are regulated secretory proteins released?
Regulated secretory proteins are concentrated and stored until secretion is signaled
71
When/how are constitutively secreted proteins released?
Constitutively secreted proteins are continuously delivered to the plasma membrane, regardless of signal presence or absence
72
How are regulated secretory proteins different from constitutive secretory proteins?
Constitutive secretory proteins are delivered to the plasma membrane continuously, whyle regulated secretory proteins are only delivered to the plasma membrane when secretion is signaled
73
What are M6P residues?
Mannoses that have been phosphorylated on their 6' carbon
74
What is the significance of M6P residues being the signal that a protein is a lysosomal protein (i.e. acid hydrolase) and needs to be trafficked to the lysosome?
Proteins only get mannoses added to them in the ER.
The acid hydrolase first needs to be co-translationally inserted into the ER, then N-linked glycosylated once in the ER, moved to the golgi, then marked with the phosphate (M6P) to go to the lysosome
75
How are lysosomal enzymes routed to their final destination?
Lysosomal enzymes (acid hydrolases) are first co-translationally inserted into the ER
N-linked glycosylated once in the ER
Transported to the golgi in a COPII vesicle
The protein has a specific shape, which is recognized by a GlcNAc phosphotransferase in the golgi
GlcNAc transfers the phosphate, creating the M6P residue
A phosphodiesterase removes the GlcNAc group, leaving behind the 6-P-mannose
76
What golgi enzyme puts the phosphate on a lysosomal enzyme?
GlcNAc
77
What enzyme removes GlcNAc from the lysosomal enzyme to leave behind the 6-P-mannose?
A phosphodiesterase (still in the golgi)
78
How does GlcNAc recognize the lysosomal enzyme?
It recognizes a specific shape, shape motif
79
What protects the ER and golgi from destruction by the lysosomal enzymes?
The lysosomal enzymes are acid hydrolases. They express their enzymatic activity only in low pH environments
The 6-P-mannose hinders the lysosomal enzyme’s activity while in transit to the lysosome The phosphate is removed only once the enzyme is in the lysosome, activating its enzymatic activity only after the removal
80
What activates the lysosome's acid hydrolases?
Low pH environments
Removal of the M6P phosphate residue (happens only in the lysosome)
81
What (seems to be) the first step in regulated and constitutive vesicle secretory pathways?
Calcium-mediated cargo aggregation seems to be the first step in vesicle assembly, for both regulated and constitutive secretory pathways
82
What effect would you expect to see if your cell's ER has high Ca2+ and low pH?
Temporary protein (cargo) aggregation, stimulating vesicle budding in the ER
83
What is the function of lipid rafts in epithelial cells?
Selective targeting of proteins to the apical membrane of the polarized epithelial cells may involve co-packaging in lipid rafts
84
LDL
Bad cholesterol. Distributes junk out to the body.
85
HDL
Good cholesterol. Pulls junk out of the body.
86
(Briefly) describe the process of receptor mediated endocytosis
Uptake vesicles are coated in AP + clathrin
Hsp7- ATP hydrolysis uncoats the vesicle
SNARE mediated fusion with the late endosome
Receptors are sent via transport vesicles to be recycled
The late endosome matures into a lysosome
87
What kind of vesicle coat is used in receptor mediated endocytosis?
Clathrin+AP coats
88
How do cells trigger vesicle uptake for receptor mediated endocytosis?
There are some options
1) "Ski lift" model
2) Ligand-induced dimerization
3) TMD shifting
89
Ski lift model of triggering vesicle uptake
Cells uptake vesicles regardless of whether anyone's inside. Continuous uptake
90
Ligand-induced dimerization as a method to trigger vesicle uptake
Receptors dimerize in response to a ligand, triggering the ligand's uptake in a vesicle
Receptors that do this often have large extracellular N-terminal domains, a single TMD, a short cytoplasmic C-terminal domain, and a cytoplasmic targeting motif
91
TMD shifting method of triggering vesicle uptake
A ligand binding to a receptor with multiple TMDs might cause the TMDs to shift, change conformation, alter cytoplasmic side patterns --> triggering uptake of the ligand in a vesicle
92
What kind of vesicle uptake is a cell most likely to do for a receptor with a large extracellular N-terminal domain, a single TMD, a short cytoplasmic C-terminal domain, and a cytoplasmic targeting motif?
Ligand-induced dimerization
93
What kind of vesicle uptake is a cell most likely to do for a receptor with multiple TMDs?
TMD shifting to alter cytoplasmic side patterns
94
What concentration should normal LDL receptors be at?
At or below 3.4 mmol/L
95
How do cells synthesize cholesterol?
Synthesized from acetyl-CoA, with HMG-CoA as the rate limiter for the synthesis reaction(s)
96
What enzymes to statins deal with?
HMG-CoA and/or HMG-CoA reductase
