Module 2 Flashcards
(178 cards)
1
Q
what part of a substrate gets ubiquitinated? what part of Ub is it linked to?
A
lysine residue (K48/K63) on substrate is linked to the C-terminal GG of ubiquitin
2
Q
how long is ubiquitin?
A
76 amino acids (8.5kd)
3
Q
what gives ubiquitin its different functions?
A
the different lysine residues
4
Q
explain the pathway of how ubiquitin gets conjugated to substrates
A
- E1 enzyme is attached to ubiquitin through a thioester bond
- E1 transfers Ub to E2 enzyme
- E2 binds E3 ligase which is coupled to the substrate and facilitates the final covalent conjugation
5
Q
how many different E2 enzymes and E3 ligases are there?
A
about 35 E2 enzymes.
over 100 E3 ligases.
6
Q
what are RING fingers and HECT domains?
A
E3s structures: RING links E2 to the substrate to get it ubiquitinated; HECT domain has direct catalytic roles
7
Q
name the 6 different ways a ub chain can be formed (different conjugates)
A
monoubiquitylation, multimonoubiquitylation, homogenous chain, mixed chain, branched chain, unanchored chain (no substrate)
8
Q
name examples of what can increase Ub chain complexity
A
- Ub-like proteins: SUMO, NEDD8
- phosphorylation, acetylation
9
Q
additional modifications of Ub chains increase complexity, but also what else?
A
increase specificity and reversibility (phosphorylation and acetylation is reversible)
10
Q
what proteins are called “readers” and read the Ub code? name 4.
What is the functions?
A
ubiquitin binding proteins.
They recognize specific Ub conformations and facilitate complex assembly and binding.
11
Q
what part of UBP binds ubiquitin?
A
conserved residues in UBP binds amino acid patches in UB
12
Q
what can interactions of Ub binding proteins with Ub chain lead to?
A
oligomerization
regulation of single, chain specific interactions
higher affinity interactions (ex in a UBP can bind multiple monoUb at once)
13
Q
what is UBD?
A
Ub binding domain. found in Ub binding proteins.
14
Q
what is UIM?
A
Ub interacting motif
15
Q
approximately how many different Ub-binding domains are there?
A
200
16
Q
what are DUBs?
A
Deubiquitinases: “erasers” of the Ub code (via hydrolysis)
17
Q
what was the first DUB to be discovered?
A
DUB that clips away Ub to the substrate can thread through the proteasome
18
Q
what kind of different aspects of Ub can DUBs specifically recognize?
A
Ub species, Ub-like proteins, types of Ub chains, substrates, phosphorylation.
They can cleave at a specific emplacement of a chain (Exo (distal/proximal) or endo).
19
Q
What does ubiquitin have to do with vesicle transport?
A
Signaling to enter! ex signal to internalize a receptor destined for the lysosome
20
Q
at first, what mutations were identified in endocytosis (END) mutants?
A
actin/actin binding genes mutations and actin cytoskeleton mutations
21
Q
What did End screens identify?
A
yeast homologs of amphiphysin, epsin, and EPS15, core elements of the endocytic machinery, and clathrin-coated vesicles
22
Q
what led ppl to wonder how are yeast surface proteins recognized by the internalization machinery and are endocytosed?
A
The tyrosine- and di-leucine based motifs that act as internalization signals in animal cells are not used in yeast
23
Q
what is Ste2p?
A
a 7 TM protein that binds a-factor, which activates a signal transduction pathway for yeast mating
24
Q
what sequence of Ste2P was identified by structure-function and deletion to possibly mediate a-factor dependent internalization?
A
SINNDAKSS intracellular sequence
25
what happened to one of the K residue in sinndakss?
ubiquitinated in an a-factor dependent manner
26
what molecule was found to be required for the studied protein (Ste2p) ubiquitination?
Rsp5P E3 Ub ligase! It was found to be required for Ste2p ubiquitination which led to its internalization
27
what did they later find about Rsp5P?
It mediates virtually all cell surface ubiquination events in yeast.
28
How can Ub chains be edited?
by DUBs degrading it while it is still growing
29
what is the first step of Ste2P internalization?
ligand binding to Ste2p which causes its phosphorylation, creating a binding site for Rsp5p
30
what happens to Ste2P when you remove its phosphorylation sites? what does that mean?
blocks ubiquitination and internalization. meaning phosphorylation of Ste2P tail is necessary for ubiquitination.
31
what are Yck1p and Yck2p and their role?
pair of kinases that mediate constitutive and a-factor induced Ste2p phosphorylation
32
where is WW domain found and what is its role?
Rsp5p (E3 Ub ligase) domain that binds phosphorylated motifs in Ste2p
33
name the 3 Rsp5p domains
C2 (non-specific, bind PIPs), WW, Hect
34
what does the C2 (Rsp5p domain) - PIP interaction do?
brings Rsp5p to the membrane to ubiquitinate the membrane protein
35
put it all together; explain how Ste2P gets internalized
- ligand binds Ste2p
- Ste2P cytoplasmic (intracellular) tail gets phosphorylated by Yck1p and Yck2p kinases (induced by alpha-factor)
- Rsp5p C2 domain bind to PIP (non specific) which brings Rsp5p to the membrane
- Rsp5p WW domain recognizes the Ste2p phosphorylated tail
- Rsp5P E3 Ub ligase mono-ubiquitinates the K in SINNDAKSS sequence of Ste2p -> internalization signal
36
how did they show that ubiquitin itself was the internalization signal?
they replaced the SINNDAKSS sequence of Ste2P that usually gets ubiquitinated for internalization with Ub itself -> it didn't affect Ste2p internalization
37
what else is required for Ste2P internalization? (apart from phosphorylation and ubiquitination) what are these proteins?
Ent1p and Ent2p proteins that contain UIM (yeast equivalent of mammalian epsin proteins)
38
what happens to ste2p when you delete Etn1p and ent2p proteins? why?
block Ste2p internalization; they are adaptors that couple Ste2P PI(4, 5)P2 to AP2/clathrin complex to allow internalization
39
in what only circumstance does Ent1p and Ent2p bind Ste2p?
only when ste2P is mono-ubiquitinated (specific)
40
what do epsin/enth proteins do?
they are adaptor proteins that bind Ub on signaling receptor (signal to internalize) and also bind NPF motifs to couple an activated receptor to AP2 to build the clathrin coat
41
name the important domains of Ent1p and Ent2p and what they bind/their function
ENTH: PIP
UIM: binds Ub on Ste2P SINNDAKSS
EH: binds NPF motif in the endocytic complex that links to clathrin
Clathrin binding domain: to build the coat
42
what kind of proteins are Ent1p and Ent2p? why are they needed?
they are AP2 adaptors: Ub specific single adaptor proteins that bind to active signaling receptors only once it is bound to the ligand and ready form internalization. Also binds AP2 to build the clathrin coat and are sometimes called Clasps.
43
what happens when you delete Ent1p and Ent2p?
stops Ste2P internalization
44
what happens once the receptor is internalized and must enter the MVB?
ubiquitination signal must be shut down!
45
what are the ESCRT complexes for?
machinery to signal when its time to internalize and degrade ubiquitinated receptors
46
what are the 3 fates of an internalized receptor once it is in the MVB?
1. degradation in lysosome
2. can become a unique intracellular signal
3. released in intralumenal vesicles in the form of exosomes
47
what is ESCRT0 protein and what does it bind?
HRS/Vps27; binds PI(3)P, clathrin, cargo (VHS), Ub, ESCRT1, all at the same time!
48
what is PI(3)P?
an endosome specific lipid
49
where can you find PI(3)P?
in endosomes (this is why ESCRT0 protein HRS/Vps27 binds PI(3)P to direct proteins to degradation
50
HRS (ESCRT0) association with clathrin forms what?
is forms flat lattices, not vesicles or cage
51
what is clathrin's role if not to make vesicles?
sequester/concentrate ligands
52
what main thing is required to start the budding of a vesicle? what protein helps acquire that?
ATP is needed; VPS4 is an oligomeric ATPase part of ESCRT complex that provides energy to seal the inward budding vesicle
53
what is thought to be Vps4 effect on ESCRT?
disassembles ESCRT
54
what is the other name for ESCRTI?
Tsg101
55
are DUBs involved in ESCRT machinery?
yes DUBs are necessary to cleave Ub before cargo enters the intraluminal vesicle MVB.
DUBs are recruited to ESCRT-0 and ESCRT-III.
56
how does it seem that the budding of intraluminal vesicles happen?
many hypotheses, the best one is that there's a spiralling and tightening of ESCRTIII filaments via VPS4 action
57
what are the different roles of ESCRT0-1 vs ESCRT3?
ESCRT0-1: sorting event (recognize ubiquitination, knowing who to put in MVB)
ESCRTIII: mechanical "popping in" of vesicle
58
what are other functions of ESCRTIII
sealing! seal ending of autophagy, seal nuclear pores, PM holes, lysosomal membrane holes, involved in cytokinesis, finish autophagy.
Basically seal stuff away from cytosol.
59
what do we know about ESCRTIII filaments?
they define the surface to be pinched
60
how is MVBs size summarized in the notes?
they are small endosomes of 100-60nm diameter that contain intralumenal vesicles (ILVs) of 25-50nm
61
What are MVBs role?
- membrane protein degradation
- cell signaling regulation in many ways
- exosome formation (part of cell-cell communication)
62
what controls the formation of ILVs and MVBs?
ESCRT machinery
63
what is the master regulator of the ESCRT machinery and ILV formation?
Vps4 ATPase
64
to what kind of disease do MVBs and ESCRTs contribute significantly?
cancer, neurological, infectious diseases
65
DUB are coupled to _____ ________ on the late endosome
ESCRT machinery
66
why do errors in ESCRT pathway lead to cancer?
causes constitutive proliferation
67
what is the basic pathway of ligand-binding activating receptors to send signals to the nucleus?
ligand binds -> receptor dimerizes -> phosphorylation -> signaling effectors are recruited -> nucleus translocation -> translation of new protein
68
epidermal growth factor (EGFR) activation leads to what? how?
leads to proliferation; via adaptor proteins that when phosphorylated go to the nucleus and affect transcription
69
Does diffusion make sense in complex cell types like neurons?
no! distance between cell body and dendrites can be super long, molecules can't be diffusing freely
70
what did the mathematical model of the random diffusion vs transport trajectory show?
phosphorylation of a signaling protein is lost around 750 nm = protein is turned off, doesn't go far
71
what protein causes dephosphorylation of the signaling protein?
phosphatases
72
what happens to signaling proteins in the diffusion model if there are no phosphatases?
proteins still don't go far and can't reach nucleus
73
how is dephosphorylation blocked on a transport endosome from membrane to the nucleus?
the receptor is still present on the endosome, it can re-phosphorylate the signaling protein
74
what is EGFR signaling adaptor?
Grb2
75
what does EGF addition do to Grb2? what does this finding mean?
EGF activates EGFR, which recruits Grb2 to the endocytic compartment..
This shows that colocalization and binding of signaling adaptors to their receptors occurs ON THE ENDOSOME, not at the surface.
76
what is APPL and what is special about it?
rab5 effector that ONLY gets selectively recruited to SIGNALING endosomes when rab5 is GTP-bound, not to other endosome types
77
what does APPL does once recruited to the early/signaling endosome?
it is released at a specific time to translocate to the nucleus where it participates in chromatin remodeling and transcription
78
what is Cbl? what does its mutation lead to?
E3 ligase; mutation blocks its ubiquitination properties -> can't tag receptor for degradation, accumulates -> signal amplification
79
more specifically what is the Cbl mutation and what disease does it lead to?
mutation creates a viral version without a ring finger sequence (can't ubiquitinate).
causes myeloid neoplasm of the hematopoietic system (leukemia)
80
what % of myeloid cancer have a Cbl mutation?
5%
81
what happens to Cbl mutants that lack ligase?
can still bind the receptor and act as a signaling adaptor, recruiting SH3-domain containing proteins, making the mutation dominant active (mutation stops downregulating the receptor AND actively recruits a bunch of proteins)
82
what part of ligase-deficient Cbl recruits a bunch of proteins, increasing the cancer?
TKB domain
83
opposite to Cbl mutation, what does Cbl total loss cause in mice?
not cancer because no signal amplification
84
these effects of Cbl mutation (recruitment of proteins by TKB doamin) show what?
Cbl is integral to the active signaling cascade, AND to the downregulation of the
signal
85
how do errors in ESCRT proteins lead to cancer and other diseases?
the activated receptors remain active (are not degraded)
86
what are the 3 main steps of the ESCRT degradation pathway
step 1: Cargo recognition & ESCRT-mediated sorting in endosome
step 2: Ub removal & membrane invagination
step 3: Membrane fission and ILV formation OR MVB docking and fusion with lysosome
87
what are 2 modifications in cancer cells that allow them to metastasize / grow? (other than signaling receptors trafficking)
changes in the recycling of integrins and actin remodeling machinery
88
how is exosome secretion in cancer? why (2 reasons)
elevated:
1. the release of microRNAs can reprogram the environment to promote ANGIOGENESIS
2. exosomes release PROTEASES to degrade the extracellular matrix
89
what is the role of the Notch pathway
determining the cell fate in the right place at the right time
90
explain how the notch pathway works
assymatric cell division leads to daughter cells with different levels of “notch” and “delta” proteins, predisposing the cells to specific fates
91
what can errors in the notch pathway lead to?
leads to human diseases spanning cancer to developmental abnormalities
92
explain how the notch pathway works in defining boundaries in development
(Ex) the cell with delta on its surface would signal to the notch-expression neighbour not to become a hair cell
93
how does delta cell signal to notch cell?
it has an activated delta ligand on its surface that activates the notch receptor on a neighbouring cell by triggering its cleaving and the cleaving of the NICD
94
what is NICD?
Notch intracellular domain (its a receptor)
95
name 2 diseases caused by the dysregulation of Notch signaling
- acute T-cell lymphoblastic leukemia (50% of patient have notch mutation)
- CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy): most common form of stroke caused by mutations in Notch3 receptor
96
how does CADASIL disease happen?
its a progressive deterioration of the smooth muscle cells in the blood vessels. TIAs start at age 40-50
97
how many times is notch cleaved? explain each time
3!
1: in the golgi during biogenesis
2 (S2): at cell surface when Delta binds. ADAM family metalloprotase
3 (S3): cytosolic fragment is cleaved and transported to the nucleus to regulate transcription and differentiation
98
what protein does the S2 cleavage at the cell surface?
an ADAM family metalloprotease
99
where is the 3rd (S3) cleavage thought to happen?
probably context specific: either on cell surface, but most agree its in the ENDOSOME, meaning endocytosis is essential for the activation of Notch
100
what protein transports notch intracellular to nucleus?
y-Sec
101
what is required for Delta activation?
endocytosis triggered by ubiquitination
102
compared to what we previously learned about Ub, how does ubiquitination affect Delta?
instead of targeting it for degradation, it "activates" it in the early endosome
103
how do they think delta gets "activated"?
via polarized redistribution of membrane proteins
104
in the notch pathway, what is the signal-sending cell vs the signal-receiving cell?
signal-sending = Delta/DSL-expressing cell
signal-receiving = Notch-expressing
105
what happens to Notch if it doesn't encounter a ligand? what is the ligand?
ligand = active DSL (delta)
if notch doesn't encounter a ligand it gets degraded
106
what degrades Notch when no ligand is encountered?
Itch/Nedd4 E3 ligase
107
what is recruited after S2 cleavage that triggers S3 cleavage?
and E3 ligase (Deltex, might be cell type specific)
108
what happens to the remaining notch fragments that don't go to nucleus?
degraded
109
putting it together: what are the 4 steps of the notch pathway?
1. recycling and activation of ubiquitinated delta
2. mechanical forces from ligand (active delta) binding to notch
3. Notch cleavage
4. Notch signaling to nucleus or notch degradation
110
what actually decides if a cell is activating or signaling? (how is asymmetry established in the first place)
the presence of numb proteins. it is enriched in activating cells and downregulate notch to let delta dominate.
111
the asymmetric division of the sensory organ precursor SOP leads to what lineages?
plla (signaling) and pllb (activating)
112
what protein is enriched in the pllb activating cell?
NUMB: adaptor that recruits AP2 and clathrin to Notch receptor, and binds E3 ligase Itch (down-regulates Notch)
113
where is Delta activated?
in the endosome!
114
what happens to delta in the signaling (Notch+) cell?
after being internalized, delta is either degraded or maintained in another endocytic compartment (don't know why yet)
115
what is the difference between delta and and notch ubiquitination?
Ub-Delta gets recycled and activated, Ub-Notch gets degraded by itch (like most ubiquitinated proteins)
116
what is mind bomb?
a E3 Ub ligase that is essential for efficient activation of Delta
117
name other secreted proteins (morphogens) that provide additional signaling cues that drive notch/delta biology
Wnt, Fgf, retinoic acid
118
what is the next thing to find out about how morphogens work?
how it works in a tissue, not just between two cells
119
they created a model in which a few mm of of cells could be defined in what time?
morphological transition of a few mm were defined over one hour
120
what happens when mind bomb (delta E3 ligase) gets phosphorylated and degraded?
it can not ubiquitinate delta and the cell loses its polarity, and changes the cell fate of neuroprogenitor cells
121
what happens to delta/notch polarization in cancer cells?
activating Notch mutations block polarization because of lack of Notch downregulation; cells just keep dividing randomly, CAN NOT DIFFERENTIATE between activating (Numb+) or signaling
122
how could drugs solve the lack of notch downregulation in cancer?
by altering the dose of Notch with anti-notch drugs that target Numb to increase Notch degradation (remember numb binds itch)
123
what is the usual faith of signaling endosomes?
carry the signaling proteins to the nucleus, then mature in late endosomes and fuse with lysosomes
124
REMEMBER Errors in the downregulation of active receptors can lead to what?
cancer
125
_______ is essential in the activation of Notch receptor signaling during development
endocytosis
126
what was the main question of the 2nd paper (on vacuole) ? what was the answer?
how are resident lysosomal membrane proteins degraded?
answer: ESCRT
127
what is the lysosome?
organelle that sense nutrient status and degrade proteins
128
when are vacuolar amino acid membrane transporters degraded?
when there is low level of the amino acid in the cytosol (no need to degrade the aa)
129
Emr did a genome-wide screen of what?
fo mutants defective for Ypq1 degradation
130
What happened in Ssh4KO mutants? what does this mean?
Ypq1-SBP could not be trafficked to the vacuolar lumen. It stayed on the membrane, showing that Ssh4 is necessary for its trafficking to the lumen.
131
why is Ssh4 needed to Ypq1 trafficking to the lumen?
to recruit Rsp5 that ubiquitinates Ypq1
132
what were the 2 contradicting theories of how vacuolar transporters were degraded?
1. transporters are ubiquitinated, sorted out of the vacuole membrane into MVBs, then sorted into vacuole for degradation
2. transporters are digested when vacuoles fuse together
133
what are the main players of the study
- Ypq1: 7 TMD tightly regulated vacuolar transporter for lysine. Gets degraded when lysine is low.
- Ssh4: single TMD adaptor protein that recruit Rsp5 Ub E3 ligase to transporter.
- Rsp5: HECT domain containing E3 Ub ligase. Binds Ssh4.
134
in the abstract they say that they already know that Ypq1 gets degraded after ______?
after lysine withdrawal which triggers its ubiquitination by Rsp5
135
what is Cot1?
vacuolar zinc transporter that like Ypq1, gets ubiquitinated and sorted in the vacuole lumen for degradation upon zinc depletion
136
where is Ypq1 transporter in the presence of lysine?
on the vacuolar membrane
137
how long after lysine withdrawal does Ypq1 get degraded?
full degradation takes 8 hours
138
why did they do the lysine deprivation experiment with Pmc1 calcium transporter?
to show that the lysine deprivation specifically affect Ypq1 because it in fact did not affect
139
how did they test that only the lost of specifically lysine led to degradation of Ypq1?
by doing the deprivation of other amino acids than lysine (His, Arg, Trp, Leu)
140
what did the addition of 38 amino acid streptavidin-binding-peptide SBP to Ypq1 led to?
quick constitutive degradation of Ypq1 even without lysine
141
why did Erm fuse Ypq1-SBP to a pH sensitive fluorophore?
so that fluorescence was lost when it enters the acidic vacuole (only the KO mutant cells in which Ypq1degradation is affected will fluoresce -> can identify genes needed for Ypq1 degradation)
142
how did they do the live death screen to find the proteins involved in Ypq1 degradation?
fusion of Ypq1 with an enzyme essential for histidine -> only the KO mutant cells in which Ypq1 degradation is affected will survive. KO mutant cells of proteins that aren't involved will die because Ypq1 will get degraded normally.
143
what components did they identify as being involved in Ypq1 turnover in the death screen? what protein of interest?
ESCRT, Rsp5, HOPS/CORVET, SNARES, PI(3)K Vps34 and Vps15.
protein of interest: Ssh4.
144
what was the problem about identifying so many proteins that when KO blocked Ypq1 degradation?
they could not distinguish the KO mutant causing issues of biogenesis from issues of turnover of Ypq1 itself
145
what are vps27 mutants?
ESCRT mutants
146
what happens to Ssh4 in ESCRT mutants? (found by immunofluorescence) what does this mean?
Ssh4 is blocked at aberrant endosomes and can not be recruited to the vacuole membrane and lumen, like it is in WT cells.
Means that ESCRT is required for Ssh4 delivery to the vacuole and therefore required for Ypq1 ubiquitination.
147
why did they use doa4KO cells?
doa4 is a major DUB and its deletion stabilizes ubiquitinated forms of Ypq1
148
they did immunostaining of myc-Ub (UB) after lysine withdrawal. what did they find in WT and in vps27 mutants? what does that mean?
WT: Ypq1 becomes polyubiquitinated
vps27: no polyubiquitination, showing that without ESCRT, Ypq1 can not be ubiquitinated because Ssh4 can't be delivered to the vacuole and recruit Rsp5
149
how did Erm stabilize the ubiquitination that is usually transient?
by doing the experiments in Ddoa4 cells that lack the DUB.
150
en resume why were so many ESCRT components identified by the death screen as being necessary for Ypq1 degradation process?
just because Ssh4 requires ESCRT to get to the vacuole. but ESCRT has no direct link with Ypq1.
151
why did they invent the rapideg system?
to bypass the ssh4 transport issue and look only at events on the vacuole by controlling Ypq1 ubiquitination
152
how does rapideg system work?
Ypq1-GFP is linked to FKBP. FKBP binds FRB ONLY in the presence of rapamycin. FRB is tagged with 3 Ubs.
they can now control Ypq1 ubiquitination without Ssh4, ESCRT, or RSP5
153
what kind of cells did they need to use for rapideg? why?
a rapamycin resistant yeast strain so that it doesn’t activate autophagy
154
indeed, what happened to Ypq1-FKBP after addition of rapamycin?
internalization in vacuole and degradation after 30 min
155
what is the vph1-cherry tag used for?
to tag the vacuole membrane
156
did rapideg system work in Ssh4KO cells?
yes! meaning no more need for Ssh4 for Ypq1 degradation
157
what did they find when they tested Ypq1-FKBP degradation in KO mutants of the HOPS/tether, SNARE, and NFS proteins identified in the death screen as important for Ypq1 degradation?
the proteins had no effect in Ypq1 degradation therefore were just there for ssh4 recruitment
158
so is membrane fusion required for Ypq1 degradation?
no!
159
remember: what is vps4?
main ATPase for ESCRT machinery
160
what happened to Ypq1-FKBP in Vps4 mutant cells after rapamycin addition?
it was stuck at the membrane and did not get degraded
161
what does it mean that vps4 mutation blocks degradation and transport of Ypq1?
ESCRT machinery is needed for Ypq1 sorting in vacuole
162
what is PI(3)P required for?
required for ESCRT recruitment to endosomes
163
what is FYVE domain on Vps27 ESCRT subunit?
PI(3)P binding domain
164
what did they find with immunostaining of FYVE domain, and what does this mean?
FYVE, and therefore vps27 (ESCRT0) is localized at the vacuolar membrane
165
what happened to FYVE when Vps34 (PI3kinase) was lost? why?
GFP-FYVE reporter becoming diffuse and cytosolic. Shows that the vacuole membrane is amenable for ESCRT recruitment.
166
how did they test the effect of ATP depletion on Ypq1 sorting?
inhibited mitochondrial respiration with sodium azide/fluoride
167
why were they interested in the effect of ATP depletion on Ypq1 sorting?
ESCRT machinery requires ATP, and depleting ATP will slow down the process of Ypq1 internalization so they can visualize it better
168
what happened to Ypq1 in cells treated with NaN/NaF 0 min after rapamycin addition? 15 min after?
0 min: Ypq1 at vacuolar membrane (duh)
15 min: Ypq1 forming puncta at the membrane like its about to bud in, but it DOES NOT bud in. NO INTERNALIZATION without ATP
169
what happened to Ypq1 in cells treated with NaN/NaF without rapamycin?
nothing. shows that the puncta are not just due to ATP depletion but to slow ESCRT activity
170
what happened to Ypq1 when you deplete ATP in vps27 cells (ESCRT mutant) and add rapamycin?
no internalization, no puncta formation
171
why didnt the ATP depletion by sodium azide/fluoride kill the cells?
it could be reverse by washing the drug away.
172
what happened when you washed sodium azide/fluoride away and added rapamycin?
Ypq1 was internalized
173
how did they test if ESCRT0 and 1 are both needed for Ypq1 internalization?
they used a tag for each (Hse1 and Vps23) and did immunofluorescence and saw that they colocalized with Ypq1 puncta after ATP depletion and Rapamycin addition
174
what control did they use to make sure that the puncta seen after ATP depletion are not endosomes?
they tagged endosomes with FM4 and showed with immunofluorescence that it is NOT colocalized with Ypq1
175
what was the problem with visualizing the Ypq1 internalization system with EM?
they will see ILVs, not just the MVBs that they are looking for
176
how did they eliminate all the random ILVs so they could only see and study the Ypq1 MVBs?
they deleted pep12, MVB sorting machinery which decreases ILV formation, and they added a DUB so no ubiquitination = no internalization
177
why did the DUB not affect Ypq1 internalization if Ypq1 needs to be ubiquitinated to be internalized?
because Ypq1 has the FKBP tag that bypasses the requirement for normal ubiquitination machinery with rapamycin
178
why is there no internalization without ubiquitination of a receptor?
because ESCRT machinery can't be degraded
