Module 1 Flashcards
lecture 1 ,2 ,3 (paper)
what are rafts?
cholesterol enriched microdomains
lipids in membrane make up what % of cellular proteins?
25%
are rafts and lipid clusters created randomly?
no; there exists a calculated partition coefficient, selectivity in lipids, different packing abilities, and it required a specific temperature
proteins with lipid conjugates have more chances of ending up where in a membrane?
in microdomains (rafts)
where will TM proteins mostly enrich in a membrane?
in non-raft, disordered domains
what does cholesterol do to a membrane?
lowers fluidity/flexibility, increases and stabilizes raft formation, increases melting temperature
what is found in ordered domains of membranes?
glycosphingolipids, sphingomyelin, cholesterol, saturated lipids
what is found in liquid disordered domains?
various forms of PC, PE (usually shorter and unsaturated lipids)
what can cluster rafts together?
antibodies, lectin, crosslinking proteins
what happens to yeast vacuoles when you leave them in stationary phase (starving)? what does this show?
they partition to allow lipid droplet entry and digestion.
shows that the generation of raft-like domains can be tightly regulated
where are GPI usually found in membranes compared to VSVG?
GPIs are found in ordered rafts domains.
VSVGs are TM proteins found in disordered domains.
describe the structure of cholesterol
4 ringed structure with a short hydrocarbon side chain
how long are carbon chains in phospholipids of unsaturated lipids vs in saturated lipids (sphingolipids, glycosphingolipids)?
phospholipids: 16-18 carbons
saturated lipids: 16-26 carbons
where are diacylglycerol vs ceramide backbone found?
diacylglycerol backbone is phospholipids of disordered domains.
ceramide backbone in glycosphingolipids of ordered domains.
What are DIGs? describe them
Detergent resistant microdomains: they include rafts which do no solubilized at 4 degress in triton X-100
what is SDS?
a harsh denaturing agent
Following solubilization of cells with detergent in a tube, where are raft domains? why?
they float at the top of the gradient because they don’t solubilize well and they are fat, which floats.
what does adding sucrose or optiprep to your tube do when isolating raft proteins?
create a sucrose gradient in which raft proteins will float after 6 hours centrifugation.
what is VSVG?
membrane-spanning protein
because of where they are found (ordered vs disordered), would PLAP float? what about VSVG?
PLAP is found in ordered domain and would float.
VSVG is found in disordered domain and would not float.
PLAP is a GPI anchored protein and is post-translationally conjugated to what?
GPI is post-translationally conjugated to a lipid at the C-terminus
how could you make PLAP not float?
by heating the sample to 30 degrees to melt the rafts
what can you do to keep PLAP floating even after incubation at 30 degrees
by crosslinking it to an antibody prior to fixation to cluster rafts
what are raft’s role?
play a central role in many cellular processes, including membrane sorting and trafficking, cell polarization, and signal transduction processes
what underlies protein hopping between distinct domains?
cytoskeleton
how did they find that the cytoskeleton has a role in lipid movement?
Kusumi traced the trajectory of a lipid PE conjugated to gold particle at 40 000 frames/s
what are PIPs and what is their role?
Phosphorylated phosphatidylinositol phosphate.
PIP patches recruit cytosolic proteins with selective affinities to distinct cellular locations depending on the position of their phosphorylation.
How are PIPs regulated?
the enzymes that generate PIPs are regulated by signaling pathways that can act as timers
“SO a PIP ______ will change from
the budding vesicle at PM through
the endocytic compartment, for
example.”
surface
what is special about PIPS inositol rings?
they are phosphorylated at specific sites by a series of lipid kinases and phosphatases with specificity
what are the 3 types of enzymes that generate asymmetry in the lipid bilayer? and their role
flippase: ATP dependent, selectively flips lipids in
floppase: ATP dependent, selectively flips lipids out
scramblase: Ca2+ dependent, non specific, flips in and out
name a lipid that is mostly found in the outer bilayer and one in the inner bilayer
PC is outside
PS is inside
is there more cholesterol inside or outside the bilayer?
more cholesterol outside
where are the membrane asymmetry generating enzymes found?
in PM, endosomes, golgi
what happens when a membrane curves? (what triggers rearrangement of lipids?)
the energy imbalance of crowded lipids on the inner curvature decreases the activation energy for trans-bilayer flipping
what is special about PA (phosphatidic acid)?
its shape makes it more stable in negative curvature
what is PLD? what does it mostly do?
Phospholipase D: enzyme that converts lipids to induce curvature.
Mostly converts PC to PA through HKD motif.
what gets PLD recruited and activated?
PIP domains recruit it.
Rho/Rac GTPases activate it.
what is sphingomyelinase and one of its function? what does the product of said function doÉ
enzyme that converts SM to ceramide, which can cluster rafts and increase rigidity
apart from lipid transitions, what is the other thing that drives membrane bending?
integral and peripheral membrane proteins, ex amphipathic helix, hydrophobic domain, protein coat assembly
what can peripheral membrane proteins do?
They exert force onto the bilayer to curve
very briefly name the steps of vesicle budding?
receptor activation
PLD activation
PC -> PA
neck curvature
PI(4)P5K generates PI(4,5)P2
Dynamin recruitment
neck constricts -> budding
what happens in cytosolic ribosome? what stops this activity?
translation of membrane proteins and proteins meant to be secreted. arrested by SRP.
what is SRP?
signal recognition particles: ER import signal that stops translation in ribosome
after being translated in ribosome, where do proteins go?
to the ER to get folded by chaperones
about how many proteins transit through the ER to their final destination?
6000
what happens to misfolded/unassembled proteins?
they are re-exported and degraded by the proteasome
where do proteins go once they exit the ER via vesicles?
to ER to Goldi Intermediate Compartment
why was the bulk-flow hypothesis proposed as a way of soluble proteins to exit the ER?
because there is no obvious “exit tag” on secreted proteins
what kind of proteins are transleted in the ribosome?
secreted (soluble) and membrane proteins
what is SRP and its function?
signal recognition particle: stops translation in the ribosome. acts as an ER import signal.
how do mature (fully folded and secreted) soluble proteins exit the ER?
selectively bind TM cargo receptors that bring them in budding COPII vesicles; or by bulk flow
What happens to TM cargo receptors once they delivered the proteins to the golgi?
they are recycled to the ER in COP1 coated vesicles
can membrane proteins exit the ER by bulk flow?
no they can only exit the ER selectively.
how are membrane proteins recognized to exit the ER?
they have aa signals exposed in the cytosol that recognizes the COPII coat machinery, which gets them enriched 2-50x in vesicles
name generic ER exit signals for membrane proteins
dephenylalanine FF, diacidic aspartic or glutamic acid D/E x E/E
how do GTPase work? name one GTPase involved in COPII assembly.
they bind GDP in their inactive form; then a GEF (GTP exchange factor) activates it; GTPase is now binding GTP and is active; a GAP (GTPase activating factor) triggers hydrolysis of GTP and makes the GTPase inactive.
example: Sar1
what do GTPases allow for?
reversibility and temporal regulation
name the GEF and the GAP involved in COPII assembly? what activates the GAP activity?
Sec12 is the GEF.
Sec23 is the GAP. Sec13/31 complex activates the GAP.
what is sec24? what does it form a complex with?
cargo binding protein.
form a complex with Sec23 (GAP).
what does sec23 GAP activity allow for after the COPII assembly?
Allows for the hydrolysis of GTP within Sar1 so Sar1 can be recycled.
what does it mean that Sec12 (GEF) is more active than Sec23 (GAP)?
the COPII coat can only disassemble when it is away from sec12, which is not in the vesicle.
what molecule is present at ER exit sites and what does it do?
PI(4)P: regulates Sar1 recruitment.
what protein forms the COPII cage?
sec13/31
what else is in the COPII structure other than Sec13/31?
Sec23 (Sar1 GAP) /24
what is COPII cage size range?
500-800A (50-80nm)
what differentiates the soluble proteins that need to return to the ER after exiting it, from the other proteins?
KDEL (lysine-aspartic acid-glutamic
acid-leucine) sequence at their C-terminus
how do KDEL receptors and returning TM proteins recruit COP1 coat?
through conserved di-lycine motif (xxKK) in C-terminal or di-arginine (xRRx or RxR) in N-terminal
give examples of proteins that need to return to the ER
receptors that carried soluble cargo forward, the Sar1 GEF (Sec12), fusion machinery, or “leaked” ER chaperones like BIP carried by bulk flow
in the COPI vesicle assemble, what is the GTPase, the GEF and the GAP?
Arf1 is the GTPase
Gea1 is the GEF
ARFGAP is the GAP
what makes the coat of vesicles that return to the ER?
COPI coatomer complex
what is ARFGAP required for?
COPI assembly, no required for COPI maintenance
what protein modifications happen in the TGN? why?
glycosylation, mannosylation, sorting.
Sorts proteins to their destination.
how can resident golgi enzymes be recycled back to earlier stacks?
via COPI vesicles
via what structures can proteins exit the TNG?
clathrin-coated vesicles and tubulovesicular structures
what is special about vesicles traveling to apical/axonal domains?
they are enriched with cholesterol-based rafts that contribute to selective transport
name 3 examples of where proteins go after exiting the ER
storage granules (for regulated secretion)
lysosome (to deliver hydrolytic enzymes)
recycling endosomes (intermediate in cell surface delivery)
what are clathrin adaptor proteins? what is their function? name specific ones
APs: they are multiple forms of this complex with distinct subunit specificity.
AP1 = TNG to PM
AP2 = PM to endosomes
AP3 = endosome budding
AP4 = TNG to basolateral domains
what do clathrin APs bind to to get their specificity? How else can they increase specificity?
PIPs, clathrin itself, cargos.
They also increase the number of binding partner, have subunit isoforms (each subunit binds smtg different), get phosphorylated
do clathrin APs have low or high affinity?
low; so the complex can be easily disassembled
what is special about proteins/enzymes destined for lysosomes?
they get tagged with a mannose-6-phopshate sugar in the golgi
explains how the mannose-6-phosphate sugar system works
M6P receptor in the golgi has a YxxO motif that binds AP1 -> recruitment of clathrin to exit the golgi -> fusion with late endosome -> pH drop -> M6P cargo is delivered.
Then M6P receptor returns to golgi via retromer complex
what happens to ALL incoming structure that enter cell from PM?
fuse with early (sorting) endosomes
what are the 2 fates of proteins in sorting endosomes?
Receptors to be recycled to the surface (or elsewhere) are sorted into tubulovesicular structures.
Luminal cargo and ubiquitinated receptors stay within the endosome which matures into a late endosome
how do endosomes become more acidic? from what pH to what?
via ATP dependent proton transport channels; 7.5 to 5.5
what is the other name for late endosomes?
multivesicular bodies
explain the steps of the generation of multivesicular bodies?
- early endosome recruits Rab5 GTPase
- when GTP bound, Rab5 GTPase recruits PI(3)K Vps34
- early endosome gets enriched in PI(3)P
- PI(3)P recruits ESCRT complex which concentrates ubiquitinated receptors
- invagination of vesicles, internalization of cargo
- signaling stops, degradation
what happens as the endosome acidifies further?
PI(3)P- 5Kinase Pik-FYVE is recruited, generating PI(3,5)P2, causing the release of a series of factors, and recruitment of new proteins
explain ESCRT complex function
vital role in the generation of multivesicular bodies by binding and clustering ubiquitinated proteins and/or receptors on the surface of a cell
what happens as the endosome acifies further, once proteins are degraded?
Pik-FYVE is recruited, generating PI(3,5)P2, causing the release of a series of factors, and recruitment of new proteins.
name the difference between exosomes and ectosomes
Exosomes come from multivesicular bodies, Ectosomes are secreted from the surface in vesicles or blebs
contrarily to what the cartoons picture, where is the ER?
the ER is spreaded through the whole cell like a net.
other than via vesicles, how else can organelles communicate/exchange metabolites?
via direct contact
what is the base of fluorescent microscopy? name a pro and cons
fluorescent proteins/antibodies
pro: can image in real time
con: only see what you are looking at & bad resolution
what wavelength is used in fluorescent microscopy?
500nm
what is a chimeric protein?
fusion fluorescent protein cDNA with cDNA of protein of interest
whats the difference between confocal and wide field microscopy?
confocal: lasers emitting specific wavelengths of light excite fluorophores introduced into cells.
wide field: filters allow selection of excitation light to hit the sample
what is the function of a dichroic mirror?
filters out the excitation light and allows the lower energy longer wavelength emitted light to be detected
what kind of proteins are stained by immunofluorescence?
endogenous proteins
how can you validate the specificity of your antibody?
by testing the antibody on cells where the protein is knocked out by CRISPR or gene editing techniques (should see nothing)
can you see dynamics with immunofluorescence?
no. cell needs to be fixed.
what are the 4 steps of antibody recognition
- inject protein into animal
- let animal generate antibodies to the protein
- purify the antibodies
- purchase a secondary antibody conjugated to a fluorophore
what are the 2 main types of electron microscopy?
transmission EM: electron beam passes
through a section, patterns detected depend on sample absorption of electrons.
scanning EM: scanning of surface structures only
difference between normal EM and cryoEM?
normal: sample is fixed and embedded in resin. can get slices of 50-100 nm
cryo: sample is flash frozen at -80.
how does focused ion beam electron microscopy work?
image electrons of a layer and then remove the layer by burning it, repeat (you can get depth this ways)
what is electron tomography?
tilting the grid on which the sample is to get a little bit of depth
how can you avoid chemical fixation?
plunging a sample into liquid nitrogen to freeze, and then freeze substitude to get it resin embedded
what is cryo-SIM? what can it be followed by?
cryo-Structured Illumination Microscopy: imaging of frozen samples to capture immunofluorescence.
Can be followed by FIB-SEM (Focused Ion Beam Scanning Electron Microscopy)
what did they find doing cryo-SIM/FIB-SEM characterization of ERES?
each ERES was composed of vesicular-tubular membranes devoid of ribosomes. Each ERES is connected to ER via a narrow neck and tubular network.
how were they able to follow a cargo through ER-Golgi?
RUSH system:
1. put a streptavidin binding domain into target cargo
2. have a streptavidin “hook” anchored to the ER in the cell
3. add biotin to compete this interaction (pulse proteins forward)
4. stop reaction at any time by fixation or freeze and analyze
what did they tag to follow the cargo?
TNF-a
the presence of Sec23 at ERES says what?
defines it as a COPII exit site from ER
how does photobleaching work?
you loose all fluorescence of fluorescent protein; recovery of fluorescence = cargo comes back in
the photobleaching experiment showed what?
rapid refilling of ERES (in WT)
Cholesterol being colocalized with TNFalpha-RUSH at the ERES showed what?
cholesterol can create a membrane domain that can dynamically retain membrane proteins and lipids by a partitioning mechanism (helps sorting cargo at ERES)
what movement did they see after the RUSH experiment?
2-3 minutes long stationary phase; 10-15 minutes of anterograde movement with tubules; minute 18: cargo in golgi
what did they find when studying the accumulation of cargo at ERES?
ERES double in size during RUSH (moving cargos) but their organization doesn’t change
what cargo did they mainly use in the ERES paper?
TNFalpha-RUSH
they analyzed the RUSH cargo at 8 min. what did they find?
long microtubules emanating from ERES
what did they find out about how different cargos exit ERES?
different timing of exit for different cargos (regulation based on cargo type)
what does it mean if the time to accumulate at ERES of a cargo is 0s?
the cargo is initially already present in ERES
what is H89? what was its effect on RUSH?
established chemical inhibitor of Sar1: blocked the enrichment of cargos in ERES (cargos dissipate from ERES after 30s) and the release of cargos, causing cargos to accumulate at the neck and tubule
why did they want to study Sar1?
to study COPII’s potential gating function at ERES
the H79G mutation locks Sar1 with Sec23 on the membrane to block cargo release. How did it affect RUSH? what can we conclude?
block enrichment/recovery of cargo, and blocks release of cargo, just like H89.
Concludes that the recycling of Sar1 is essential to cargo enrichment.
what did the photobleaching experiment with H89 and Sar1-H79G mutation show?
Both showed the lack of refilling of ERES (different from wild type who’s ERES filled back up)
what did the paper find COPII’s primary role to be?
gate protein entry into ERES. Confocal imaging found that COPII stays at ERES upon cargo exit
graph shows that the % of Sec23 with BCOP is highly decreases in H79G mutant Sar1. what does this mean?
BCOP is a subunit of COPI. So, we can conclude that COPI acts after COPII and requires dynamic cargo enrichment
unlike COPII, what happens to COPI after biotin addition?
COPI exits ERES with the cargo.
what did they find about cargo ERES entry and exit rates?
Different types of cargos (TNF-alpha-RUSH, GPI-RUSH, TfR-RUSH, and Gp135-RUSH) have different rates
what is BCOP?
antibody to the endogenous COPI subunit (used to localized COPI)
where did they find BCOP (COPI)?
colocalized with Sec23 (COPII)
what happened to BCOP (COPI) in H79G mutant (Sar1 mutant) / H89+ cells? what does this tells us about the order of things?
BCOP/COPI subunits are lost from ERES, no more colocalization with COPII. COPII mutation affects COPI, therefore COPI acts after COPII
what is BFA?
something that inactivates Arf1 to interfere with COPI (cus Arf1 is COPI GTPase)
what happens to the cargos when BFA was added?
BFA (Arf1 blocker; Arf1 is main COPI GTPase) did not alter ERES assembly but blocked ERES exit. ERES enlarged because cargo was accumulating and can’t exit. Shows that COPI acts downstream of COPII
the results of adding BFA (blocking COPI formation) implacte what?
blocket ERES exit = implicates that COPI is necessary for differentiation of transport intermediates after ERES
how did the paper hypothesize cargo move to/from ERES?
via tubules and microtubules with a dynactin motor (p150glued)
how did the paper find microtubules?
with FIB-SEM imaging, found pearled transport intermediates going to the golgi
why do they think the ERES have a pearled appearance?
the pearled appearance could reflect tensions induced by motor pulling
what is p150glued? where did they find it?
a subunit of the dynactin motor complex. they found it associated with transport intermediates containing cargos
what did they find when localizing cholesterol and cargo?
colocalization; to be expected because remember the intermediates are cholesterol rich
what does the paper suggest that Sar1 function is?
COPII Sar1 acts as a gate at the neck of ERES. It doesn’t coat the budded vesicles like we thought!
what is the ERES cholesterol enrichment hypothesized to do?
facilitate stabilization of cargoes there
the paper hypothesized that what molecule is the determinant of cargo exit and recycling?
COPI
what is the main characteristic of the neck of ERES (ER to ERES neck)
it’s narrow (def needs proteins to hold it tight)
where is COPII vs COPI localized in ERES?
COPII is closer to the neck and acts first, COPI is closer to the rims (exit site)
last part of discussion
