Midterm 2 Difficult Topics Flashcards
lipid shape that forms micelles? bilayers?
single tail/cone, double tail/cylinder
why do membranes need cholesterol?
it decreases local membrane fluidity by tightly binding adjacent hydrocarbons close to the polar head
how it cholesterol like antifreeze?
prevents membrane from freezing when temps are low, but prevents it from boiling when temps are high
cytosolic leaflet lipid ratios vs extracellular leaflet lipid ratios
phosphatidylserine stays on exterior side but flips to cytosolic during apoptosis (to signal that it needs to be eaten)
ATP dependent flippases cover hydrophilc head group so the lipid can pass through hydrophobic interior to other leaflet
Why are hydrophobicity plots bad at detecting beta barrels?
The amino acids in a beta barrel alternate hydrophobicity every other, it won’t show up on this test
why should you use non ionic detergent to remove proteins from a membrane?
they are polar but not charged, so they bind to hydrophobic residues and outcompete the bound surrounding lipids while stabilizing the protein (need lots of detergent to achieve)w
why do lysosomes have a ~100x higher H+ concentration inside them?
The high pH in lysosomes supports its acid hydrolases, enzymes that break stuff down can only funciton at highly acidic pHs
is GLUT1 (and other uniporters like it) saturable?
Yes: Vmax (max transport rate) limited by number of transport protein
What does digoxin do? why is this bad if you’re healthy but good for weak hearts?
suppressed sodium ATPases which indirectly makes cells worse at pumping out calcium
it’s good for weak hearts because they need the calcium for muscle contraction
MRD1 cancers
tumor cells that express ABC transporters can pump out more of the anti-cancer and chemo drugs resulting in patients with MRD1 cancers (fitness advantage for tumor cells)
what makes y spin in ATP synthases?
a steep proton gradient made with the ETC drives the proces and F0 acts as a merry. go round with the aspartate acting as the H+ carrier… arginine (+ charged) swings between H+ entry and exit sites and is displaced by the incoming proton which facilitates exit of outgoing proton
what causes cells cytoplasmic calcium concentration to change?
plasma membrane channels let calcium ion in or ER channels released of of their stored calcium to the cytoplasm
how do we know what ER looks like?
transmission electron micrographs
do secretory proteins enter the ER lumen?
What experimental techniques proved this?
YES
give pancreatic cells radioactive leucine (labels newly synthesized proteins) then isolate rough microsomes from density technique… treat samples with and without detergent then add proteases to all samples; if proteins are in the ER, protease can’t digest them without detergent, will digest with detergent
proteins not in ER digest regardless of detergent presence
how would you determine whether proteins are co translationally inserted into the ER?
run samples of protein synthesis with one with microsomes containing ER that was added after and one with microsomes containing ER that was there from the start… if proteins from both samples end up in the ER, they are inserted after translation (no cotranslationally) and if proteins from the sample containing microsomes from start then they are inserted co translationally
Functions of sec61 translocon
helps polypeptides cross the ER membrane
enables TMDs to pass sideways through walls and guides proper orientation
binds to and releases ribosomes
has tight seal preventing things (ATP or calcium) from leaking
what does BiP do in co translational insertion?
acts as a clamp to pull the peptide into the lumen –> molecular ratchet
single pass transmembrane proteins into ER membrane
nascent polypeptide in translocon, N terminal signal sequence is cleaved
new n terminus is now in ER lumen
TMD in polypeptide stops it from going through the translocon (transfer stopped)
TMD slips through translocon walls into lipid bilayer where it is anchored and translation resumes
remaining c terminal domain now in cytosol with the rest of the translated protein
What if a nascent polypeptide does not have an N terminal signal sequence?
secondary rules
SRP recognize internal hydrophobic sequence and brings it to the translocon
Sec61 looks for positive charges in polypeptide adjacent to hydrophobic TMD
the side with adjacent + charges will be oriented to remain in the cytosol and side without will be oriented to be in ER lumen
hydrophobic TMD slips through translocon’s walls to be anchored in the lipid bilayer (no cleavage)
what about when a polypeptide is being cotranslationally inserted into the ER without an N terminal signal sequence and with 2 TMDs?
Translocon encounters the first hydrophobic TMD and orients it based on positive charges, translation continues, translocon encounters second hydrophobic TMD and passes it to lipid bilayer (without orienting it)
rest of peptide is translated in the cytosol
what happens when a polypeptide is being co translationally inserted into the ER without an N terminal signal sequence and with three or more TMDs?
follows same pattern as with 1 or 2 TMDs
first TMD provides orientation
every TMD after the first is put straight into the membrane without additional orientation
how are tail anchored proteins co translationally inserted into the ER?
TMD at c terminal but too late for SRP recognition…
interacts with Get3 ATPase complex and a distinct translocon, Get3 hydrolyzes 2 ATP to ADP to stick the c terminal hydrophobic TMD in the ER membrane (rest of protein remains in cytosol)
how are GPI anchored protein co translationally inserted in the ER?
protein starts with cleavable N terminal signal sequence and c terminal TMD, N terminal signal sequence is cleaved by translocon then c terminal TMD anchors it in membrane… instead of staying there the c terminal TMD is cleaved and protein is linked to GPI anchor
what does the GPI anchor allow for in the membrane?
Increased mobility
functional relevance of phospholipid asymmetries in a plasma membrane?
glycolipids on exterior side like PI and PS… exoplasmic PS signals to other cells that it is in apoptosis and that nearby cells need to eat the dying guy
in ERAD, what recognizes the problem?
ER Hsp70 chaperones, ER lectins, or other factors inside ER; PTC does retrotranslations which moves unfixable proteins from ER lumen to cytosol
to be degraded, proteins must be… because there are no proteasomes in the ER lumen
retrotranslocated
what happens when BiP is busy?
theres lots of unfolded proteins in the ER and this indicates stress so in response general translation is shut down and transcription of specific proteins is activated to mitigate stress… emergency mode
Ire1 UPR
when cells are happy, it’s bound to BiP (exists as monomer) but when BiP is busy (stress) Ire1 homodimerizes and autophosphorylates and can now function as endonuclease and cut/splice mRNA
PERK UPR
exists as monomer when BiP is bound
when BiP is busy it autophosphorylates and homodimerizes which then phosphorylates elF2alpha which blocks elF2alpha from helping the small ribosomal subunit bind to charged tRNAs –> phosphorylated elF2alpha blocks a lot of translation
some mRNAs that can be translated without needing elF2alpha (ATF4: enters nucleus once translated)
ATF6 UPR
BiP bound to ATF6 it’s a homodimer bound together with disulfide bonds
dimer reduced/disulfide bonds broken with BiP is busy and it breaks into monomers
monomers then trafficked from ER to golgi where it is cut to become ATF6 TF which enters the nucleus and promotes ER chaperone transcription (creates feedback pattern)
Defective CFTR
usually an ABC transporter that exports chloride ions out of cells
detect causes buildup of chlorine ions inside cells so they import more sodium to mitigate the negative charge which then forms to salt and causes cell to import more water…. this cycle dries out extracellular fluids preventing it from clearing out as usual
F508del
deletion in cystic fibrosis that slows folding of CFTR as it’s being synthesized causing it to be degraded before it can leave the ER (can be helped by inhibiting ERAD)
how does a phospholipids head group contribute to overall lipid shape?
larger head groups make the molecule cylinder shaped and smaller head groups make it cone shaped
FG nucleoporins
free ends of nuclear pore complex central channel proteins… mostly extend IDRs with some phenylalanine glycine (FG) repeats interspersed throughout
proteins larger than 40 kDa enter nucleus?
importin binds to cargo and brings it through NPC
GEF phosphorylates RanGDP to RanGTP in nucleus
RanGTP replaces cargo and binds to importing (both exit nucleus)
once back in cytosol, RanGTP hydrolyzed by GAP to RanGDP
proteins larger than 40 kDa exit nucleus?
cargo bound to exportin and RanGTP inside nucleus
exit nucleus through NPC together
RanGTP hydrolyzed to RanGDP in cytosol by GAP
exportin and RanGDP re-enter nucleus
RanGDP must be phosphorylated by GEF to become RanGTP and begin again
