Sunday, March 20, 2011

Book Notes: Membranes Part 2

:D my use of colors has no learning significance, except to attract and wake up your eyes so you hopefully don't nap during studying.
  • endoplasmic reticulum: netlike organelle of tubules and flattened sacs, lipid bilayer membrane encloses "lumen" or "cisternal space" and is continuous with nuclear envelope
    • site of lipid/protein synthesis: makes all transmembrane proteins/lipids, mitochondrial/peroxisomal membrane lipids, secreted proteins, lumenal proteins for ER/Golgi/lysosomes
    • stores intracellular calcium ions
  • differences in structure give ER different functions
    • rough ER: ribosomes bound to the ER cytosolic surface feed in polypeptide chain during translation (co-translational process)
    • smooth ER: regions that lack bound ribosomes, not as populous but has subtypes
      • transitional ER: form and fuse with transport vesicles, close to Golgi
      • synthesize lipids, lipoproteins, cholesterol
      • membrane enzymes catalyze detoxification reactions
      • contain regions of high concentration of Ca2+ binding proteins to store calcium
  • microsome: resealed vesicles of ER fragments when cell is homogenized, 100-200 nm diameter
    • can be easily isolated and studied like miniature, functional ERs
      • rough microsomes are obviously from ER, but smooth microsomes also come from plasma membrane, Golgi fragments, mitochondria, and endosomes (solve by just getting microsomes from muscle/liver cells, who have lots of smooth ER or SR)
  • co-translational import of proteins into ER based on signal sequences
    • proteins destined to be transmembrane don't fully come through ER pore and gets embedded in ER membrane
    • proteins destined to be lumenal or water-soluble fully come through and hang around inside
    • discovered signal sequence by having isolated ribosomes translate mRNA with or without microsomes nearby: polypeptides made in the presence of microsomes came out shorter than the ones made without
    • ER was cleaving the N-terminus leader peptide: the signal sequence on the polypeptide that directed it to the ER.
      • it's cleaved by signal peptidase in the ER membrane (co-translational)
  • signal-recognition particle (SRP): protein that shuttles between ER membrane and cytosol, binds to signal sequence, is composed of 6 separate polypeptide chains bound to 7SL small RNA
    • binds to SRP receptor on the ER membrane
    • binds to variable ER signal sequences, who all have 8+ nonpolar A.A. at center
    • binding site is large hydrophobic pocket lined with Met: Met side chains are extremely flexible for accommodating different sequences
  • SRP is a long rod that wraps around ribosome, one end binding to signal sequence, one end blocking the E site (remember EPA?)
    • this halts protein synthesis until SRP binds to the receptor to ensure that the finished protein will head directly into the ER
    • because multiple ribosomes tend to translate one mRNA simultaneously, for a given mRNA that encodes, say, a secretory protein, the entire polyribosome is bound to the ER
      • as soon as a ribosome is done, it leaves and other ribosomes take over and keep the mRNA associated to the ER
  • Sec61 complex: aqueous pore protein in the ER membrane, has a short helix for a gate to keep out everything except polypeptide and to prevent lumen stuff from escaping
    • incoming polypeptide pushes aside the gate to enter
    • the side of the complex can open to integrate a transmembrane protein or to release the cleaved signal peptide
  • most proteins get imported cotranslationally, but some are done so post-translation
    • SecA ATPase: bacterial motor protein that attaches to the cytosolic side of the channel and hydrolyzes ATP to push in polypeptide bit by bit
    • BiP: eukaryotic binding protein associate with ER lumenal side of membrane and cycles between binding to incoming polypeptide and release to drag it in
  • signal peptide must be recognized twice: SRP binds and brings it to ER, Sec61 binds and opens the pore
    • start-transfer signal: leader signal peptide when it binds to Sec61 b/c it initiates pore opening and allows polypeptide import, is cleaved
    • stop-transfer signal: a different signal peptide within the polypeptide that initiates lateral gate opening so the protein can integrate into the membrane rather than be fully imported, N term inside, C term out
    • separate method of integration involves the start-transfer signal being in the middle of the polypeptide chain
      • SRP brings it over to the ER like normal, sequence binds in one of two ways (either resulting in N term in or N term out), half of the polypeptide goes through and the other half stays out, this sequence also serves to open lateral gate, is never cleaved but stays as the transmembrane domain
    • these are called single pass transmembrane proteins because only one sequence is bound to the channel at one time
  • multipass transmembrane: polypeptide chain loops back and forth through membrane, have alternating start and stop signals within the chain
    • stop and start sequences are actually very similar and can swap function
    • SRP defines first hydrophobic segment as start and decides the following hydrophobic segments' function based on position (it alternates)
    • recombination of the sequences change the function of each sequence based on their resulting position
      • example: in a sequence ABCD, A and C are start signals, B and D are stop signals
      • in a recombined sequence DCAB, D and A are start signals, C and B are stop signals
  • all proteins are inserted from cytosolic side, so transmembrane proteins tend to be asymmetric, and their asymmetry is preserved b/c of how systematic protein import is
    • asymmetry preserved with budding and transport events (cytosolic side always faces cytosol)
    • is not based on protein characteristics (fragmenting and allowing microsomes to form shows embedded proteins facing random directions) but only on how they were inserted in the first place
  • ER retention signal: four A.A. sequence at C-terminus on ER resident proteins
    • examples include BiP and protein disulfide isomerase (PDI) which catalyzes formation of disulfide bonds (aids in folding)
      • in cytosol, exposed Cys are not bonded, while Cys in ER are almost ALL bonded
  • most ER proteins are glycosylated, as opposed to cytosolic proteins who are mostly not
    • precursor oligosaccharide: contains N-acetylglucosamine, mannose, glucose, 12 other sugars
      • oligosaccharyl transferase: membrane enzyme facing the lumen that attaches the precursor to the NH2 side chain of Asn (N-linked)
      • associates with Sec61 so it transfers as soon as Asn comes through
    • dolichol: membrane lipid that holds the precursor (phosphate bond) until it's time to transfer
      • sugars are attached to cytosolic side one by one, and then a transporter protein flips the dolichol to the lumenal side
    • precursors are trimmed of 1 mannose and 3 glucose and modified/decorated
      • trimming/decoration occurs in ER and in Golgi
    • exist O-linked oligosaccharides (O-linked) that bind to hydroxyl group of Ser, Thr, or hydroxy-Lys
      • formed in Golgi
  • oligosaccharides identify state of protein folding: some proteins need it for proper folding but location of oligosaccharide attachment doesn't matter
    • calnexin and calreticulin are calcium-using chaperones and lectins because they bind to the carbohydrate part
      • they bind to incompletely folded proteins to prevent them from (1) running amuck and (2) aggregating
      • promote association with another chaperone to properly fold it
      • bind only to oligosaccharides with 1 glucose (meaning 2 have already been removed) and dissociate when the last is removed
    • glucosidase continuously trims glucose, while glycosyl transferase keeps adding a last glucose to unfolded proteins with oligosaccharides that have no more glucoses
      • keeps unfolded proteins associated to calnexin and calreticulin until they are fully folded (receive no more extra glucose and are free to go!)
  • dislocation (retrotranslocation): sending failed proteins outside of the ER to get degraded
    • misfolded proteins are deglycosylated once outside and ubiquitylated by and ER-bound ubiquitin ligase
  • unfolded protein response: similar to heat-shock response when too many misfolded proteins accumulate in cytosol, this response occurs when too many misfolded proteins accumulate in ER
    • ER sends signal to nucleus to increase expression of ER chaperones, dislocation proteins, and other folding helpers
    • signal is sent via three different pathways:
      • misfolded proteins activate ER transmembrane kinase, who oligomerizes and self-phosphorylates, activating its cytosolic endoribonuclease portion
        • this domain cleaves a certain cytosolic RNA and excises an intron
        • ligase connects the exons and the new mRNA is translated into a gene regulatory protein, who goes to activate transcription
      • misfolded proteins activate a 2nd ER transmembrane kinase, who phosphorylates/inhibits a translation initiation factor
        • certain mRNAs are still translated w/o the initiation factor, and one of these is a gene regulatory protein that goes to activate transcription
      • a gene regulatory protein exists embedded in the ER membrane
        • misfolded proteins cause it to be exported to the Golgi, where proteases cleave off its cytosolic domain
        • the free domain goes to the nucleus and activates transcription
  • glycosylphosphatidyl-inositol anchor (GPI): lipid attached to the C-terminus of proteins destined for plasma membrane, attached to lumenal side of ER membrane
    • proteins facing lumen of ER but bound to the membrane at C-term is cleaved from its embedded C-term and binds to the N2H of the lipid at the cleaved end
    • protein bound this way can only be released from membrane if lipid is cleaved by phospholipase in the membrane
  • ER assembles most lipid bilayers by synthesizing most all of the lipids required
    • phosphatidylcholine is major one, formed from choline, 2 F.A., and glycerol phosphate
    • steps catalyzed by ER membrane proteins facing cytosol
      • F.A. binding protein brings F.A. to ER membrane and F.A. is activated by an attached CoA
      • acyl transferase put 2 F.A. on glycerol phosphate = phosphatidic acid, which cannot be extracted from the bilayer and represents the step of membrane growth
      • latter steps are addition/modification of head group
    • lipids are flip-flopped btwn ER membrane leaflets by scramblase, who does this randomly so that both sides get evenly distributed with all types of phospholipids
    • plasma membrane uses flippases that spend ATP to move phospholipids with A.A. in head group to the cytosol leaflet
      • plasma membrane scramblase is regulated and active only during apoptosis or with activated platelets (subsequent exposure of phosphatidylserine is "eat me signal")
  • ER makes cholesterol and ceramide (precursor for sphingolipids)
    • serine is condensed with F.A. to make sphingosine, plus one more F.A. = ceramide
    • ceramide goes to Golgi
      • addition of oligosaccharides makes glycosphingolipids
      • addition of choline head groups from phosphatidylcholine makes sphingomyelin
      • both are exclusive on lumenal leaflet b/c the Golgi enzymes are in the lumen
    • mitochondria and plastid membranes receive lipids by import, not by membrane fusion like with Golgi or plasma membrane
      • use phospholipid exchange/transfer proteins
pg. 723-745 (from Chapter 12)
F.A. = fatty acid

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