Meeting 6

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  1. it appears that the translation of mRNA by ribosomes in the cytosol can have a number of different fates depending on the targeting sequence in the proteins:
    • 1) no targeting sequence means the protein is released into cytosol and stays there
    • 2) if the targeting sequence directs protein to a) mitochondrion/chloroplast (pass through outer then inner membranes to sit in matrix/stroma space), peroxisome (through membrane into matrix) or nucleus (enter/exit through envelope pores) then that's what happens!

    3) proteins in the secretory pathway have signaling sequence that directs them to rough ER; after translation finishes on ER, the protiens can move via transport vesicle to golgi where they undergo further processing and are either exported to plasma membrane or lysosome
  2. signal sequence
    the information to target a protein to a particular organelle destination is encoded within the amino acid sequence of the protein itself; 6-50 aa long

    • -this is the first part of protein to be synthsized, on the 5' end, on the N-TERMINUS
    • -translation is briefly halted, and the complex is brought to where the signal sequence directs it to go
  3. translocation channel
    allows the protein to pass through it's specific target organelle's membrane bilayer

    each organelle carries a set of receptor proteins that bind only specific kinds of signal sequences
  4. polysome
    mRNA molecule with a succession of ribosomes translating that mRNA molecule (use e. microscopeto visualize it)

    -membranes are electron dense: polar groups absorb electrons, they're darker)
  5. Pulse-Chase Experiment
    Image Upload
    what this experiment showed: newly made proteins are inside the microsome, essentially the equivalent of the lumen of the rough ER right after they're made

    • -cells are incubated with radiolabeled amino acids real quick --- only newly made proteins glow
    • -cells are homogenized; plasma membrane is broken and the ER reforms into small vesicles: microsomes
    • -these remnants of ER membrane have ribosomes still attached so they are higher in density than other parts of cell; can be centrifuged out
    • -purified microsomes are either treated with detergent THEN protease or just protease
    • -labeled secretory proteins are digested by proteases only if the membrane is first destroyed by the detergent: this shows where the newly made proteins are (INSIDE the lumen)
  6. signal recognition particle (SRP)
    a cytosolic ribonucleoprotein particle that transiently binds to both the ER signal sequence (in nascent protein) + as the large ribosomal subunit forming a complex

    -SRP then targets the nascent protein-ribosome complex to the ER membrane by binding to the SRP receptor on the membrane
  7. P54 subunit
    a subunit in SRP responsible for binding the ER signal sequence; is homologous to the bacterial Ffh protein, which contains a large, cleft lined with hydroPHOBIC amino acids

    the signal sequence on the N-term of a ER directed protein is also composed of hydrophobic amino the aqueous environment of the cytosol these two hydrophobic entities will likely find each other
  8. translocation and translation occur simultaneously:
    Image Upload
    secretory protein is synthesized in the absence of microsomes but is translocated across the vesicle membrane and loses its signal sequence ONLY if microsomes are present during protein synthesis
  9. SRP & SRP Receptor
    together, they form a GTP domain; when GTP is hydrolyzed 1) SRP is released and subsequently 2) translation restarts 3) upon GTP hydrolysis, the translocon OPENS! newly synthesized peptide squeezes through it

    -a lot of this energy comes from hydrolysis of GTP &! translation
  10. signal peptidase
    cleaves the signal sequence after the N-term of the peptide has made it through the 'open' translocon (Sec61 complex); needs to be cleaved so hydrophobic-ness doesn't affect the peptide's ability to move fully into the ER lumen
  11. single-pass proteins
    topological classes I, II, & III; have only 1 membrane-spanning alpha-helical segment
  12. Type I proteins
    have a cleaved N-terminal ER signal sequence; anchored in teh membrane with their hydroPHILIC N-terminal region on the luminal (exoplasmic) face & their hydroPHILIC????? C-terminal region on the cytosolic face
  13. Type II proteins
    opposite of type I; don't have cleavable ER signal sequence & are oriented w/ hydroPHILIC N-terminal region on the cytosolic face and their hydroPHILIC C-terminal region on the exoplasmic (luminal) face
  14. Type III proteins
    have same orientation as Type I proteins but do not contain cleavable signal sequence

    SA sequence acts like an STA sequence
  15. Type IV proteins (multipass proteins)
    contain 2 or more membrane spanning segments; ex's include membrane transport proteins & G protein-coupled receptors
  16. In the cytosol is where you find:
    the +++ parts of the peptide; using this you can predict topologies of sequencies
  17. STA (internal stop-transfer anchor sequence)
    the hydrophobic sequence close to the N-terminal region of a nascent peptide that 1) stops the passage of its polypeptide chain through the translocon closer to the lumen and 2) becomes a hydrophobic transmembrane segement in the bilayer
  18. SA (internal signal-anchor sequence)
    found in type II & III proteins (not I); functions as both an ER signal sequence AND membrane-anchor sequence
  19. GPI Anchor
    always on exterior of cell: exoplasmic side (luminal)
  20. type IV protein with EVEN v. ODD number of transmembrane helices:
    even: BOTH its N-term and C-term will be oriented toward the same side of the membrane

    odd: its N-term and C-term will be oriented toward opposite sides of the membrane
  21. Image Upload
    • just because
    • P54 binds to the signal sequence
Card Set:
Meeting 6
2013-08-24 15:51:19

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