Meeting 7 & 8

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Meeting 7 & 8
2014-01-15 13:12:11

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  1. cis v. trans
    • cis: on the side of the ER
    • trans: on the opposite side

    proteins move from cis to trans-golgi network; this later area is where decisions are made as to where to send the proteins
  2. anterograde v. retrograde transport
    • anterograde transport: the right way; cis to trans (ER to golgi)
    • retrograde transport: the opposite, vesicles can move in multiple ways, eg. trans to cis (Golgi to ER)
    • -from more mature compartments back to initial compartments
  3. cisternal maturation
    when a cis-Golgi cisterna, with its protein content, physically moves from the cis to the trans face of the Golgi complex
  4. VSVG-GFP Protein

    • -VSV G protein: is a viral membrane glycoprotein and it was 1) linked to GFP and 2) transfected into cultured cells
    • -a mutant version of the gene is the one used where at 40 degrees it stays in the ER (b/c it's misfolded) and at 32 degrees it's released for transport (b/c protein folds properly)

    -graph shows the level of VSVG-GFP in each individual organelle; basically goes ER --- Golgi --- PlasmaMem.

    -generally the whole process takes 1 hour; overall flouresence decreases after a certain amount of time
  5. Yeast Temperature Sensitive Sec Mutants
    • -how you identify successive stages in the secretory pathway
    • -these yeast mutants were temp. sensitive, so when their environement changed from the (permissive) lower temp. to the (non-permissive) higher temp., secreted proteins will accumulate at the point in the pathway where the mutation takes place
    • -to determine the order of the steps in the pathway, double sec mutants were analyzed: ex. if a mutant had both the B & D mutations and proteins were found accumulated in the rough ER (but not the Golgi cisternae), one could say class B mutations acted earlier in the pathway than did the D ones!

    • -stidies confirmed that AS A SECRETED PROTEIN IS SYNTHESIZED/processed, it moves from:
    • cytosol --- rough ER --- ER-to-Golgi transport vesicles --- Golgi cisternae --- secretory vesicles & finally is exocytosed
  6. coated vesicles
    three types, each characterized by their different type of protein coat; function to transport cargo proteins from particular parent organelles to particular destination organelles
  7. COPII
    vesicles that trasport proteins from the rough ER to the golgi
  8. COPI
    vesicles that mainly transort proteins in the retrograde direction between Golgi cisternae and form the cis-Golgi back to the rough ER

  9. Clathrin
    vesicles that transport proteins from the plasma membrane (cell surface) and the trans-Golgi network to late endosomes
  10. ARF family proteins
    type of GTPases; change conformation when bound to either GDP (inactive) or GTP (active)

    GEF: guanine-exchange factor: changes GDP to GTP (activator of small GTPases)

    GAP: GTPase activating protein; will hydrolyze GTP and convert to GDP state
  11. v-SNAREs
    membrane proteins in a budding vesicle that are responsible for fusion of the vesicle with the correct target membrane

    -ex. VAMP; can dimerize with SNAP-25 (t-SNARE): forms a very stamble complex; sufficient to tether the two membranes
  12. t-SNAREs
    proteins found in the TARGET organelle membrane that are cognate to v-SNAREs; facilitate the fusion of the two membranes

    -ex. SNAP-25 dimerizes with VAMP (v-SNARE); forms a very stamble complex; sufficient to tether the two membranes
  13. the model of the Sar1: the assembly/disassembly of COPII coats
    • 1) Sar1 bound 'around' a GDP binds to the membrane, where Sec12 (membrane protein) catalyzes the exchange of GDP for GTP!
    • -when Sar1's bound to GTP, it's hydrophobic N-term sticks out and binds to the ER membrane
    • 2) Sar1's attachment to the membrane acts as a BINDING SITE for Sec23/24 coat proteins
    • -the coat is completed when secondary coat proteins (Sec13/31) are attached
    • 3) after ALL that/the vesicle's been transported from ER to Golgi, Sec23 subunit promotes GTP hydrolysis (BY Sar1)!
    • 4) now that Sar1's complexed w/ GDP not GTP, it detaches from vesicle's membrane --- leading the whole coat to disassemble!
  14. (cis)-SNARE complex
    v and t-SNARE protein complexdelivery of the cargo occurs at this point'
  15. NSF and alpha-SNAP
    proteins required to unwind the SNARE complex (so the proteins, especially v-SNARE can be recylced and used over)

    -NSF-catalyzed hydrolysis of ATP drives dissociation of the SNARE complexes
  16. Rab*GTP & the Rab effector
    a second set of GTP binding proteins; participate in the targeting of vesicles to the appropriate target membrane (like Sar1 and ARF)
  17. the binding affinity of the KDEL receptor is very sensitive to pH:
    -proteins with KDEL (c-term) sequence will BIND to receptors in Golgi-derived vesicles; Golgi has lower pH (higher [H+]); KDEL binds strongly in low pH

    -proteins with KDEL (c-term) sequence will be RELEASED from KDEL-receptors in the ER; ER has high pH (low [H+]); KDEL dissociates in high pH
  18. C-terminal KDEL sequence (Retrograde Transport of Proteins that Belong in ER lumen)
    a sequence found on many ER luminal/soluble proteins that are sometimes passively incorporated into COPII (the right way ones) vesicles and transported into the Golgi; the sequence alerts KDEL receptors (found in cis-Golgi network, and both COP I/II vesicles) to return them to the ER

    -retrieval using KDEL identification prevents depletion of ER proteins that help with, say, the proper folding of secretory proteins
  19. post-translation modifications in the secretory pathway
    • -disulfide bond formation
    • -lipid modifications
    • -protein folding complex assembly
    • -proteolytic cleavages
  20. Glycosylation
    -there are 2 types: depend a lot where on the protein the sugars are going to be attached; occurs on the lumen

    • 1) O-linked sugars: are sugars linked to –OH in serine or threonine; added in Golgi, by glycosyltransferases
    • 2) N-linked sugars: sugars linked to the amide N in the asparagine residues; process starts in ER & is completed (modified) in Golgi

    -commonalities: always starts with same residue (1st to be added to proteins, and is then modified)
  21. dolichol and mature residues HAVE to be on the ER luminal (exoplasmic) side

    -also if the protein isn't properly folded, a glucose is added which is a signal to send it back to a chaperone to be refolded
  22. Protein Disulfide Isomerase
    • the enzyme responsible for creating disulfide bonds in proteins in the ER lumen; does this by undergoing an oxido-reduction reaction
    • -protein starts out reduced (has 2 SH's sticking out)
    • -PDI enzyme is in oxidized state
    • -so it works by accepting the 2 protons/electrons FROM the protein, becoming reduced and the protein becomes oxidized

    -cells wants to reuse PDI though, so it needs to return to its oxidized state: Ero1 carries extra e- and H+, eventually reduces? oxygen to water?
  23. Lectins (calnexin, calreticulin)
    carbohydrate binding proteins; double checks to see whether protein is being properly folder (if there are glucose residues recognize and work as a check)
  24. BiP/Misfolded Proteins in the ER Lumen
    BiP acts as a chaperone in the ER lumen that's tethered at the ER membrane by a transmembrane protein (Ire1)

    • -when there are misfolded proteins, BiP is released to chaperone them all
    • -Ire1 not attached to BiP will dimerize (w/ another Ire1)
    • -the dimerized form works as an endonuclease that splices specfic mRNA: Hac1
    • -Hac1 when spliced INCREASES the amount of chaperones (eg. BiP) present
  25. Permanently Misfolded Proteins
    • -removed from ER lumen
    • -tagged serially by ubiquitin in the cytosol
    • -degradation by the Proteasome
  26. Clatharin Coats
    • -the beginning of endocytosis
    • -have the triskelion strcuture: heavy chains + light chains
    • -globular heads pointed inward are important for which proteins will be carried by the clatharin

    -there's modularity because the coat can be the same, but the acceptors within the coat can be altered to tailer to the specific cargo
  27. Dynamin
    • -located at the neck of the vesicle
    • -+ GTP hydrolysis are NECESSARY for coated vesicles to pinch off
    • -(remember the experiment with non-hydrolyzable ATP, the coat can't pinch off)
  28. chromogranin
    enzymes that respond to a change in pH