Test Review Session

Here is the review for Naren, again. Fain gave us a review sheet and he reviewed at the end of each individual lesson. Having been through this stuff before, I would recommend FLOW CHARTING the processes, what goes in and comes out, enzymes, cofactors, etc. for Fain's lectures.
Naren-powerpoints and movies have been updated and it does work. There will be six questions from the lectures.
1-The importance of ER in protein translocation- What is co translational and post-translational translocation? Cotranslational is critical.
-How are proteins inserted into ER membranes?2 questions here. Questions are A(6pts),B, C (4pts each). What do we mean by cotranslational translocation? Signal sequence indicates that a protein is destined for the secretory pathway. As it is translated, it is translocated at same time. Most common process in secreted proteins. Protein in ER may be soluble or could be destined to be membrane associated protein. How are they inserted into membrane?
Cotranslational translocation has 4 or 5 major steps.
1. Ribosome bound to RNA- ss is stretch of hydrophobic AAs recognized by SRP. SRP binds SS and determines that SRP can bind receptor.
Receptor has alpha and beta subunit. Translocon slides closer and GTP hydrolysed. Gives fidelity for process. SRP comes off polypeptide through channel of three subunits, Signal peptidase cleaves SS, protein is synthesized, folds, ribosome comes off, starts over.
Protein insertion in membrane (look up and study these items) :
topogenic sequence- defined sequence of hydrophobic 20-22 AAs for alpha helix, classes (stop transfer anchor, GPI anchor,) hydropathic profile. Look at 4 major topological classes.
Look at the simple experiments he explained in class. Study pages 3-8 of the file for Lecture 1.
2- Glycosylation, Disulfide bond formation
Proper folding, and degradation of misfolded protein
Protein can fold in cytosol or in lumen. Without proper glycosylation, protein does not leave ER.
3-Nuclear transport: Import (NLS signal) and export
import is well-worked out pathway. Remember NLS (positively charged residues anywhere in sequence recognized by importins or import receptors). Remember RAN cycle, GEF, GAP. Outer nuclear membrane is continuous with ER. Ions and small molecules can diffuse passively, but importins are essential for large proteins. How regulated? By pore by means of NLS. 4-5 positive AAs. KKKRK is like a ticket.Mutate any charged amino acid- alter movement of protein into nucleus. Alter protein to make chimera with NLS- protein goes into nucleus.
RAN cycle – membrane slide. Know which side of the membrane is which.
Export not well known yet.
4-mitochondrial and peroxisomal protein transport.
Translocons in mitochondria- TOM and TIM in regions of contact outer membrane and inner. SKL on C terminal tail is important. Peroxisomes have one membrane and one set of translocon proteins.

5-Vesicular traffic:Secretion/exocytosis: Sar1 & COPII,
docking and fusion of vesicles
2 concepts to remember: how Sar plays an essential role in assembly of proteins
How are mislocalized proteins relocalized correctly? See his slides.
Sar1 is molecular switch. Sec 12 binds Sar1. Tail anchors Sar, which starts whole process. Upon anchoring, the COPII assembly with Sec 23 and 24 and bind SAR protein. As coat is formed, GTP hydrolysis happens and tail retracts, and Coat disassembles. Then membrane can bind where it needs to go. See slides on blackboard for structure.

Anterograde and retrograde transport: Coat formation helps in anterograde transport from ER to Golgi. Retrograde helps bring proteins that do not belong in Golgi back to ER. KDEL receptor recognized misssorted protein, COPI coat forms, protein brought back to ER. Step must take place precisely. Minimal components: KDEL receptor, KDEL sequence in protein , vesicle with COP coat.

6-Vesicular traffic, Endocytosis
Receptor mediated endocytosis
KNow receptor mediated endocytosis and work of Brown and Goldstein (LDL receptor and clathrin-dependent endocytosis, and the importance of environmental pH to the function of the receptor protein propeller domain). SPecific bound ligands are brought into the cell. Clathrin, adaptors, and dynamin are important.
Sorting signals include (npxy, YXXphi), with phi being a hydrophobic amino acid.
Be aware of lysosomic pathways.
See Slide for pathway.
LDL is recognized by LDL receptor. binds. AP2 forms clathrin coated pit. Pinches off in dynamin dependent way. Get noose with GTPgamma S (a nonhydrolysable analog of GTP). Early endosome sheds coats and fuses with low-pH late endosome. Receptor is transported back to neutral pH and ligand-binding arm is free to bind something else. lowpH, propeller domain does not interact. Ligand kicked out by charged residues at low pH.