Naren Notes

Lecture 1: ER translocation
Start with protein secretion movie.
Movie is what we will talk about.
Imagine a cell. mRNA comes from nucleus. Protein synthesis happens in the cytosol. The ER transports proteins to go outside, or to organelles inside cell, or into nucleus. What determines the fate of protein?
Protein destined to be secreted is secreted into ER. Protein goes to Golgi, which add sugars, then passes thorugh trans golgi and out.
Sorting and targeting:
Protein sorting: protein can go through secretory pathway or cytosolic, or to peroxisome or mitochondria.
There is no well-defined ER signalling sequence. Contains lots of leucine, other hydrophobic residues.
Experimental proof of signal sequence:
Give cells in media without methionine or cysteine a pulse with radioactive 35S met/cys, grow 30-40 min, homogenize, treat with detergent and trypsonize or monitor protein.
Without microsomes in a cell-free system, protein is made, but signal sequence stays on. Add microsome afterward, protein deos not enter.
Add microsome at beginning- protein translocates as it is translated. This is cotranslational translocation. Know this.
1. signal sequence- SRP is a signal recognition particle.
3. translocon is a ligand-gated channel
Details on individual slides.
SRP is ribonuclear protein complex.
Converting type 2 to type 3: (page 7) If you mutate + to – on type II, protein flips as translocation is taking place, lacking interaction with translocon.
What starts in ER lumen winds up on outside cell.
Lecture 2:
Protein modification, folding, and quality control in the ER
As protein is folded, prcesses go through quality control. This is not on test.
Just study slides.
X on page 2 slide 1 is any amino acid except proline.
Look up J.Clin. Invest. volume 108 p. 1579-1582.
PDI is used as standard to monitor ER during organelle isolation.
Page 5 slide 3 hemaglutinin
Lecture 3: Nuclear Transport
Warning: This file got corrupted, so I reconstructed this part from memory and the text. Lodish pages 509-517. Reference in our little booklet is incorrect.
2:
You will see this slide several times. Today we are focusing on the red box.
3. This is the nuclear membrane structure. Notice the nuclear pores. Notice what needs energy to get through and what does not. An RNP is a ribonuclear protein complex, a complex of mRNA and associated proteins. See slides 15-17 for more detailed structure of nuclear pores.
5. In eukaryotes, DNA transcription and translation occur in the nucleus, but protein synthesis occurs in the cytoplasm.
Slide 10:
Note the roles of nuclear pores.
11: We will be tested over the nuclear localization signal. It is usually internal. Also know the experiment described on slides 12-14. Pyruvate kinase normally stays in cytoplasm, but can be localized to nucleus with NLS.
Slide 19: Digitonin acts like a detergent to break up cell membranes. If you permeabilize the cell membrane so the cytoplasm leaks out, but the nucleus and nuclear pore complexes are intact, and put in a protein with an NLS signal, it will not localize to the nucleus. With cell lysate, it will. The NLS is not sufficient on its own- there must be cytosolic components involved in localizing proteins to the nucleus.
Slide 23: Remember these proteins.
Slide 24: He will ask about the RAN cycle. Remember where it is associated with GDP and where with GTP. Slide 25 illustrates.
Slide 29: Fuse a Hela (human) cell with a xenopus cell in the presence of PEG. Their nuclei come close together. RNP-C stays localized in the Hela nuclei (no NES). RNP A1 is exported to the xenopus nucleus.
32: mRNA exporter is a protein that interacts with hydrophobic FG (rich in phenylalanine and glycine) repeats in the FG nucleoporins.
Slides 33-36: Balbiani ring mRNA codes for a glue that helps the larva stick to a leaf. It uncoils from the associated proteins as it enters the cytoplasm and is transcribed.
Slide 39: HIV Rev protein enables unspliced mRNA to be exported. Rev binds a rev response element on the mRNA and contains an NES that interacts with exportin 1 and Ran-GTP.
Slide 40 is a summary of the important things to remember.