Recapitulation slides and corrected nuclear transport conclusions slide

1. Recapitulation slidesandcorrected nuclear transport conclusions slide

2. Recapitulate: Proteins destined for ER are translated on membrane bound polysomes. Proteins are targeted to ER by a signal peptide. SRP binds to the signal peptide, stops translation and sends the polysome to ER. Polysome docks on ER by binding to SRP receptors. Translocation is through the sec61 translocase.

3. Recapitulate: Proteins that enter the ER encounter: Signal peptidase, Chaperones, Protein disulfide isomerase (PDI), Prolyl isomerase (PPI) and Glycosylation enzymes Protein complexes require additional chaperones (calnexin, calreticulin). Unfolded proteins are removed from ER and degraded by proteasomes (viruses have subverted this pathway) Mitochondrial proteins often have an amphipathic leader as signal for targeting. Upon import, this peptide is cleaved by MPP and the protein is folded by hsp10/hsp60. The mitochondrial import machinery consists of Toms and Tims. There are multiple destinations within mitochondria.

4. Recapitulate: Peroxisomal import utilizes C-terminal SKL or N-terminal leader sequence. ER retention utilizes C-terminal KDEL(HDEL) for lumenal proteins or KKXX for membrane proteins. ERD2 is the KDEL receptor and this resides in the cis Golgi Rothman developed cis-medial Golgi complementation assay Successful labeling requires donor membrane, acceptor membrane, cytsol and ATP. Cytosol contains an NEM sensitive factor (NSF)

5. Recapitulate Rothman’s cis-medial Golgi complementation assay is blocked by GTPgS and by NEM The block in vesicle uncoating caused by GTPgS made it possible to purify coated vesicles This led to the discovery of coat proteins (COPs) and a GTPase named ARF Schekman’s lab isolated yeast mutants with secretion defects. Early acting sec-mutants affect vesicle budding from the ER or they affect vesicle fusion to Golgi. The vesicle budding mutants helped discover a second class of coat proteins, termed COPII, which also has a small GTPase. Rab proteins are small GTPases that help direct vesicle traffic. The NEM sensitive factor, NSF, was purified by adding back column fractions: it proved to be a large ATPase. NSF binds to Golgi membranes but only in the presence of SNAPs.

6. Recapitulate: NSF, SNAPs and SNAREs for the 20 S particle, frozen by ATPgS myc-tagged NSF, along with ATPgS, was used to purify SNAREs tSNAREs and vSNAREs form a strong coiled coil that drives fusion NSF dissociates this coiled coil after fusion Receptor mediated endocytosis helps concentrate external ligands Clathrin triskelia form a basket that helps vesicle budding AP1 at the TGN and AP2 at the plasma membrane serve as adaptors that collect receptors within the coated pit.

7. Import carriers like Kap-b bind to their cargo and RanGDP in the cytoplasm. Upon import into the nucleus, a Ran GEF (RCC1) triggers release of GDP and dissociation of the import complex. Export carriers in the nucleus bind to their cargo and RanGTP in the nucleus. Upon export to the cytoplasm and subesquent contact with RanGAP, Ran hydrolyzes GTP, leading to dissociation of the export complex. Import and export carriers travel in opposite directions along the nucleoporins.