Question

1. Using cells mutant for the gene Sec61, which encodes the translocon, researchers found that VSVG-GFP accumulates in the cytoplasm. Why is that? Do you expect the protein to be efficiently translated? Why? 2. Where do you expect to find VSVG-GFP if you over-express a dominant negative form of the small GTPase Sar1? 3. You are studying vesicular transport using the VSV-G system. Brefeldin A, inhibits the ARF GEF involved in COPI vesicle formation. a. What would happen with VSVG if you treat cells with Brefeldin A some time before placing the cells at the permissive temperature? b. How would you detect VSV-G to verify your predictions?

Answer 1

Question 1

Answer: Sec61 is an essential sub-unit of heterotrimeric protein Sec61 complex or translocon. It form a channel in the membrane of endoplasmic reticulum (ER) and play a major role in the translocation of both membrane and secretory proteins synthesized on the ribosomes into ER. It also mediates the retrograde transport of misfolded proteins out of the ER to the cytoplasm for degradation. Cells with mutation in Sec61 gene does not produce functional Sec61 protein complex or translocon and hence inhibits protein translocation.

Vesicular Stomatitus virus G protein (VSVG) is a virus glycoprotein. VSVG-GFP (green fluorscent protein tagged to VSVG) used to study protein transport between ER- golgi. Cell expressing VSVG-GFP are synthesized on the ribosomes in the cytoplasm and get tranlocated to ER for further processing with the help of Sec61complex. As the reseacher used cells mutant for Sec61 to express VSVG-GFP protein, translocation of VSVG-GFP is inhibited due to mutant sec61 protein complex and hence, VSVG-GFP gets accumulated in the cytoplasm.

The VSVG-GFP protein is efficently translated becuase protein synthesis occurs on the ribosomes present in the cytoplasm and Sec61 has no role in translation becuase its function is to translocate proteins to ER.