please answer A! thanks! In preliminary studics (shown bclow) you compare the pr
ID: 255760 • Letter: P
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please answer A! thanks!
In preliminary studics (shown bclow) you compare the processing of VSVG in WT cells (left) to that in Palbo cells (right. In pulse-chasc expcriments, you pulsc labecls with S methionine for 5 hours followed by washout of the labeled amino acid (lhe "chase"). You then lyse the cells and mix the membrane Iraction (which includes the ER and Golgi membranes) with cytoplasm. At dilTerent time points over a course of 16 minutes you solubilize the membranc and immunoprecipitatc VSVG with a specific antibody and run the precipitates on a gel followed by cxposur usng Xray film. The results are shown: VSV-infected WT HeLa cells VSV-infected PaBo cells . (membranes +cytoplasm) (membranes cytoplasm) Defect in Protein Immunoprecipitation results:- Unprocessed Processed VSV-G 6 8 10 12 14 16 35 2 46 8 10 12 14 16 35 Time post S Met washout (min) Time post''s Met washout (min) . A. Plcase briefly summarize the results shown above and explain why there is a differencc in the data between WT HeLa cells and PuBo cells. 5 ptExplanation / Answer
Ans:
PaBo cells are essentially mutant HeLa cells, which have a mutation in the enzyme mannosidase which carries out trimming of the mannose. When a protein is co-translationally translocated into the ER, it sometimes receives attachments of eight mannose residues, which are added to a core carbohydrate unit consisting of GlcNAc (N-acetylglucosamine). The mannose residues are trimmed down from a unit of eight to a unit of five either in the ER prior to transport to the Golgi, or in the cis-Golgi after transport. The removal of three mannose residues can be detected using gel electrophoresis because proteins modified in this way will migrate faster on a gel than untrimmed proteins.
VSV upon infection, causes cells to translate a GTPase called VSV-G protein. VSVG is translated at the ER membrane, imported into the lumen, and undergoes the mannose processing steps described above.
Now in WT HeLa cells, the protein gets processed either in Golgi or ER, which requires some time. Hence, after 6 minutes we are getting a thin band of processed VSV-G. After 8 and 10minutes we can see a thicker processed VSV-G band and a thinner unprocessed VSV-G band. After 10 minutes we can find no band of unprocessed VSVG, as all the protein have been trimmed by this time.
PaBo cells lack the mannosidase enzyme which is required for the processing of the protein, so over time we only find a single band of the unprocessed VSVG.
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