When a protein is co-translationally translocated into the ER it sometimes recci

Question

When a protein is co-translationally translocated into the ER it sometimes reccives attachments of eight mannose residues, which are added to a core carbohydrate unit consisting of GleNAc (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 trans 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. As a dedicated undergraduate student with a brain that is literally larger in volume than a normal sized brain, you decide to take advantage of this carbohydrate processing step as a means to develop a cell-free assay to determine where the mannose processing takes place (in the ER or in the Golgi). Luckily you also have access to a mutant HeLa cell line (called PaBo cells) with a mutation in the enzyme that carries out the trimming (called mannosidase). In addition, you have access to a virus called VSV, which, 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. In preliminary studies (shown below) you compare the processing of VSVG in WT cells (left) to that in PaBo cells (right). In pulse-chase experiments, you pulse label live cells with S methionine for 5 hours followed by washout of the labeled amino acid (the "chase"). You then lyse the cells and mix the membrane fraction (which includes the ER and Golgi membranes) with cytoplasm. At different time points over a course of 16 minutes you solubilize the membrane and immunoprecipitate VSVG with a specific antibody and run the precipitates on a gel followed by exposure using Xray film. The results are shown: VSV-infected WT HeLa cells (membranescytoplasm) VSV-infected PaBo cells (membranescytoplasm) Immunoprecipitation results: Unprocessed Processed VSV-G 2 4 6 8 10 12 14 16 2 4 6 8 10 12 14 16 Time post S Met washout (min) Time post S Met washout (min) Please summarize the results shown above and explain why there is a difference in the data between WT HeLa cells and PaBo cells.

Explanation / Answer

Ans. Proteins are glycosylated in endoplasmic reticlum and trimming of excess manose units from proteins is done in ER or golgi appartus.Here we are provided with two types of cell lines mutant HeLa cell line called as PaBo cell line and wild type cell line.Now it is mentioned that PaBo cells are having mutant enzyme mannosidase in it.So there will be no trimming of protein VSVG provided to us because the enzyme will be unable to process this protein.This can be seen in the results of gel electrophoresis.To study the process of trimming pulse and chase experiment is done in which at pulse state the radiolabelled methionine is incorporated for 5 hours so that the we can see the what all changes occur during the time period.The two results are displayed.The PaBo cell line cannot process the protein so no trimming is observed.In case of WT cell line we can see that as the time increases during the 16 min period the whole protein being processed is visible because of S35 methionine because WT cell line has intact cell enviroment and intact enzyme mannosidase which trimms the protein.

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