Question

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 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 cell:s (left) to that in PaBo cells (right). In pulse-chase experiments, you pulse label live cells with 35S 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 VSV-infected PaBo cells (membranes +cytoplasm) (membranes cytoplasm) Defect in Extrast glyooprote in VSV-G Protein ManglGkNA Immunoprecipitation results: Unprocessed VSV-G Processed VSV-G 2 46 8 1012 14 16 2 468 10 12 14 16 35 35 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.

Answer 1

A) The pulse labeling experiments were conducted with labeled S-35 methionine to analyze the post -translational modification of the VSVG protein in the endoplasmic reticulum and golgi apparatus of two cell lines one was the normal cell line HeLa and other was mutant mannosidase cell line Pabo. In one experiment, the mixture of VSVG infected cell sample of ER and golgi was analyzed by western blotting for the unprocessed and processed protein by pulse labeling assays. In the other experiment the immunoprecipitation was done in the samples containing the samples of golgi and ER with wild type cell line instead of the experiment in Pabo cell line which yielded the same results. The processing of the protein as observed by the pulse labeling studies indicated the start of processing after 6 minutes of infection with the VSV virus.

B) The addition of a non hydrolyzable GTP analog which did not convert GTP to GDP indicated that the processing of the VSVG protein in the sample could not takes place as indicated by the presence of the western blotting bands in the S-35 labeled methionine.