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

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 cells (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:

Please summarize the results shown above and explain why there is a difference in the data between WT HeLa cells and PaBo cells in two paragraphs.

VSV-infected WT HeLa cells (membranes + cytoplasm) VSV-infected PaBo cells (membranes+cytoplasm) Mannose Defect in Extract glycoprotein VSV-G Protein ? Mannose Man GcNAc Immunoprecipitation results: Unprocessed VSV-G-1 Processed VSV-G 2 46 8 10 12 14 16 Time post S Met washout (min) 2 4 6 8 10 12 14 16 Time post S Met washout (min) 35 35

Explanation / Answer

In WT cells, the protein gets processed over time as it undergoes processing in the ER or Golgi apparatus. So, intially only unprocessed protein is present but at six minutes, the processing of VSV-G proteins has started (since thin band of processed protein is seen implying less amount of processed protein). After ten minutes, the processing of all the proteins is complete since no unprocessed band is observed. In case of PaBo cells, due to the absence of enzyme, mannose trimming does not occur, because of which only unprocessed proteins are present within the cell throughout the entire period. As, a result only one band is observed at all times.

The difference in the data of WT HeLa cells and PaBo cells is because there is no mannose trimming step to generate processed VSV-G protein. In case of VSV infected WT cells, the VSV-G protein undergoes processing in which one of the steps is removal of mannose residues. Mannose removal allows the proteins to move faster as compared to the VSV-G proteins which are yet to be processed. As a result two set of bands are observed in the case when both processed and unprocessed proteins are present in the cells. On the other hand, PaBo cells cannot generate processed protein because of the absence of enzyme which helps in mannose trimming. As, a result only one band is observed in second case.

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