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 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.

Explanation / Answer

1.

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.

A.

The result observed is due to the presence of active mannosidase enzyme in the membranes of WT cells. when we mixed VSV infected membranes of PaBo cells and membranes of uninfected cells, the mannosidase present in WT membranes start processing VSVG protein resulting in the processed VSVG protein in the blot.

B.

Mannosidase requires GTP for its processing activity. Even though membranes were mixed just like above experiment, mannosidase cannot process the VSVG protein. This results in unprocessed VSVG protein, thus single high molecular VSVG protein band.

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