When a protein is co-translationally translocated into the ER it sometimes recei
ID: 254572 • Letter: W
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
Q.1 the difference between HeLa and PaBo is due absence of mannosidase in PaBo cells which gives only band( right immunoprecipitation result) for untrimmed mannose residue as described in the question the mannosidase is present in the HeLa cells so it will gives two bands as shown in left immunoprecipitation result.
Q.2. or A. when his labmate accidantly added HeLa cells membrane fractions to PaBo which makes fusion of mannose containg ER and golgi compartments of HeLa cells fraction to PaBo cells fraction which makes availability of mannosidase in mannosidase mutant PaBo cells so now there will we two bands one untrimmed and one trimmed.
Q.3. Or B. when we added unhydrolysable GTP
Related Questions
drjack9650@gmail.com
Navigate
Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.