Figure 4. To test whether these amino acids are important for ER retention/retri
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Figure 4. To test whether these amino acids are important for ER retention/retrieval in the context of the untagged, full-length acids identified in Figure 2 were mutated in the DNA encoding the full-length US3. The mutants were then expressed in c (these are modified HeLa cells) and the sub-cellular localization of each mutant was US3 protein, the amino a modified cervical carcinoma cell called HtTA examined using co-immunofluorescence. PDI US3 Merge GM130 Merge GM130 Merge GM130 Merge mutations were of homolopous US3, as was done for 4123/GFP. Colocalization of the US3 mutants with macker was analyzed by confocal laser microscopy. HtTa cells stably expressing wild-type US3 orA A or A mutants were fixed, mcabilized, and double immunostained with anti-US,3 for cach mutant (middle columa) and with anti-PDI or anti-GM130 anribody for FIG. 4. Essential role of three amino acid residucs for ER retention in the coetext of homologous US3. Alanine directly introduced into the region endogenous marker proteins (left column). The right column shows merged images 10. (1pt) What is the purpose of looking at PDI (protein disulfide isomerase) and GM130 (Golgi matrix protein) expression in this experiment? a. These proteins are directly involved in CMV infection b. These proteins are involved in trafficking of US3 c. These proteins process US3 d. These proteins are markers for different cellular compartments. e. These proteins are markers for CMV infection.Explanation / Answer
Q10 Answer: d (These proteins are markers for different cellular compartments)
Q11 Answer b It acts as a tether for vesicular transport to golgi
Q12 Where do mutants localize compare to wildtype normal US3 protein?
Answer: Wild type normal US3 protein is localized in ER beacuse it colacilzes with ER marker protein PDI. However the other three mutants colocalize with cis golgi marker protein GM130 so they are localized in Golgi.
Q13 What is Brefeldin A?
Answer: Brefeldin A inhibits protein transport from the endoplasmic reticulum to the Golgi apparatus indirectly by preventing association of COP-I coat to the Golgi membrane.
Q14 What would happen to subcellular localization of each of the constructs in fig. 4 if cells are treated with Brefeldin A (include WT US3 in your answer)
Answer: In secretory pathway of protein targetting proteins are transported from ER to golgi and then to lysosome or plasma membrane. Brefeldin A inhibits protein transport from ER to golgi. so mutants (when treated with Brefeldin) which are localized in golgi will now be localized in ER. localization of wild type US3 protein is normally in ER only so Brefeldin will produce any effect on its localization
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