Question

4) A cell line is transfected with a plasmid encoding either a secreted glycoprotein, X, or a variant of X that contains the amino acids KDEL at its C terminus, both of them under control of an inducible promoter, KDEL motifs are normally found on proteins located in the lumen of the ER. At 30 minutes after induction, cycloheximide, an inhibitor of protein synthesis, is added to the culture medium. At 30, 45, and 60 minutes after induction (that is, 0,15, or 30 min in cycloheximide), the cells are lysed, rough microsomes are isolated and solubilized, and X or X- KDEL is immunoprecipitated from the solubilized material and run on SOS-PAGE gels. The following gels are obtained (9). 1 2 3 4 5 6 Minutes After Induction: 30 45 60 Protein X 30 45 60 Protein X-KDEL 1. The most likely reason why there is less material in lanes 2 and 3 than there is in lane 1 is that protein X? a. is digested in cycloheximide b. is less stable than protein X-KDEL c. is translocated into the lumen of the endoplasmic reticulum within 15 minutes of its synthesis d. exits the endoplasmic reticulum within 15 minutes of its synthesis e is incorporated into COPI vesicles within 15 minutes of its synthesis State Rational for your answer?

Answer 1

1. Is incorporated into COP1 vesicles within 15 minutes it's synthesis (protein X is a secretory protein. After synthesis in on the ER, it is supposed to be transferred from ER into COP1 coated secretory vesicles and eventually secreted from the cell.)

2. X-KDEL becomes modified in the ER after protein synthesis is completed (in lane 5 and 6, the previous X-KDEL protein is seen in a higher level, meaning it's weight has got increased, which can only happen if it's chemically modified and some group is added to it. Oligomerization would have produced higher molecular weight bands.)

3. The total amount of protein observed in lanes 2 and 3 and 5 and 6 would increase (cycloheximide blocks further protein synthesis, so whatever protein is coming up in bands, is the protein already produced in ER and fresh protein is not being produced anymore. So if cycloheximide is not added, fresh proteins would be allowed to synthesize, so protein intensity would have been more in the later time points.)