Question

need help with 4i, ii, and iii please.

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 SDS-PAGE gels. The following gels are obtained (9). I. 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? II. The most likely explanation for the additional bands seen in lanes 5 and 6 is that a. the signal sequence is not removed from some of the X-KDEL molecules b. X-KDEL oligomerizes after protein synthesis is completed C. X-KDEL becomes modified in the endoplasmic reticulum after protein synthesis is completed d. some of the X-KDEL molecules are modified in the Golgi and then returned to the endoplasmic reticulum e. the quality control mechanism in the endoplasmic reticulum recognizes X-KDEL as being aberrant and has targeted it for degradation State Rational for your answer? III. If cycloheximide had been omitted from the experiment, then a. no protein band would be observed in lane 2 b. bands at higher molecular weight equivalent to those observed in lanes 5 and 6 would be observed in lanes 2 and 3 c. only the lower-molecular-weight bands would be observed in lanes 5 and 6 d. the total amount of protein observed in each gel lane would increase e. the total amount of protein observed in lanes 2 and 3 and 5 and 6 would increase State Rational for your answer?

Answer 1

(i) a.

There is less material in lane 2 & 3, i.e. 15 & 30 minutes respectively because the protein is gradually digested. At 0 min, digestion is going to start hence protein is seen in gel. At 15 min, some protein is digested, but not whole, hence little protein is seen in gel. At 30 min no protein is seen as all the protein is digested.

(ii) c.

Additional bands are seen in lane 5 & 6 as X-KDEL modifies in ER afterprotein sysnthesis is completed. The protein which is not modified is digested gradually at 15 min & 30 min same as lane 2 & 3.

(iii) e.

Toatal amount of protein present in lane 2, 3, 5 & 6 would increase if cycloheximide was absent from the experiment as no inhibitor to protein sysnthesis would remain.