In a forward genetic screen to investigate the heat stress response in Arabidopsis, a team of researchers identified an uncharacterized gene 'x' that shows some sequence homology to alpha-subunit of heterotrimeric G-protein. Since a typical alpha-subunit of heterotrimeric G-protein localizes at the membrane in a eukaryotic cell, researchers sought to validate whether the protein coded by gene 'x' localizes to membrane in tobacco protoplasts. To achieve this, they cloned the gene in fusion with GFP at its N-terminus, under the control of the CaMV promoter; however, upon expression of this GFP-gene ‘x’ fusion construct, they did not observe any membrane localization of GFP-signal in tobacco protoplasts. Based on this, they made a few assumptions:

A. N-terminally tagging of protein ‘X’ with GFP may block membrane localization of protein X

B. Tagging of protein ‘X’ with GFP may alter the conformation of protein X because of its bigger size

C. Tobacco protoplasts are a heterologous system for the expression of gene ‘x,’ and thus, the protein X does not localize to the membrane

D. Gene 'x' is not getting transcribed because of the wrong promoter choice

Which one of the following options represents all correct assumptions?

Correct option is D

Explanation-

Analyzing Each Statement-
A. N-terminal tagging with GFP may block membrane localization of protein X - Correct.  Many membrane proteins rely on their N-terminus for membrane targeting signals. GFP tagging at N-terminus can mask these signals.

B. GFP may alter the conformation due to its large size -  Correct.  GFP (~27 kDa) can sterically hinder proper folding or localization signals, altering the normal trafficking of protein X.
C. Tobacco protoplast is a heterologous system  - Correct.  Expression of Arabidopsis gene X in tobacco may not perfectly mimic its native expression/localization due to differences in cellular machinery or targeting sequences.
D. Wrong promoter -  Not likely.  They used CaMV 35S promoter, which is commonly used and strong for expression in tobacco, so transcriptional failure is unlikely here.

Correct Assumptions:
A, B, and C

Correct option - d

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