- 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?
(1) A and B only (2) B and D only
(3) C and D only (4) A, B and CThe correct option is (1) A and B only.
Understanding the experiment
-
Gene x from Arabidopsis shows homology to a Gα subunit, which typically localizes to the plasma membrane via N‑terminal lipid modifications and interaction motifs.
-
Researchers expressed GFP fused to the N‑terminus of protein X (GFP–X) under the strong CaMV 35S promoter in tobacco protoplasts.
-
The fusion protein showed no membrane localization of GFP signal.
Evaluating each assumption
A. “N-terminal tagging with GFP may block membrane localization of protein X.” – Correct
-
Many Gα proteins require an unobstructed N‑terminus for myristoylation/palmitoylation or for binding to βγ subunits and the membrane.
-
Fusing GFP at the N‑terminus can mask these signals or sterically hinder lipidation, preventing membrane association.
-
So A is a reasonable assumption.
B. “Tagging protein X with GFP may alter its conformation due to its bigger size.” – Correct
-
GFP is ~27 kDa and forms a rigid β‑barrel; when fused to another protein, it can:
-
Distort folding,
-
Interfere with interaction domains,
-
Restrict conformational changes needed for proper localization.
-
-
Thus B is also a valid concern.
C. “Tobacco protoplasts are a heterologous system, so protein X does not localize to the membrane.” – Not fully justified here
-
Protein X is from Arabidopsis, another plant; tobacco is also a plant, and many Arabidopsis proteins (including membrane proteins) localize correctly when expressed in tobacco cells.
-
Unless specific interacting partners or lipidation enzymes were missing (unlikely across higher plants), merely being a heterologous plant system does not generally abolish membrane targeting.
-
This is a weaker, less well‑supported assumption.
D. “Gene x is not transcribed because of the wrong promoter choice.” – Unlikely
-
The construct uses the CaMV 35S promoter, a standard strong constitutive promoter that drives robust expression in a wide range of dicot species, including tobacco and Arabidopsis.
-
GFP fluorescence is seen (even if cytosolic), which already indicates that the fusion gene is transcribed and translated.
-
So transcription failure due to promoter choice is not a reasonable explanation.
Why option (1) is correct
-
The lack of membrane localization is best explained by tag-related issues:
-
A (N‑terminal GFP masking targeting signals).
-
B (conformational/structural interference from large GFP).
-
-
Assumptions C and D are not well supported given that tobacco is a suitable plant expression system and CaMV 35S is an appropriate promoter.
Therefore, the set of all correct assumptions is A and B only (option 1).
-
