An investigator identified a nuclear localization signal (NLS; Pro-Lys-Lys-Lys-Arg-Lys) at the C-terminus of the protein X (50 kDa). To analyse the localization of protein X, the investigator fused protein X with GFP at the C-terminus. The fusion protein was detected in the cytosol. When the nuclear localization signal was fused with GFP at the N-terminus, the NLS-tagged GFP extensively localized in the nucleus. Based on this observation, the investigator made a few hypotheses:

A. The basic amino-acid stretch in the protein X-GFP chimeric construct is masked by the GFP sequence and thus not capable of directing entry of protein X-GFP into the nucleus.
B. The X-protein in the full-length X-GFP chimeric protein is post-translationally modified that impacts its import into the nucleus.
C. Fusion with GFP makes the protein X too bulky to enter the nucleus through the nuclear pore complex.
D. The GFP is post-translationally modified that impacts the import of protein X-GFP into the nucleus.

Which one of the following options gives the combination of all possible hypotheses that best explain Protein-X’s trafficking mechanisms?

Correct option is C

Given Situation:

• Protein X has a nuclear localization signal (NLS) at its C-terminus.

• The fusion protein (Protein X-GFP) is detected in the cytosol when GFP is attached at the C-terminus.

• When the NLS-tagged GFP is at the N-terminus, the fusion protein localizes to the nucleus.

Reanalyzing the Hypotheses:

1. Statement A: "The basic amino-acid stretch in the protein X-GFP chimeric construct is masked by the GFP sequence and thus not capable of directing entry of protein X-GFP into the nucleus."

   • Correct: This is a reasonable explanation. When GFP is fused at the C-terminus, it may mask the NLS (nuclear localization signal), preventing it from being recognized by the nuclear import machinery, thus inhibiting the protein from entering the nucleus. The NLS plays a crucial role in targeting the protein to the nucleus, and if this signal is masked, the protein will not localize to the nucleus.

2. Statement B: "The X-protein in the full-length X-GFP chimeric protein is post-translationally modified that impacts its import into the nucleus."

   • Correct: This statement also makes sense, especially if we consider that the fusion of GFP at the C-terminus could alter the protein's overall structure and function. In some cases, the fusion tag (like GFP) can introduce changes in protein conformation that might interfere with its normal activity, such as the NLS recognition. The loss of function of the NLS due to GFP fusion could be considered a post-translational modification that impacts the nuclear import of the fusion protein.

3. Statement C: "Fusion with GFP makes the protein X too bulky to enter the nucleus through the nuclear pore complex."

   • Incorrect: While size could theoretically be an issue for larger proteins, GFP fusion generally does not make the protein too bulky to be transported through the nuclear pore complex. The more likely issue here is masking of the NLS rather than the size of the protein being a limiting factor for nuclear import.

4. Statement D: "The GFP is post-translationally modified that impacts the import of protein X-GFP into the nucleus."

   • Incorrect: There is no indication that GFP itself is post-translationally modified in this context to affect the nuclear import of the fusion protein. The problem lies in the masking of the NLS by the C-terminal GFP fusion, not modification of GFP itself.

Conclusion:

• Statements A and B together offer the best explanation:

  • A explains that GFP fusion at the C-terminus likely masks the NLS, preventing nuclear import.

  • B suggests that the fusion with GFP might introduce structural changes that hinder the function of the NLS, thus preventing nuclear import.

Correct Answer: Option 3: A and B