I have isolated a novel protein designated Xer15. After textensive computer anal

Question

I have isolated a novel protein designated Xer15. After textensive computer analysis, I am convinced that the Xer15 protein contains a signal sequence that targets the protein to the LYSOSOME via the endomembrane system. My colleague disagrees and is convinced that Xer15 has nothing to do with the endomembrane system and is instead targeted to the MITOCHONDRIA.

1. How would you determine the presence and nature of a protein-localized targeting signal?

2. What would one look like?

3. How would you PROVE that the sequence you identify is actually a localization signal?

Explanation / Answer

1. The protein-localized targeting signal is located at the N-terminal of the proteins that determine the location of the protein in the cell. The proteins destined to remain in the cytosol are translated by the free ribosomes in the cytosol. Whereas, the protein destined either to cross or to be incorporated into the membranes of cellular vesicles or the outer plasma membrane.

2. The protein-localized targeting signals are 15-30 amino acid long peptide containing at least one positive residue and followed by a hydrophobic peptide of 6-12 amino acids at the N terminal of the proteins.

3. To prove that the sequence we identified is actually a localization signal by:

a), Ascertaining whether the peptide satisfies the characters of a signal peptide mentioned in answer 2. A number of online tools are available to predict a signal sequence, such as TargetP, PredSIGNAL, and PredLIPO to name a few.

b). The signal peptides are clipped off once they reached the target destination. The absence of the signal peptide in the mature peptide may be indicative of it as a signal peptide. [This step is not a fool-proof step unless care is taken to avoid the misidentification of the propeptide sequence as the signal peptide. The former is also at N terminal and is clipped off in an ACTIVE protein. ]

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