Question 1

Does the targeting signal go at the amino or carboxyl terminus?

Accepted Answer

It depends on the destination. Mitochondrial matrix presequences and secretory signal peptides sit at the amino terminus and are usually cleaved after import, so nothing may be appended before them. Peroxisomal targeting most often uses a carboxyl-terminal tripeptide that must remain the final residues. Placing a tag at the wrong end silently abolishes targeting.

Question 2

What evidence is needed beyond fluorescence colocalisation?

Accepted Answer

Imaging shows proximity at diffraction-limited resolution, which cannot distinguish surface association from genuine import. Protease protection on isolated organelles, with and without detergent, demonstrates that the protein is enclosed. Subcellular fractionation with marker enzymes for each compartment gives quantitative distribution. Two independent methods make the claim considerably stronger than either alone.

Question 3

How should colocalisation be quantified?

Accepted Answer

Overlap coefficients should be calculated per cell across a substantial population, with the number of cells and independent experiments reported. Randomised or rotated image controls establish the coefficient expected by chance at the given density. A single representative image, however clear, cannot support a quantitative statement about targeting efficiency.

Question 4

Why does expression level affect localisation results?

Accepted Answer

Import machinery has finite capacity, so strong overexpression floods the pathway and leaves protein in the cytosol that would otherwise be imported. Comparing several expression levels, or using an inducible system with a titration, distinguishes a poor targeting signal from saturation. Reporting expression level alongside localisation is expected practice.

Question 5

Do targeting signals transfer between organisms?

Accepted Answer

Only partly. Core recognition motifs are often conserved, but flanking context, membrane insertion requirements and receptor specificity differ between yeast, plant and mammalian systems, and plastid targeting has no mammalian equivalent. A signal working in one host should be validated in the intended host rather than assumed to carry over.

Question 6

Why might a correctly localised enzyme still show no activity?

Accepted Answer

Each compartment has its own pH, redox potential, ion composition and cofactor supply, and folding conditions differ too. An enzyme requiring a cytosolic cofactor may find none in the organelle lumen, and disulphide-dependent folding fails in reducing environments. Activity should be measured in situ rather than inferred from correct arrival.

Biotechnology

Biotechnology

Research Frontiers in Protein Compartmentalization and Localization