Effect of the orientation of the GFP-tag relative to the ORF. (A) For 340 proteins of 567 tested, both orientations resulted in the correct localization of the fusion proteins (same). Another 219 proteins localized differently in the two orientations. Of these, 120 localizations were correctly localizing with the ORF-GFP construct, and 99 fusion proteins localized correctly in the GFP-ORF orientation. Eight expression constructs did not show any detectable expression (none). (B) An example of a mitochondrial protein (upper image). The fusion protein mislocalized (lower image) when the signal peptide at its N terminus was blocked by the GFP-tag. The cytoplasmic and nuclear staining of the GFP-ORF fusion protein is also the default localization of GFP alone. The bar indicates 10 μm.
