Functional expression and cellular localization of a green fluorescent protein-tagged proline transporter in Aspergillus nidulans

The PrnB protein is a highly specific proline transporter that belongs to a n amino acid transporter family conserved in both prokaryotes and eukaryote s. In this work, we detected and analyzed the cellular localization of PrnB in vivo by means of green fluorescent protein (GFP) fusions. Several pmB-g fp gene fusions, driven by prnB native promoter sequences, were constructed and targeted to the genomic locus of a prnB null mutant. Chimeric proteins containing GFP fused to the C terminus of PrnB through a linker of two, fo ur, or eight amino acids, with low potential to form secondary structure el ements, were shown to be functional in vivo. A two-linker fusion results in partial complementation at both 25 and 37 degreesC. A four-linker fusion a ffords almost full complementation at 25 degreesC but partial complementati on at 37 degreesC, whereas the eight-linker fusion results in partial compl ementation at both temperatures but in no GFP fluorescence. These results s how that the number of linker amino acids is critical for the correct expre ssion and/or translocation of PrnB-GFP fused proteins to the plasma membran e and for the fluorescence of the GFP. The expression of the four-linker Pr nB-GFP transporter was detected and analyzed in vivo by both conventional f luorescence and confocal laser microscopy. This chimeric protein is localiz ed in the plasma membrane, secondarily in large vacuoles found in the swoll en conidial end of the germlings, and in other small intracellular structur es observed as fluorescent granules. A strong correlation between known pat terns of PrnB expression and intensity of PrnB-GFP fluorescence was observe d. This work also demonstrates that the GFP fusion technology is a unique t ool with which to study the expression and cellular localization of low-abu ndance transmembrane transporters expressed from their native promoters. (C ) 2001 Academic Press.