A novel strategy for constructing N-terminal chromosomal fusions to green fluorescent protein in the yeast Saccharomyces cerevisiae

A novel rapid polymerase chain reaction (PCR)-based technique for N-termina l attachment of green fluorescent protein (GFP) to a yeast protein is descr ibed. Genomic integration of a PCR-generated loxPkanMX4loxP-yEGFP fusion ca ssette immediately upstream of the open reading frame (ORF) allows for sele ction of G418 resistant transformants carrying GFP fused N-terminally to th e protein of interest. In a subsequent step, the loxPkanMX4loxP selection m arker that is inserted between the tagged ORF and the endogenous promoter i s excised upon site-specific recombination between the loxP sites by Cre re combinase, leaving behind in the promoter one loxP site, immediately upstre am of the GFP start codon, The essential protein Ydl193wp of unknown functi on and the oleate-inducible fatty acid activation protein, encoded by FAA2, were N-terminally tagged using the novel technique. Both experiments yield ed viable haploid strains with growth phenotypes indistinguishable from the wild type strain. The subcellular localization pattern for the chromosomal ly expressed GFP-Ydl193wp to the endoplasmic reticulum and lipid particles was identical to the pattern observed for a plasmid-borne GFP construct exp ressed under control of the MET25(P) promoter, albeit at a lower level and with a more homogeneous distribution among the cell population. GFP-FAA2 wa s inducible by oleate, as is the mild type gene, demonstrating that specifi c expression patterns are not grossly affected by the promoter manipulation . In agreement with previous reports, GFP-Faa2p was found to localize to pe roxisomes. (C) 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.