Subcellular localisation of an active green fluorescent protein-tagged GUP1 glycerol transporter in Saccharomyces cerevisiae.

The GUP1 (Glycerol Uptake Protein 1) gene plays an important role in Saccharomyces cerevisiae utilising glycerol as a carbon source and providing glycerol-mediated recovery from salt stress to deficient strains (Holst et al., 2000). Green fluorescent protein gene (GFP) from Aequora victoria was employed as an in vivo reporter protein when fused to the GUP1 cistron. The GUP1 gene C- and N-terminus was separately tagged with GFP. The two obtained chimerical genes were ligated to the pYES2 vector and the recombinant plasmid used to transform S. cerevisiae W303-1A (wild type) and BHY66 (isogenic to W303-1A but gpd1-, gut1-, gup1- and unable to produce, to metabolise and to import glycerol) strains. The transformation of BHY66 with both GUP1-GFP and GFP-GUP1 chimerical proteins restored wild-type growth under osmotic stress, indicating that both chimerical proteins acted as functional transporters. Localisation at subcellular levels of GUP1-GFP and GFP-GUP1 chimeras was carried out by confocal laser scanning microscopy. In W303-1A cells expressing GFP fused to either 5' or 3' end of GUP1 gene, green fluorescence was localised in two specific compartments, endoplasmic reticulum and plasma membrane. The GUP1 gene was then genetically fused at its C-terminal terminus with the GFP gene. Functional expression, quantification and cellular localisation of GUP1 protein will be discussed.