Subcellular localization and functional expression of the glycerol uptake protein 1 (GUP1) of Saccharomyces cerevisiae tagged with green fluorescent protein

GFP (green fluorescent protein) from Aequorea victoria was used as an in vivo reporter protein when fused to the N- and C-termini of the glycerol uptake protein 1 (Gup1p) of Saccharomyces cerevisiae. The subcellular localization and functional expression of biologically active Gup1-GFP chimacras was monitored by confocal laser scanning and electron microscopy, thus supplying the first study of GUP1 dynamics in live yeast cells. The Gup1p tagged with GFP is a functional glycerol transporter localized at the plasma membrane and endoplasmic reticulum levels of induced cells. The factors involved in proper localization and turn-over of Gup1p were revealed by expression of the Gup1p-GFP fusion protein in a set of strains bearing mutations in specific steps of the secretory and endocytic pathways. The chimaerical protein was targeted to the plasma membrane through a Sec6-dependent process; on treatment with glucose, it was endocytosed through END3 and targeted for degradation in the vacuole. Gup1p belongs to the list of yeast proteins rapidly down-regulated by changing the carbon source in the culture medium, in agreement with the concept that post-translational modifications triggered by glucose affect proteins of peripheral functions. The immunoelectron microscopy assays of cells expressing either Gup1-GFP or GFP-Gup1 fusions suggested the Gup1p membrane topology: the N-terminus lies in the periplasmic space, whereas its C-terminal tail has an intracellular location. An extra cytosolic location of the N-terminal tail is not generally predicted or determined in yeast membrane transporters.