The study of the endocytic trafficking of the yeast monocarboxylate transporter Jen1 from Saccharomyces cerevisiae

Abstract

The monocarboxylate transporter Jen1 of the yeast Saccharomyces cerevisiae has proven to be an excellent model system for genetically dissecting mechanisms that regulate trafficking of a eukaryotic plasma membrane protein, according to physiological constraints. Glucose triggers a rapid endocytosis of Jen1 being the main signal ubiquitylation through Rsp5p ubiquitin ligase (Paiva et al., 2002; Paiva et al., 2009) and the arrestin-like adaptor Rod1 (Becuwe et al., 2012). In an attempt to identify domains that are important for the subcellular localization, activity and turnover of Jen1, domain swap experiments were carried out. It was used a novel strategy, based on the gap repair technique to construct 9 chimeric proteins by the replacement of either the N- or the C-terminal half of Jen1 by the N- or the C-terminal half (or both terminals) of the Gap1 general aminoacid transporter, Fur4 uracil transporter and Hxt6 hexose transporter. Gap1 and Fur4 are regulated by ammonium and excess of substrate, respectively (Hein et al., 1995) in contrast to Hxt6 that are regulated by glucose just as Jen1. Each gene was also C-terminally fused with the ORF of the green fluorescent protein (GFP). Here, we will present the successful genetic strategy and the characterization of two chimeras at the protein and subcellular levels.