Pinpointing intracellular trafficking determinants in the Jen1 yeast lactate transporter by domain swap

Abstract

The intracellular trafficking of plasma membrane proteins, such as receptors and transporters, in eukaryotic cells is a highly regulatedprocess. Changing environment conditions (nutrients, substrates, hormones) can trigger endocytosis of unwanted transporters. The monocarboxylate transporter Jen1p of the yeast Saccharomyces cerevisiae has proven to be an excellent model system forgenetically dissecting mechanisms that regulate trafficking of a eukaryotic PM protein, according to physiological constraints. Ourgroup has demonstrated that glucose acts as a signal to induce endocytic down-regulation of Jen1 within minutes, a processdependent both on phosphorylation and ubiquitylation (Paiva et al, 2002; Paiva et al, 2009).In an attempt to identify domains that are important for the subcellular localization, activity and turnover of Jen1p, domain swapexperiments were carried out. The hybridtransporter genes also carry a C-terminal fusion with the ORF of the green fluorescent protein (GFP), which enabled the in vivomicroscopic investigation of the trafficking, membrane localization and turnover of Jen1p protein. This strategy is ideal to identify whether the Jen1 terminals include molecular determinants necessary and sufficient for glucoseelicitedendocytosis and sorting to endosomes. Studies regarding the rates of endocytosis and/or direct sorting of the chimerictransporters, under various physiological conditions, will be presented. Uptake assays using radiolabeled lactic acid were also employed to measure the activity of the chimeric transporters, under specific conditions.