title: Utilization of green fluorescent protein as a marker for studying the expression and turnover of the monocarboxylate permease Jen1p of Saccharomyces cerevisiae.
abstract: Green fluorescent protein (GFP) from Aequorea victoria was used as an in vivo reporter protein when fused to the C-terminus of the Jen1 lactate permease of Saccharomyces cerevisiae. The Jen1 protein tagged with GFP is a functional lactate transporter with a cellular abundance of 1670 molecules/cell, and a catalytic-centre activity of 123 s(-1). It is expressed and tagged to the plasma membrane under induction conditions. The factors involved in proper localization and turnover of Jen1p were revealed by expression of the Jen1p-GFP fusion protein in a set of strains bearing mutations in specific steps of the secretory and endocytic pathways. The chimaeric protein Jen1p-GFP is targeted to the plasma membrane via a Sec6-dependent process; upon treatment with glucose, it is endocytosed via END3 and targeted for degradation in the vacuole. Experiments performed in a Deltadoa4 mutant strain showed that ubiquitination is associated with the turnover of the permease.
authors.affiliation: Centro de Ciências do Ambiente, Departamento de Biologia, Universidade do Minho, 4710-057 Braga Codex, Portugal.
authors.forename: Sandra
authors.initials: S
authors.lastname: Paiva
authors.forename: Arthur L
authors.initials: AL
authors.lastname: Kruckeberg
authors.forename: Margarida
authors.initials: M
authors.lastname: Casal
mesh_terms: D015153:Blotting, Western; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic; D005947:Glucose / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006863:Hydrogen-Ion Concentration; D019344:Lactic Acid / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism*; D027501:Monocarboxylic Acid Transporters / Q000096:biosynthesis / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D013050:Spectrometry, Fluorescence; D027981:Symporters / Q000096:biosynthesis / Q000235:genetics*
chemical_list: C118697:JEN1 protein, S cerevisiae; D008164:Luminescent Proteins; D027501:Monocarboxylic Acid Transporters; D029701:Saccharomyces cerevisiae Proteins; D027981:Symporters; D049452:Green Fluorescent Proteins; D019344:Lactic Acid; D005947:Glucose