Utilization of green fluorescent protein as a marker for studying the expression and turnover of the monocarboxylate permease Jen1p of Saccharomyces cerevisiae

• Published in: Biochemical journal 2002-05, Vol.363 (3), p.737-744
• Main Authors: PAIVA, Sandra, KRUCKEBERG, Arthur L., CASAL, Margarida
• Format: Article
• Language: English
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj3630737

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 123s−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 Δdoa4 mutant strain showed that ubiquitination is associated with the turnover of the permease.