Utilization of green fluorescent protein as a marker for studying the expressio...

• Author(s): Paiva, Sandra ; Kruckeberg, Arthur L. ; Casal, Margarida
• Date: 2002
• Persistent ID: http://hdl.handle.net/1822/2881
• Origin: RepositóriUM - Universidade do Minho
• Subject(s): Yeast; Transport; Secretion; Ubiquitination; Endocytosis
• Document Type: Article
• Language: English

Description

Green Fluorescent protein (GFP) from Aequorea ictoria 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 catalyticcentre 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 Ddoa4 mutant strain showed that ubiquitination is associated with the turnover of the permease.