Functional expression of plant plasma membrane H+-ATPase in yeast endoplasmic reticulum

• Published in: The Journal of biological chemistry Vol. 267; no. 17; pp. 12341 - 12349
• Main Authors: Villalba, J.M. (Facultade de Ciencias, Cordoba, Spain), Palmgren, M.G, Berberian, G.E, Ferguson, C, Serrano, R
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 15.06.1992
• Subjects: Adenosine Triphosphate - metabolism; ADN RECOMBINADO; ADN RECOMBINE; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Cell Membrane - enzymology; CRUCIFERAE; Electrophoresis, Polyacrylamide Gel; Endoplasmic Reticulum - metabolism; Enzymes and enzyme inhibitors; ESTRUCTURA CELULAR; Fluorescent Antibody Technique; Fundamental and applied biological sciences. Psychology; HIDROLASAS; HYDROLASE; Hydrolases; Hydrolysis; Kinetics; LEVADURA; LEVURE; Microscopy, Electron; Phenotype; Phosphorylation; Plants - enzymology; Plants - genetics; PLASMIDE; PLASMIDIOS; Plasmids; Proteolipids; Proton-Translocating ATPases - genetics; Proton-Translocating ATPases - metabolism; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; RNA, Messenger - metabolism; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae - ultrastructure; STRUCTURE CELLULAIRE; Yeast; Purification; Enzyme; F0F1-ATPase; Gene expression; Fungi; Arabidopsis thaliana; Enzymatic activity; Cruciferae; Dicotyledones; Angiospermae; Ascomycetes; Spermatophyta; Kinetics; Localization; Saccharomyces cerevisiae; Thallophyta
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(19)49845-1

Recombinant plant plasma membrane H+-ATPase has been produced in a yeast expression system comprising a multicopy plasmid and the strong promoter of the yeast PMA1 gene. Western blotting with a specific monoclonal antibody showed that the plant ATPase is one of the major membrane proteins made by the transformed cells, accounting for about 1% of total yeast protein. The plant ATPase synthesized in yeast is fully active. It hydrolyzes ATP, pumps protons, and the reaction cycle involves a phosphorylated intermediate. Phosphorylation is possible from both ATP and Pi. Unlike the situation in plants, however, most of the plant ATPase is not expressed in the yeast plasma membrane. Rather, the enzyme appears to remain trapped at a very early stage of secretory pathway: insertion into the endoplasmic reticulum. This organelle was observed to proliferate in the form of stacked membranes surrounding the yeast nucleus in order to accommodate the large amount of plant ATPase produced. In this location, the plant ATPase can be purified with high yield (70 mg from 1 kg of yeast) from membranes devoid of endogenous yeast plasma membrane H+-ATPase. This convenient expression system could be useful for other eukaryotic membrane proteins and ATPases