title: Functional expression of plant plasma membrane H(+)-ATPase in yeast endoplasmic reticulum.
abstract: 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.
authors.affiliation: European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: J M
authors.initials: JM
authors.lastname: Villalba
authors.forename: M G
authors.initials: MG
authors.lastname: Palmgren
authors.forename: G E
authors.initials: GE
authors.lastname: Berberián
authors.forename: C
authors.initials: C
authors.lastname: Ferguson
authors.forename: R
authors.initials: R
authors.lastname: Serrano
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006868:Hydrolysis; D007700:Kinetics; D008854:Microscopy, Electron; D010641:Phenotype; D010766:Phosphorylation; D010944:Plants / Q000201:enzymology* / Q000235:genetics; D010957:Plasmids; D011510:Proteolipids; D006180:Proton-Translocating ATPases / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D011510:Proteolipids; D012333:RNA, Messenger; D011994:Recombinant Proteins; C020485:proteoliposomes; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases