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

J.M Villalba,M.G Palmgren,G.E Berberián,C Ferguson,R Serrano

doi:10.1016/s0021-9258(19)49845-1

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.

Highlights

RecombinantplantplasmamembraneH+-ATPase the fungal plasma membrane H+-ATPases, the animal has been produced in a yeast expression system com- Na+,K+, H+,K+- andCa2+-ATPases,and the bacterial K+
At the early stages of the project, we were concerned with the fact that yeast cells contain aplasma membrane H+-ATPasewith properties similar to the plantenzyme (Serrano, 1989).to avoid the co-existence of both ATPases in the same cell, we introduced the plant ATPase expression plasmid into the S. cereuisiae strain RS-72 (Cid et al, 1987)
This is a genetically engineered strain with the constitutive promoter of the yeast plasma membrane H+-ATPase gene replaced by a galactosedependent promoter

Summary

THEJOURNAOLF BIOLOGICACLHEMISTRY

Q 1992 by The American Society for Biochemistry and MolecularBiologv, Inc. VOl. 267, No., Issue of June 15, pp. 12341-12349,1992 Printed in U.S.A. The plant ATPase synthesized in yeast is fully active It hydrolyzes ATP,pumps protons, and with other enzymes of the the structure of the plant biological function. Inaddition, afull undermembranes devoid of endogenous yeast plasma mem- standing of the structure-function relationship of the plant brane H+-ATPase. This convenient expression systemATPase is unlikely to be achieved without knowledge of its could be useful for other eukaryotic membrane pro- three-dimensional structure. We reporton the synthesis of plant plasma membrane H+-ATPasein S. cereuisiae.In thisheterologous system, the enzyme is not delivered to theplasma membrane but is retained at theyeast ER.’ In this location, the plant ATPase is fully active and can be purified with high yields and without contamination of endogenous yeast ATPases.

MATERIALS ANDMETHODS

RESULTS

Yeast ATPase

Glycerol gradient

DISCUSSION

Methods