HIGH-YIELD EXPRESSION OF THE NEUROSPORA-CRASSA PLASMA-MEMBRANE H-ATPASE IN SACCHAROMYCES-CEREVISIAE()

A simple system for high yield expression of the neurospora plasma mem brane H+-ATPase is described Two neurospora H+-ATPase cDNAs differing only in a few bases preceding the coding region were cloned into a hig h copy number yeast expression vector under the control of the constit utive promoter of the yeast plasma membrane H+-ATPase, and the resulti ng plasmids were used to transform Saccharomyces cerevisiae strain RS- 72, which requires a plasmid-borne functional plasma membrane H+-ATPas e for growth in glucose medium (Villalba, J. M., Palmgren, M. G., Berb erian, G. E., Ferguson, C., and Serrano, R. (1992) J. Biol. Chem. 267, 12341-12349. Both plasmids supported growth of the cells, indicating that the neurospora ATPase is expressed in functional form in yeast. W estern blots of membranes from the transformants confirmed that the ne urospora ATPase is expressed in the yeast cells, with production in th e range of several percent of the yeast membrane protein, Importantly, when the expressed, recombinant neurospora ATPase molecules are solub ilized from the membranes with lysolecithin and subjected to glycerol gradient centrifugation, they migrate to a position indistinguishable from that of the native ATPase and display a comparable specific ATPas e activity, indicating that the great majority of the recombinant neur ospora ATPase molecules produced in yeast fold in a natural manner, Th is expression system thus appears to be ideal for site-directed mutage nesis studies of the neurospora ATPase molecule.