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 membrane H(+)-ATPase is described. Two neurospora H(+)-ATPase cDNAs differing only in a few bases preceding the coding region were cloned into a high copy number yeast expression vector under the control of the constitutive promoter of the yeast plasma membrane H(+)-ATPase, and the resulting plasmids were used to transform Saccharomyces cerevisiae strain RS-72, which requires a plasmid-borne functional plasma membrane H(+)-ATPase for growth in glucose medium (Villalba, J. M., Palmgren, M. G., Berberian, 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. Western blots of membranes from the transformants confirmed that the neurospora ATPase is expressed in the yeast cells, with production in the range of several percent of the yeast membrane protein. Importantly, when the expressed, recombinant neurospora ATPase molecules are solubilized 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 ATPase activity, indicating that the great majority of the recombinant neurospora ATPase molecules produced in yeast fold in a natural manner. This expression system thus appears to be ideal for site-directed mutagenesis studies of the neurospora ATPase molecule.
Mahanty
S K
SK
Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill 27599.
Rao
U S
US
Nicholas
R A
RA
Scarborough
G A
GA
D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D004271:DNA, Fungal; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000201:enzymology*; D010957:Plasmids; D006180:Proton-Translocating ATPases / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D004271:DNA, Fungal; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases