Abstract
A high-yield yeast expression system for site-directed mutagenesis of the Neurospora crassa plasma membrane H(+)-ATPase has recently been reported (Mahanty, S. K., Rao, U. S., Nicholas, R. A., and Scarborough, G. A. (1994) J. Biol. Chem. 269, 17705-17712). Using this system, each of the eight cysteine residues in the ATPase was changed to a serine or an alanine residue, producing strains C148S and C148A, C376S and C376A, C409S and C409A, C472S and C472A, C532S and C532A, C545S and C545A, C840S and C840A, and C869S and C869A, respectively. With the exception of C376S and C532S, all of the mutant ATPases are able to support the growth of yeast cells to different extents, indicating that they are functional. The C376S and C532S enzymes appear to be non-functional. After solubilization of the functional mutant ATPase molecules from isolated membranes with lysolecithin, all behaved similar to the native enzyme when subjected to glycerol density gradient centrifugation, indicating that they fold in a natural manner. The kinetic properties of these mutant enzymes were also similar to the native ATPase with the exception of C409A, which has a substantially higher Km. These results clearly indicate that none of the eight cysteine residues in the H(+)-ATPase molecule are essential for ATPase activity, but that Cys376, Cys409, and Cys532 may be in or near important sites. They also demonstrate that the previously described disulfide bridge between Cys148 and Cys840 or Cys869 plays no obvious role in the structure or function of this membrane transport enzyme.
Highlights
The plasma membrane Hϩ-ATPase of Neurospora crassa is an electrogenic [1] proton pump [2], that belongs to the P-type family of ion translocating ATPases that form an aspartyl phosphate intermediate in their reaction cycle [3, 4]
As described previously [18], in the yeast expression system used for these studies, both the wild type yeast Hϩ-ATPase and the plasmid-encoded Neurospora Hϩ-ATPase are produced when the cells are grown in medium containing galactose as the carbon source
The results indicated the presence of substantial amounts of the mutant ATPases in the membranes of all of the mutants except C376S, C532S, and D378A
Summary
Site-directed Mutagenesis and Plasmid Constructions—For mutagenesis, we began with the plasmid pSKMHA2 [18], which contains the full-length N. crassa plasma membrane Hϩ-ATPase cDNA. Isolation of Membranes and Purification of the Expressed Hϩ-ATPase Molecules—Membranes from different yeast transformants were prepared essentially as described [18], except that cell homogenization was carried out in a medium bead beater chamber and all of the solutions contained 2 g/ml chymostatin. For densitometric analysis of the amounts of mutant Neurospora ATPases present in the glycerol gradient fractions, aliquots of the pooled peak activity fractions (numbers 9, 10, 11, and 12 from the top) were first subjected to SDS-PAGE as described above, and the gels were silver-stained and photographed. The sources of all other reagents were as described [18]
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