Site-directed mutagenesis of the cysteine residues in the Neurospora crassa plasma membrane H(+)-ATPase.
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.
Mahanty
S K
SK
Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill 27599, USA.
Scarborough
G A
GA
D002462:Cell Membrane / Q000201:enzymology; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009492:Neurospora crassa / Q000201:enzymology*; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D006180:Proton-Translocating ATPases; D003545:Cysteine