Cloning and characterization of the plasma membrane H(+)-ATPase from Candida albicans.
The Candida albicans PMA1 gene was isolated from a genomic library by using a hybridization probe obtained from the PMA1 gene of Saccharomyces cerevisiae. The gene was localized to chromosome III of the Candida genome. An open reading frame of 2,685 nucleotides predicts an amino acid sequence of 895 amino acids that is 83% homologous at both the DNA and protein levels to its S. cerevisiae equivalent. A polyadenylated mRNA transcript of about 4,000 nucleotides contains a highly folded AU-rich leader of 242 nucleotides. The structure of the gene, codon bias, and levels of approximately 100-kDa H(+)-ATPase protein recovered in plasma membranes indicate a highly expressed gene. The plasma membrane ATPase was purified to about 90% homogeneity and appeared to be blocked at the amino terminus. Three hydrophobic membrane sector tryptic fragments from the partially digested ATPase provided internal sequence information for over 50 amino acids, which agrees with the sequence predicted by the cloned gene. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the C. albicans enzyme is about 3 kDa smaller than its Saccharomyces counterpart and was consistent with a predicted Mr of 97,398. Antibodies to the S. cerevisiae whole ATPase or its carboxyl terminus bound to the C. albicans enzyme but with lower avidity. Kinetic analysis showed that the Candida and Saccharomyces ATPases respond to glucose activation-starvation in nonidentical fashions. The amino-terminal domain of the C. albicans ATPase is marked by a net deletion of 23 amino acids in comparison with the S. cerevisiae ATPase. These differences maintain net charge, occur in nonconserved regions of fungal ATPases, and are sufficient to account for the observed difference in electrophoretic mobility between the two yeast ATPases.
Monk
B C
BC
Public Health Research Institute, New York, New York 10016.
Kurtz
M B
MB
Marrinan
J A
JA
Perlin
D S
DS
D000595:Amino Acid Sequence; D001483:Base Sequence; D005075:Biological Evolution; D015152:Blotting, Northern; D002176:Candida albicans / Q000201:enzymology* / Q000235:genetics; D002462:Cell Membrane / Q000201:enzymology; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004271:DNA, Fungal; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D006180:Proton-Translocating ATPases / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D012331:RNA, Fungal / Q000302:isolation & purification; D015183:Restriction Mapping; D016415:Sequence Alignment; D014357:Trypsin
D004271:DNA, Fungal; D012331:RNA, Fungal; D014357:Trypsin; D006180:Proton-Translocating ATPases