Sequencing and heterologous expression in Saccharomyces cerevisiae of a Cryptococcus neoformans cDNA encoding a plasma membrane H(+)-ATPase.
A cDNA containing an open reading frame encoding a putative plasma membrane H(+)-ATPase in the human pathogenic basidiomycetous yeast Cryptococcus neoformans was cloned and sequenced by means of PCR and cDNA library hybridization. The cloned cDNA is 3475 bp in length, containing a 2994 bp open reading frame encoding a polypeptide of 997 amino acids. As in the case of another basidiomycetous fungus (Uromyces fabae), the deduced amino acid sequence of CnPMA1 was found to be more homologous to those of P-type H(+)-ATPases from higher plants than to those from ascomycetous fungi. In order to prove the sequenced cDNA corresponds to a H(+)-ATPase, it was expressed in Saccharomyces cerevisiae and found to functionally replace its own H(+)-ATPase. Kinetic studies of CnPMA1 compared to ScPMA1 show differences in V(max) values and H(+)-pumping in reconstituted vesicles. The pH optimum and K(m) values are similar in both enzymes.
Gorgojo
B
B
Unidad de Micología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain.
Portillo
F
F
Martínez-Suárez
J V
JV
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D003455:Cryptococcus neoformans / Q000201:enzymology / Q000235:genetics*; D018076:DNA, Complementary / Q000737:chemistry / Q000378:metabolism; D005656:Fungal Proteins / Q000096:biosynthesis*; D015870:Gene Expression; D005800:Genes, Fungal; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D017494:Proton Pumps; D006180:Proton-Translocating ATPases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment
D018076:DNA, Complementary; D005656:Fungal Proteins; C434364:PMA2 protein, S cerevisiae; D017494:Proton Pumps; D029701:Saccharomyces cerevisiae Proteins; C092477:PMA1 protein, S cerevisiae; D006180:Proton-Translocating ATPases