Molecular evaluation of the plasma membrane proton pump from Aspergillus fumigatus.
The gene encoding the plasma membrane proton pump (H+ -ATPase) of Aspergillus fumigatus, PMA1, was characterized from A. fumigatus strain NIH 5233 and clinical isolate H11-20. An open reading frame of 3,109 nucleotides with two introns near the N terminus predicts a protein consisting of 989 amino acids with a molecular mass of approximately 108 kDa. The predicted A. fumigatus enzyme is 89 and 51% identical to H+ - ATPases of Aspergillus nidulans and Saccharomyces cerevisiae, respectively. The A. fumigatus PMA1 is a typical member of the P-type ATPase family that contains 10 predicted transmembrane segments and conserved sequence motifs TGES, CSDKTGT, MLTGD, and GDGVN within the catalytic region. The enzyme represents 2% of the total plasma membrane protein, and it is characteristically inhibited by orthovanadate, with a 50% inhibitory concentration of approximately 1.8 microM. H+ -ATPases from Aspergillus spp. contain a highly acidic insertion region of 60 amino acids between transmembrane segments 2 and 3, which was confirmed for the membrane-assembled enzyme with a peptide-derived antibody. An increasing A. fumigatus PMA1 copy number confers enhanced growth in low-pH medium, consistent with its role as a proton pump. These results provide support for the development of the A. fumigatus H+ -ATPase as a potential drug discovery target.
Burghoorn
Henriette P
HP
Public Health Research Institute, New York, New York 10016, USA.
Soteropoulos
Patricia
P
Paderu
Padmaja
P
Kashiwazaki
Ryota
R
Perlin
David S
DS
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001232:Aspergillus fumigatus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D017494:Proton Pumps / Q000235:genetics / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000378:metabolism; D012331:RNA, Fungal / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism
D004271:DNA, Fungal; D017494:Proton Pumps; D012331:RNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C092477:PMA1 protein, S cerevisiae; D006180:Proton-Translocating ATPases