Molecular evaluation of the plasma membrane proton pump from Aspergillus fumigatus

Abstract

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.

FIG. 1.

Nucleotide and predicted amino acid sequences of the A. fumigatus PMA1 gene. Untranslated regions and intron sequences are shown in lowercase letters. The deduced amino acid sequence of the PMA1 gene is shown by the one-letter amino acid designations below the nucleotide sequence. Amino acid residues are numbered beginning with the first methionine, and the translation termination codon is denoted by an asterisk. The site of phosphorylation, D⁴²⁷KTGT, is double underlined. Ten putative hydrophobic transmembrane segments are shaded, consensus intron sequences are underlined, and the three extra insertion regions are boxed. Ser-417 near the site of phosphorylation has been boxed with a double line.

FIG. 1.

Nucleotide and predicted amino acid sequences of the A. fumigatus PMA1 gene. Untranslated regions and intron sequences are shown in lowercase letters. The deduced amino acid sequence of the PMA1 gene is shown by the one-letter amino acid designations below the nucleotide sequence. Amino acid residues are numbered beginning with the first methionine, and the translation termination codon is denoted by an asterisk. The site of phosphorylation, D⁴²⁷KTGT, is double underlined. Ten putative hydrophobic transmembrane segments are shaded, consensus intron sequences are underlined, and the three extra insertion regions are boxed. Ser-417 near the site of phosphorylation has been boxed with a double line.

FIG. 2.

Alignment of conserved regions of P-type ATPases. Amino acid sequence alignment of A. fumigatus PMA1 (AfPMA1) relative to other fungal PMA genes from A. nidulans (AnPMA1), S. cerevisiae (ScPMA1), and C. neoformans (CnPMA1). The highly conserved sequence TGES and transmembrane segments TM3, TM4, TM9, and TM10 are shaded. The Aspergillus amino acid insert regions are boxed.

FIG. 3.

Hydropathy profiles for the plasma membrane H⁺-ATPases from A. fumigatus, A. nidulans, and S. cerevisiae. Highly conserved regions of the H⁺-ATPases from S. cerevisiae (A), A. nidulans (B), and A. fumigatus (C) are noted, including sequence motifs involved with phosphorylation (CSD⁴²⁷KTGT), nucleotide binding (MLTGD and GDGVN), and dephosphorylation (TGES). Transmembrane segments TM1 to TM10 are designated, and the Aspergillus insertion regions are shaded.

FIG. 4.

Effect of gene dosage on pH-dependent growth. (A) The A. fumigatus PMA1 gene was amplified by PCR from wild-type (wt) cells and transformants containing marked copies of PMA1 with a unique EcoRI restriction enzyme site. The fragments were either untreated or digested with EcoRI. The amount of native PMA1 relative to the amount of marked enzyme was used to determine whether cells contained a single copy (1×), double copy (2×), or triple copies (3×) of A. fumigatus PMA1. (B) Approximately 1,000 spores from either wild-type or polyploid PMA1 cells, as described above for panel A, were used to inoculate microdilution medium (YG) containing 50 mM acetate adjusted from pH 3.5 to 7.0. Each assay was performed in duplicate, and growth was determined after 48 h.

FIG. 5.

Electrophoretic and Western blot analyses of the A. fumigatus plasma membrane H⁺-ATPase. (A) Plasma membranes from A. fumigatus and other fungi were purified as described in Materials and Methods and were resolved by SDS-gel electrophoresis. Molecular mass markers designated by the lines show the region containing PMA1p from S. cerevisiae (lane 1; 100 kDa), C. albicans (lane 2; 97 kDa), C. neoformans (lane 3; 108 kDa), and A. fumigatus (lane 4; 108 kDa). The arrow in lane 4 shows the exact position of PMA1p from A. fumigatus (first main band). (B) Western blotting was performed with a peptide-derived polyclonal antibody specific for a unique amino acid stretch of the predicted A. fumigatus PMA1 protein. The position of the PMA1 band is indicated by the arrow.