ABSTRACT
Aspergillus fumigatus possesses a branched mitochondrial electron transport chain, with both cyanide-sensitive and -insensitive oxygen-consumption activities. Mitochondrial reactive oxygen species mediate signaling for alternative oxidase (AOX) expression. A 1173 bp-long Afaox gene encoding a 40 kDa protein has been cloned and identified. Recombinant constructs containing the Afaox ORF were transformed into Escherichia coli and Saccharomyces cerevisiae for heterologous expression. In A. fumigatus, AOX activity and mRNA expression were both induced with menadione or paraquat, suggesting an important role of AOX under oxidative stress. Therefore, positive transformants showed a cyanide-resistant and salicylhydroxamic acid-sensitive respiration, whereas in control cells the oxygen uptake was completely inhibited after KCN addition.
Subject(s)
Aspergillus fumigatus/genetics , Fungal Proteins/genetics , Oxidative Stress , Oxidoreductases/genetics , Amino Acid Sequence , Aspergillus fumigatus/drug effects , Aspergillus fumigatus/metabolism , Blotting, Western , Cloning, Molecular , Cyanides/pharmacology , Enzyme Activation/drug effects , Escherichia coli/drug effects , Escherichia coli/genetics , Escherichia coli/metabolism , Fungal Proteins/metabolism , Gene Expression Regulation, Fungal , Mitochondria/enzymology , Mitochondria/metabolism , Mitochondrial Proteins , Molecular Sequence Data , Oxidoreductases/metabolism , Oxygen Consumption/drug effects , Paraquat/pharmacology , Plant Proteins , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reactive Oxygen Species/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Salicylamides/pharmacology , Sequence Homology, Amino Acid , Vitamin K 3/pharmacologyABSTRACT
The understanding of the controlling factors of calcium homeostasis in Aspergillus fumigatus is very poor, although this ion is involved in several important events of these particular cells. We have cloned, identified and expressed for functional complementation a PMR1-like Ca(2+)-ATPase gene from A. fumigatus. The Afpmr1 gene encodes a protein of 1061 deduced amino acids, containing all the conserved subdomains found in other P-type ATPases: the phosphatase region, phosphorylation site, FITC labelling site, ATP binding domain; E(386), N871, D875 amino acid residues for calcium ion interaction and Q880, a residue that alters ion selectivity in PMR1. The expressed AfPMR1 in S. cerevisiae K616 strain functionally complemented the deficient growth in EGTA (5-20 mM)- and MnCl2 (4 mM)-containing medium. These results demonstrate the first evidence of a Ca(2+)-ATPase in A. fumigatus and strongly suggest a role for this enzyme in calcium and manganese homeostasis.
