The plasma membrane H(+)-ATPase from the biotrophic rust fungus Uromyces fabae: molecular characterization of the gene (PMA1) and functional expression of the enzyme in yeast.
To study the molecular basis of biotrophic nutrient uptake by plant parasitic rust fungi, the gene (Uf-PMA1) encoding the plasma membrane H(+)-ATPase from Uromyces fabae was isolated. Uf-PMA1 exists probably as a single gene. However, two nearly identical sequences were identified; the similarity apparently is due to two Uf-PMA1 alleles in the dikaryotic hyphae. Multiple Uf-PMA1 transcripts were observed during early rust development, and reduced amounts of a single Uf-PMA1 mRNA were observed in haustoria and rust-infected leaves. This is in contrast to elevated enzyme activity in haustoria compared to germinated spores (C. Struck, M. Hahn, and K. Mendgen. Fungal Genet. Biol. 20:30-35, 1996). Unexpectedly, the PMA1-encoded rust protein is more similar to H(+)-ATPases from plants (55% identity) than from ascomycetous fungi (36% identity). When the rust PMA1 cDNA was expressed in Saccharomyces cerevisiae, both the wild-type enzyme and a mutant derivative (delta 76) deleted for the 76 C-terminal amino acids were able to support growth of a yeast strain lacking its own H(+)-ATPases. Compared to the wild-type, the delta 76 mutant enzyme displayed increased affinity to ATP, a higher vanadate sensitivity, and a more alkaline pH optimum. These results indicate that the C-terminal region of the rust enzyme exhibits auto-regulatory properties.
Struck
C
C
Fakultät für Biologie, Universität Konstanz, Germany.
Siebels
C
C
Rommel
O
O
Wernitz
M
M
Hahn
M
M
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D001487:Basidiomycota / Q000201:enzymology*; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D018076:DNA, Complementary; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics
D018076:DNA, Complementary; D006180:Proton-Translocating ATPases