Genetic and molecular characterization of a gene encoding a wide specificity purine permease of Aspergillus nidulans reveals a novel family of transporters conserved in prokaryotes and eukaryotes.
In Aspergillus nidulans, loss-of-function mutations in the uapA and azgA genes, encoding the major uric acid-xanthine and hypoxanthine-adenine-guanine permeases, respectively, result in impaired utilization of these purines as sole nitrogen sources. The residual growth of the mutant strains is due to the activity of a broad specificity purine permease. We have identified uapC, the gene coding for this third permease through the isolation of both gain-of-function and loss-of-function mutations. Uptake studies with wild-type and mutant strains confirmed the genetic analysis and showed that the UapC protein contributes 30% and 8-10% to uric acid and hypoxanthine transport rates, respectively. The uapC gene was cloned, its expression studied, its sequence and transcript map established, and the sequence of its putative product analyzed. uapC message accumulation is: (i) weakly induced by 2-thiouric acid; (ii) repressed by ammonium; (iii) dependent on functional uaY and areA regulatory gene products (mediating uric acid induction and nitrogen metabolite repression, respectively); (iv) increased by uapC gain-of-function mutations which specifically, but partially, suppress a leucine to valine mutation in the zinc finger of the protein coded by the areA gene. The putative uapC gene product is a highly hydrophobic protein of 580 amino acids (M(r) = 61,251) including 12-14 putative transmembrane segments. The UapC protein is highly similar (58% identity) to the UapA permease and significantly similar (23-34% identity) to a number of bacterial transporters. Comparisons of the sequences and hydropathy profiles of members of this novel family of transporters yield insights into their structure, functionally important residues, and possible evolutionary relationships.
Diallinas
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Institut de Génétique et Microbiologie, Unité Associé au Centre National de la Recherche Scientifique 1354, Université de Paris-Sud, Centre d'Orsay, France.
Gorfinkiel
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L
Arst
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HN
Cecchetto
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Scazzocchio
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D000595:Amino Acid Sequence; D001233:Aspergillus nidulans / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004271:DNA, Fungal; D005800:Genes, Fungal; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D033702:Nucleobase Transport Proteins; D011687:Purines / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D004271:DNA, Fungal; D026901:Membrane Transport Proteins; D033702:Nucleobase Transport Proteins; D011687:Purines; D012333:RNA, Messenger; C022483:purine permease