Identification and characterisation of eroA and ervA, encoding two putative thiol oxidases from Aspergillus niger.
The oxidative folding of proteins in the secretory pathway involves the formation and isomerisation of disulphide bonds and is catalysed by foldases in the lumen of the endoplasmic reticulum (ER). The transfer of reducing equivalents, from disulphide bond formation, to oxygen involves the participation of thiol oxidases. Here, we describe the identification and functional characterisation of the eroA and ervA genes from Aspergillus niger, encoding functional orthologues of S. cerevisiae ERO1 and ERV2, respectively. The eroA gene encodes a product of 600 amino acids, EroA, and the ervA gene encodes a product of 215 amino acids, ErvA, both of which share common motifs and features with their S. cerevisiae orthologues. In contrast to Ero1p in S. cerevisiae, A. niger EroA appears to be retained in the ER lumen by a C-terminal retention motif. Real-time PCR analysis indicated that eroA is transcriptionally up-regulated in response to ER stress, whereas ervA is slightly down-regulated in response to DTT stress yet up-regulated in response to expression of a heterologous protein. Gene disruption studies indicated that, unlike ervA, eroA is essential for viability. When expressed in the thermosensitive S. cerevisiae ero1-1 strain, both eroA and ervA were able to complement the temperature and DTT sensitive phenotype, although a truncated eroA, missing the putative HEEL ER-retention signal was unable to complement as well as the full-length eroA gene.
Harvey
Anna R
AR
School of Biology, University of Nottingham, Nottingham, NG7 2RD, UK.
Ward
Michael
M
Archer
David B
DB
D020121:5' Untranslated Regions / Q000235:genetics; D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D017124:Conserved Sequence; D004721:Endoplasmic Reticulum / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal / Q000235:genetics; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013312:Stress, Physiological / Q000235:genetics; D014158:Transcription, Genetic; D050862:Oxidoreductases Acting on Sulfur Group Donors
D020121:5' Untranslated Regions; D005656:Fungal Proteins; D010088:Oxidoreductases; D012333:RNA, Messenger; D050862:Oxidoreductases Acting on Sulfur Group Donors