Isolation and analysis of functional homologues of the secretion-related SAR1 gene of Saccharomyces cerevisiae from Aspergillus niger and Trichoderma reesei.
The Aspergillus niger and Trichoderma reesei genes encoding the functional homologues of the small GTP-binding protein SAR1p, which is involved in the secretion pathway in Saccharomyces cerevisiae, have been cloned and characterised. The A. niger gene (sarA) contains five introns, whereas the T. reesei gene (sar1) has only four. In both cases the first intron is at the same position as the single S. cerevisiae SAR1 intron. The encoded proteins show 70-80% identity to the SAR1 protein. Complementation of S. cerevisiae sar1 and sec12 mutants by expression vectors carrying the A. niger sarA and T. reesei sar1 cDNA clones confirmed that the cloned genes are functional homologues of the S. cerevisiae SAR1 gene. Three mutant alleles of the A. niger sarA gene (D29G, E109K, D29G/E109K), generated by site-directed mutagenesis, revealed a thermosensitive dominant-negative phenotype in the presence of the wild-type sarA allele. This result contrasts with the situation in S. cerevisiae, where similar mutations have a thermosensitive phenotype. Taken together, our results indicate that the sarA gene is involved in an essential function in A. niger.
Veldhuisen
G
G
Department of Molecular Genetics and Gene Technology, TNO Nutrition and Food Research Institute, Zeist, The Netherlands.
Saloheimo
M
M
Fiers
M A
MA
Punt
P J
PJ
Contreras
R
R
Penttilä
M
M
van den Hondel
C A
CA
D000483:Alleles; D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000235:genetics*; D001483:Base Sequence; D004271:DNA, Fungal; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics*; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D020559:Monomeric GTP-Binding Proteins; D009154:Mutation; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014242:Trichoderma / Q000235:genetics*; D033921:Vesicular Transport Proteins
D004271:DNA, Fungal; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D019204:GTP-Binding Proteins; D020559:Monomeric GTP-Binding Proteins; C077395:SAR1 protein, S cerevisiae