Cloning and sequencing of arg3 and arg11 genes of Schizosaccharomyces pombe on a 10-kb DNA fragment. Heterologous expression and mitochondrial targeting of their translation products.
The Schizosaccharomyces pombe arginine anabolic genes encoding ornithine carbamoyltransferase (arg3) and acetylglutamate kinase/acetylglutamyl-phosphate reductase (arg11) were cloned by functional complementation of S. pombe arg3 and arg11 mutant strains from S. pombe DNA genomic libraries. Restriction analysis and sequencing of the two clones showed that both genes are located on a common DNA fragment. The arg3 gene encodes a 327-amino-acid polypeptide presenting a strong identity to Saccharomyces cerevisiae and human ornithine carbamoyltransferases. The arg11 gene encodes a 884-amino-acid polypeptide. The acetylglutamate kinase and acetylglutamate-phosphate reductase domains have been defined by their identity with the S. cerevisiae ARG5,6 protein. The cloned arg11 gene from S. pombe does not complement an arg5,6 mutation in S. cerevisiae, nor does the ARG5,6 gene complement the S. pombe arg11- mutation. In contrast, both ornithine-carbamoyltransferase-encoding genes function in S. pombe. However, the S. pombe arg3 gene complements only weakly an arg3 S. cerevisiae strain, which is in agreement with the low level of expression of the S. pombe gene in S. cerevisiae. The subcellular localization of both ornithine carbamoyltransferases in the two yeasts indicates that, in contrast to the S. pombe enzyme, more than 95% of the S. cerevisiae enzyme remains in the S. pombe cytoplasm. The low expression of S. pombe ornithine carbamoyltransferases in S. cerevisiae did not allow its localization. The promoters of S. pombe arg3 and arg11 genes do not present striking similarities among themselves nor with the promoters of the equivalent genes of S. cerevisiae.
Van Huffel
C
C
Laboratoire de Microbiologie, Universite Libre de Bruxelles, Belgium.
Dubois
E
E
Messenguy
F
F
D000445:Aldehyde Oxidoreductases / Q000235:genetics*; D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D005800:Genes, Fungal; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D009954:Ornithine Carbamoyltransferase / Q000235:genetics*; D010770:Phosphotransferases / Q000235:genetics*; D017851:Phosphotransferases (Carboxyl Group Acceptor); D010957:Plasmids; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012331:RNA, Fungal / Q000235:genetics; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012568:Schizosaccharomyces / Q000235:genetics* / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
D004271:DNA, Fungal; D012331:RNA, Fungal; D001120:Arginine; D000445:Aldehyde Oxidoreductases; C018722:N-acetyl-gamma-glutamyl-phosphate reductase; D009954:Ornithine Carbamoyltransferase; D010770:Phosphotransferases; D017851:Phosphotransferases (Carboxyl Group Acceptor); C018721:acetylglutamate kinase