A new yeast metabolon involving at least the two first enzymes of arginine biosynthesis: acetylglutamate synthase activity requires complex formation with acetylglutamate kinase.
Open reading frame YJL071W of Saccharomyces cerevisiae was shown to be ARG2 and identified as the structural gene for acetylglutamate synthase, first step in arginine biosynthesis. The three Ascomycete acetylglutamate synthases characterized to date appear homologous, but unlike the other enzymes of the yeast arginine biosynthesis pathway, they showed no significant similarity to their prokaryotic equivalents. The measured synthase activity did not increase with the number of ARG2 gene copies unless the number of ARG5,6 gene copies was increased similarly. ARG5,6 encodes a precursor that is maturated in the mitochondria into acetylglutamate kinase and acetylglutamyl-phosphate reductase, catalyzing the second and third steps in the pathway. The results imply that the synthase must interact stoichiometrically in vivo with the kinase, the reductase, or both to be active. Results obtained with synthetic ARG5 and ARG6 genes suggested that both the kinase and the reductase could be needed. This situation, which has completely escaped notice in yeast until now, is reminiscent of the observation in Neurospora crassa that nonsense arg-6 kinase/reductase mutants lack synthase activity (Hinde, R. W., Jacobson, J. A., Weiss, R. L., and Davis, R. H. (1986) J. Biol. Chem. 261, 5848-5852). In immunoprecipitation experiments, hemagglutinin-tagged synthase coprecipitated with a protein proven by microsequencing to be the kinase. Western blot analyses showed that the synthase has reduced stability in the absence of the kinase/reductase. Our data demonstrate the existence of a new yeast arginine metabolon involving at least the first two, and possibly the first three, enzymes of the pathway. Hypotheses regarding the biological significance of this interaction are discussed.
Abadjieva
A
A
Department of Microbiology of the Vrije Universiteit Brussel, Belgium.
Pauwels
K
K
Hilven
P
P
Crabeel
M
M
D000123:Acetyltransferases / Q000235:genetics / Q000378:metabolism*; D000483:Alleles; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D051046:Amino-Acid N-Acetyltransferase; D001120:Arginine / Q000096:biosynthesis* / Q000378:metabolism*; D015153:Blotting, Western; D002384:Catalysis; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D017931:DNA Primers / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D019143:Evolution, Molecular; D006133:Growth Substances; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid
D000596:Amino Acids; D017931:DNA Primers; D006133:Growth Substances; D010940:Plant Proteins; C085797:auxin regulated proteins, Vigna radiata; D004247:DNA; D001120:Arginine; D000123:Acetyltransferases; D051046:Amino-Acid N-Acetyltransferase; D017851:Phosphotransferases (Carboxyl Group Acceptor); C018721:acetylglutamate kinase