Characterization of two 5-aminoimidazole-4-carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase isozymes from Saccharomyces cerevisiae.
The Saccharomyces cerevisiae ADE16 and ADE17 genes encode 5-aminoimidazole-4-carboxamide ribonucleotide transformylase isozymes that catalyze the penultimate step of the de novo purine biosynthesis pathway. Disruption of these two chromosomal genes results in adenine auxotrophy, whereas expression of either gene alone is sufficient to support growth without adenine. In this work, we show that an ade16 ade17 double disruption also leads to histidine auxotrophy, similar to the adenine/histidine auxotrophy of ade3 mutant yeast strains. We also report the purification and characterization of the ADE16 and ADE17 gene products (Ade16p and Ade17p). Like their counterparts in other organisms, the yeast isozymes are bifunctional, containing both 5-aminoimidazole-4-carboxamide ribonucleotide transformylase and inosine monophosphate cyclohydrolase activities, and exist as homodimers based on cross-linking studies. Both isozymes are localized to the cytosol, as shown by subcellular fractionation experiments and immunofluorescent staining. Epitope-tagged constructs were used to study expression of the two isozymes. The expression of Ade17p is repressed by the addition of adenine to the media, whereas Ade16p expression is not affected by adenine. Ade16p was observed to be more abundant in cells grown on nonfermentable carbon sources than in glucose-grown cells, suggesting a role for this isozyme in respiration or sporulation.
Tibbetts
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AS
Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology and the Biochemical Institute, University of Texas, Austin, Texas 78712, USA.
Appling
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D000225:Adenine / Q000494:pharmacology; D002458:Cell Fractionation; D003432:Cross-Linking Reagents / Q000378:metabolism; D019281:Dimerization; D004120:Dimethyl Suberimidate / Q000378:metabolism; D000431:Ethanol / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005656:Fungal Proteins / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D005947:Glucose / Q000378:metabolism; D019877:Hydroxymethyl and Formyl Transferases / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009709:Nucleotide Deaminases / Q000235:genetics / Q000378:metabolism*; D011687:Purines / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D003432:Cross-Linking Reagents; D005656:Fungal Proteins; D007527:Isoenzymes; D009097:Multienzyme Complexes; D011687:Purines; C113120:inosine monophosphate synthase; D004120:Dimethyl Suberimidate; D000431:Ethanol; D019877:Hydroxymethyl and Formyl Transferases; D009709:Nucleotide Deaminases; D005947:Glucose; D000225:Adenine