Characterization of Two 5-Aminoimidazole-4-carboxamide Ribonucleotide Transformylase/Inosine Monophosphate Cyclohydrolase Isozymes from Saccharomyces cerevisiae *
Characterization of Two 5-Aminoimidazole-4-carboxamide Ribonucleotide Transformylase/Inosine Monophosphate Cyclohydrolase Isozymes from Saccharomyces cerevisiae *
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DOI：
10.1074/jbc.m909851199
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发表时间：
2000-07
期刊：
影响因子：
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通讯作者：
A. S. Tibbetts;D. Appling
中科院分区：
文献类型：
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作者：
A. S. Tibbetts;D. Appling
酿酒酵母ADE 16和ADE 17基因编码5-氨基咪唑-4-甲酰胺核糖核苷酸转化酶同工酶，其催化从头嘌呤生物合成途径的倒数第二步。这两个染色体基因的破坏导致腺嘌呤营养缺陷型，而单独表达任何一个基因都足以在没有腺嘌呤的情况下支持生长。在这项工作中，我们表明，一个ade 16，ade 17双破坏也导致组氨酸营养缺陷型，类似于腺嘌呤/组氨酸营养缺陷型ofade 3突变酵母菌株。我们还报道了ADE 16和ADE 17基因产物（Ade 16 p和Ade 17 p）的纯化和表征。与其他生物体中的对应物一样，酵母同工酶是双功能的，含有5-氨基咪唑-4-甲酰胺核糖核苷酸转化酶和肌苷单磷酸环化水解酶活性，并且基于交联研究以同源二聚体存在。这两种同工酶都定位于细胞质，如亚细胞分级分离实验和免疫荧光染色所示。表位标记的构建体用于研究两种同工酶的表达。Ade 17 p的表达被腺嘌呤抑制，而Ade 16 p的表达不受腺嘌呤的影响。Ade 16 p被观察到是更丰富的非发酵碳源生长的细胞比葡萄糖生长的细胞，这表明这种同工酶在呼吸或孢子形成的作用。
The Saccharomyces cerevisiae ADE16and 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 anade16 ade17 double disruption also leads to histidine auxotrophy, similar to the adenine/histidine auxotrophy ofade3 mutant yeast strains. We also report the purification and characterization of the ADE16 and ADE17gene 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.