Abstract
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.
Highlights
5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR)1 transformylase catalyzes the ninth step of the de novo purine biosynthesis pathway
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
AICAR transformylase assays of yeast crude extracts demonstrated that the ADE17 gene product is the more active of the two enzymes, but the low level of activity supplied by the ADE16 gene product is sufficient for wild-type growth rates
Summary
Materials—Dimethyl suberimidate, AICAR, and 5-formyltetrahydrofolate were purchased from Sigma. His-tagged ADE16 and ADE17 were subsequently cloned into the yeast expression vector, pVT-101U [9] to generate the plasmids pVT-HisADE16 and pVT-HisADE17 These constructs were transformed into the ATY3.1 yeast strain to test the ability of the His-tagged proteins to rescue the adenine auxotrophy of this double mutant strain. Purification of His-tagged Proteins—The pET16b-ADE16 construct and the pREP4-GroESL plasmid [10] were co-transformed into E. coli BL21(DE3) cells (Novagen, Madison, WI). Transformants containing both plasmids were used to inoculate 1 liter of 2YT medium (1.6% tryptone, 1% yeast extract, 0.5% NaCl) containing 50 g/ml ampicillin and 30 g/ml kanamycin. Oligonucleotides ADE16Xho and ADE16OE were used to amplify a PCR product containing sequence upstream of the ADE16 translation
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
