Abstract
AbstractPathway transplantation from one organism to another represents a means to a more complete understanding of a biochemical or regulatory process. The purine biosynthesis pathway, a core metabolic function, was transplanted from human to yeast. We replaced the entireSaccharomyces cerevisiaeadenine de novo pathway with the cognate human pathway components. A yeast strain was “humanized” for the full pathway by deleting all relevant yeast genes completely and then providing the human pathway in trans using a neochromosome expressing the human protein coding regions under the transcriptional control of their cognate yeast promoters and terminators. The “humanized” yeast strain grows in the absence of adenine, indicating complementation of the yeast pathway by the full set of human proteins. While the strain with the neochromosome is indeed prototrophic, it grows slowly in the absence of adenine. Dissection of the phenotype revealed that the human ortholog ofADE4, PPAT, shows only partial complementation. We have used several strategies to understand this phenotype, that point toPPAT/ADE4as the central regulatory node. Pathway metabolites are responsible for regulatingPPAT’sprotein abundance through transcription and proteolysis as well as its enzymatic activity by allosteric regulation in these yeast cells. Extensive phylogenetic analysis of PPATs from diverse organisms hints at adaptations of the enzyme-level regulation to the metabolite levels in the organism. Finally, we isolated specific mutations in PPAT as well as in other genes involved in the purine metabolic network that alleviate incomplete complementation byPPATand provide further insight into the complex regulation of this critical metabolic pathway.
Publisher
Cold Spring Harbor Laboratory
Reference45 articles.
1. The exceptionally high rate of spontaneous mutations in the polymerase delta proofreading exonuclease-deficient Saccharomyces cerevisiae strain starved for adenine;BMC Genet,2004
2. Agmon, N. , Mitchell, L.A. , Cai, Y. , Ikushima, S. , Chuang, J. , Zheng, A. , Choi, W.J. , Martin, J.A. , Caravelli, K. , Stracquadanio, G. , et al. (2015). Yeast Golden Gate (yGG) for efficient assembly of S. cerevisiae transcription units. ACS synthetic biology.
3. Low escape-rate genome safeguards with minimal molecular perturbation ofSaccharomyces cerevisiae
4. Andrews, S. FastQC a quality-control tool for high-throughput sequence data.
5. Myc-dependent purine biosynthesis affects nucleolar stress and therapy response in prostate cancer;Oncotarget,2015
Cited by
3 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献