TY - JOUR T1 - Human to yeast pathway transplantation: cross-species dissection of the adenine de novo pathway regulatory node JF - bioRxiv DO - 10.1101/147579 SP - 147579 AU - Neta Agmon AU - Jasmine Temple AU - Zuojian Tang AU - Tobias Schraink AU - Maayan Baron AU - Jun Chen AU - Paolo Mita AU - James A. Martin AU - Benjamin P. Tu AU - Itai Yanai AU - David Fenyö AU - Jef D. Boeke Y1 - 2017/01/01 UR - http://biorxiv.org/content/early/2017/06/14/147579.abstract N2 - Pathway 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 entire Saccharomyces cerevisiae adenine 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 of ADE4, PPAT, shows only partial complementation. We have used several strategies to understand this phenotype, that point to PPAT/ADE4 as the central regulatory node. Pathway metabolites are responsible for regulating PPAT’s protein 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 by PPAT and provide further insight into the complex regulation of this critical metabolic pathway. ER -