RT Journal Article SR Electronic T1 Human to yeast pathway transplantation: cross-species dissection of the adenine de novo pathway regulatory node JF bioRxiv FD Cold Spring Harbor Laboratory SP 147579 DO 10.1101/147579 A1 Neta Agmon A1 Jasmine Temple A1 Zuojian Tang A1 Tobias Schraink A1 Maayan Baron A1 Jun Chen A1 Paolo Mita A1 James A. Martin A1 Benjamin P. Tu A1 Itai Yanai A1 David Fenyö A1 Jef D. Boeke YR 2017 UL http://biorxiv.org/content/early/2017/06/14/147579.abstract AB 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.