Cross-Species Complementation of Nonessential Yeast Genes Establishes Platforms for Testing Inhibitors of Human Proteins

Cross-species complementation can be used to generate humanized yeast, which is a valuable resource with which to model and study human biology. Humanized yeast can be used as an in vivo platform to screen for chemical inhibition of human protein drug targets. To this end, we report the systematic complementation of nonessential yeast genes implicated in chromosome instability (CIN) with their human homologs. We identified 20 human–yeast complementation pairs that are replaceable in 44 assays that test rescue of chemical sensitivity and/or CIN defects. We selected a human–yeast pair (hFEN1/yRAD27), which is frequently overexpressed in cancer and is an anticancer therapeutic target, to perform in vivo inhibitor assays using a humanized yeast cell-based platform. In agreement with published in vitro assays, we demonstrate that HU-based PTPD is a species-specific hFEN1 inhibitor. In contrast, another reported hFEN1 inhibitor, the arylstibonic acid derivative NSC-13755, was determined to have off-target effects resulting in a synthetic lethal phenotype with yRAD27-deficient strains. Our study expands the list of human–yeast complementation pairs to nonessential genes by defining novel cell-based assays that can be utilized as a broad resource to study human drug targets.

Medicago truncatulacopper transporter 1 (MtCOPT1) delivers copper for symbiotic nitrogen fixation

Summary• Copper is an essential nutrient for symbiotic nitrogen fixation. This element is delivered by the host plant to the nodule, where membrane copper transporter would introduce it into the cell to synthesize cupro-proteins.• COPT family members in model legumeMedicago truncatulawere identified and their expression determined. Yeast complementation assays, confocal microscopy, and phenotypical characterization of aTnt1insertional mutant line were carried out in the nodule-specificM.truncatulaCOPT family member.•Medicago truncatulagenome encodes eight COPT transporters.MtCOPT1(Medtr4g019870) is the only nodule-specificCOPTgene. It is located in the plasma membrane of the differentiation, interzone and early fixation zones. Loss of MtCOPT1 function results in a copper-mitigated reduction of biomass production when the plant obtains its nitrogen exclusively from symbiotic nitrogen fixation. Mutation ofMtCOPT1results in diminished nitrogenase activity in nodules, likely an indirect effect from the loss of a copper-dependent function, such as cytochrome oxidase activity incopt1-1bacteroids.• These data are consistent with a model in which MtCOPT1 transports copper from the apoplast into nodule cells to provide copper for essential metabolic processes associated with symbiotic nitrogen fixation.