Abstract

Orthologous genes in diverged species tend to perform similar functions. This conjecture forms the foundation of most biomedical research and justifies the use of model organisms in research with implications in human biology. Humans and the baker’s yeast Saccharomyces cerevisiae diverged from a common ancestor approximately one billion years ago. Yet, they share several thousand genes accounting for nearly one-third of the yeast genome. Several systematic studies have shown that many human orthologs are replaceable in yeast. However, nearly half of the tested human genes are non-replaceable in yeast. This work focuses on the non-replaceable human genes; we establish a genetic suppressor screen that allows the selection for replaceability of otherwise non-replaceable human genes in yeast. We focused on the non-replaceable human catalytic beta subunit β2 (PSMB7) of the proteasome core to test this strategy.
Traditional suppressor screens are tedious and time-consuming; therefore, we developed a high-throughput strategy using a robotic setup. Using error-prone PCR, we generated a mutant pool of the non-replaceable human catalytic subunits, which were transformed in heterozygous knockout diploid yeast strains and screened for yeast colonies carrying a replaceable human mutant gene. After validation by re-transformation followed by sequencing, we identified the amino acid changes in the human genes that allow replaceability. To identify the mode of suppression, we performed structural analysis of the mutants by modelling human protein structure in the yeast proteasome. The mutants are broadly categorised to three classes. 1) The mutations close to the interacting surfaces of the neighbouring proteins in the complex, 2) The mutations that affect the catalytic activity of the protein thus, affecting the assembly in the yeast proteasome core and lastly, 3) The mutations that lie in the C-terminal region of β2. The mutants reveal divergence of regulation and function in human and yeast proteasome core assembly. Broadly, this research will create an extended resource to study human gene function in yeast by swapping even more as yet non-replaceable human orthologs in yeast. Additionally, the genetic approach allows us to discern whether the gain of replaceability is driven by changes in protein-protein interactions (PPIs); thus, uncovering core evolutionary principles. Through characterization of the essential PPIs out of many, the approach may help decode genotype-phenotype relationships in organisms, especially in the cases where aberrant phenotypes are associated with diseases in humans.