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Species-specific protein-protein interactions govern the humanization of the 20S proteasome in yeast

Yeast and humans share thousands of genes despite a billion years of evolutionary divergence. While many human genes can functionally replace their yeast counterparts, nearly half of the tested shared genes cannot. For example, most yeast proteasome subunits are humanizable, except subunits comprising the β-ring core, including β2 (HsPSMB7). We developed a high-throughput pipeline to humanize yeast proteasomes by generating a large library of Hsβ2 mutants and screening them for complementation of yeast β2 (ScPup1). Variants capable of replacing ScPup1 included (1) those impacting local protein-protein interactions (PPIs), with most affecting interactions between the β2 C-terminal tail and the adjacent β3 subunit, and (2) those affecting β2 proteolytic activity. Exchanging the full-length tail of human β2 with that of ScPup1 enabled complementation. Moreover, wild-type human β2 replaced yeast β2 if the adjacent human β3 subunit was also provided. Unexpectedly, yeast proteasomes bearing a catalytically inactive HsPSMB7-T44A variant blocking precursor autoprocessing were viable, suggesting an intact propeptide stabilizes late assembly intermediates. Our data reveal roles for specific PPIs governing functional replaceability across vast evolutionary distances.