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 ss-ring core, including ss 2c (HsPSMB7, a constitutive proteasome subunit). We developed a high-throughput pipeline to humanize yeast proteasomes by generating a large library of Hs ss 2c mutants and screening them for complementation of a yeast ss 2 (ScPup1) knockout. Variants capable of replacing ScPup1 included (1) those impacting local protein-protein interactions (PPIs), with most affecting interactions between the ss 2c C-terminal tail and the adjacent ss 3 subunit, and (2) those affecting ss 2c proteolytic activity. Exchanging the full-length tail of human ss 2c with that of ScPup1 enabled complementation. Moreover, wild-type human ss 2c could replace yeast ss 2 if human ss 3 was also provided. Unexpectedly, yeast proteasomes bearing a catalytically inactive HsPSMB7-T44A variant that blocked precursor autoprocessing were viable, suggesting an intact propeptide stabilizes late assembly intermediates. In contrast, similar modifications in human ss 2i (HsPSMB10), an immunoproteasome subunit and the co-ortholog of yeast ss 2, do not enable complementation in yeast, suggesting distinct interactions are involved in human immunoproteasome core assembly. Broadly, our data reveal roles for specific PPIs governing functional replaceability across vast evolutionary distances.