Abstract

The proteasome is a large, multi-subunit protein complex essential for degrading unneeded, misfolded or damaged proteins in eukaryotic cells. Mutations in its subunits are linked to many human diseases. How these mutations affect proteasome function remains unclear, so scientists have begun developing a budding yeast (Saccharomyces cerevisiae) model with a fully humanized proteasome to accommodate detailed study.
To date, up to 6 of 7 alpha subunits in the 20S core of the proteasome can be humanized together, all except the α5 subunit, preventing complete humanization of alpha ring in S. cerevisiae. This suggests that yeast chaperones responsible for assembling the 20S proteosome complex may be incompatible with some human subunits (e.g. α5). Thus, to achieve my aim of completely humanizing the alpha ring, I created a set of genetically engineered yeast strains expressing orthologous human chaperones and investigated whether they improved proteasome humanization in yeast.
Results show that human chaperones do not complement phenotypes associated with deleting their yeast orthologs, suggesting they may be incapable of yeast proteosome assembly. Moreover, negative genetic interactions observed between human chaperones and human α5 or β2 subunits suggest these subunits may disrupt yeast 20S assembly. Human chaperone expression failed to permit humanization of α5 in strains harboring partially humanized alpha rings, suggesting that incompatible interactions involving assembly chaperones are not likely responsible for incomplete alpha ring humanization in yeast.
In conclusion, expressing human chaperones in S. cerevisiae failed to overcome barriers preventing complete proteasome humanization, but provided several insights to direct future studies focused on developing this model to study proteasome related diseases.