Strategies and Bottlenecks Towards Humanizing the Proteasome Core in Yeast

All organism are derived from a common ancestor and share several biological processes. The principle of evolutionary conservation of genes between species enables their investigation in simpler model organisms. These evolutionary conserved genes encode essential cellular machinery whose failures are linked to diseases in humans. Despite a billion years of evolutionary divergence, budding yeast share several thousand protein coding-genes with humans. Recent systematic studies have identified many human orthologs that can individually complement a lethal growth defect confered by the loss of the corresponding yeast gene. Computational analysis of many properties of orthologous gene pairs revealed functional ability is not well-explained by sequence similarity between the human and yeast genes. Instead, it is a property of specific protein complexes and pathways "genetic modularity", that broadly defines the human protein's ability to interact with yeast proteins such that some genetic modules are entirely non replaceable (e.g., DNA replication initiation complexes, splicing machinery), whereas some are entirely replaceable, including the proteasome complex, a highly conserved, multi-protein complex comprising ~33 proteins. The modularity paradigm allows if the entire yeast and human systems are, to a first approximation, interchangeable (atleast in yeast). My thesis aims to humanize the yeast proteasome core complex comprising 14 subunits. In Chapter 1, I present a review of concepts relevant to humanized yeast and our recent efforts to use humanized yeast models to study human biology, disease, and evolution. In Chapter 2, I describe efforts to humanize the entire alpha proteasome core in yeast. In the process, I describe a novel rapid, scalable, and combinatorial genome engineering strategy, by Markerless Enrichment and Recombination of Genetically Engineered loci (MERGE) in yeast. In Chapter 3, I demonstrate the humanization of the non-replaceable yeast beta core subunits revealing the role of species specific protein-protein interactions and genetic modularity in functional replaceability. Finally, in chapter 4, I discuss future efforts to humanize the proteasome core in its entirety in yeast, including the assembly chaperones required for optimal assembly of the multi-subunit core