Human ORFeome expression in S. cerevisiae to better understand extracellular vesicle biology
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Extracellular vesicles (EVs) mediate intercellular communication by all organisms studied, from bacteria to yeast to man. Yet evolutionarily conserved mechanisms governing aspects of fundamental EV biology remain enigmatic. To address this, we sought to establish Saccharomyces cerevisiae (baker’s yeast) as a model by identifying ectopically expressed human proteins sorted into yeast EVs. Using an optimized pooled cloning method, we inserted >13,000 human open reading frames (ORFs) upstream of an EGFP tag within yeast expression plasmids. After transformation into S. cerevisiae , we confirmed expression of 3,288 EGFP-tagged human proteins with diverse cellular expression levels and subcellular localizations. Heat stress triggered release of intact, lipid-bound, EGFP-positive small EVs from all transformant pools. Proteomic analysis identified 292 human proteins within EV samples, including canonical human EV biomarkers. Over 70% had yeast orthologs also found in yeast EVs suggesting conserved sorting mechanisms. Protein–protein interaction network analysis linked these EV cargoes to ESCRT-associated pathways. Finally, validation of seven candidates showed that DEF3A, ANXA2 and CLIC1 were enriched in yeast EVs. This study establishes an omics-compatible synthetic biology framework to humanize yeast EVs, begins to uncover conserved cargo sorting mechanisms, and supports future engineering of designer EVs.
GRAPHICAL ABSTRACT and TOC BLURB
Over 13,000 human open reading frames were expressed in Saccharomyces cerevisiae to study fundamental extracellular vesicle (EV) biology. Results suggest evolutionary conservation of EV cargo protein sorting pathways and identify human protein scaffolds to engineer yeast EVs for broad applications.
