Yeast Complementation Assay

Overview

A functional genetics approach in which human gene variants are expressed in Saccharomyces cerevisiae in place of an essential yeast ortholog. Cells are grown on selective media (typically 5-fluoroorotic acid / 5-FOA, which selects against plasmids carrying the wild-type yeast gene) to assess whether the human variant can substitute for the yeast gene and restore viability. The assay classifies variants as complementing (functional) or non-complementing (loss-of-function equivalent), enabling direct functional annotation of human cancer mutations in a tractable eukaryotic system.

Used by

• Applied to five oncogenic RHOA hotspot mutants (R5Q, G17V, C16R, A161P, E40Q) substituting for essential yeast RHO1 on 5-FOA; E40Q failed complementation while the other four supported growth PMID:24816253

Notes

• Selection medium is typically 5-FOA, which is toxic to cells retaining the URA3-marked plasmid carrying the wild-type yeast ortholog; only cells that have lost the plasmid (and thus rely solely on the human variant) survive.
• Complementation is necessary but not sufficient to infer wild-type function; some gain-of-function and attenuated-output loss-of-function alleles can both complement null backgrounds.
• Results from yeast may not translate directly to mammalian contexts because higher-eukaryote effectors (ROCK kinases, mammalian-specific RhoGEFs/GAPs) are absent in yeast.

Sources

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