A yeast model for the functional analysis of SRP54 mutations associated with severe congenital neutropenia.

Mutations in the human SRP54 gene are linked to the pathophysiology of severe congenital neutropenia (SCN). SRP54 is a key protein comprising one of the six protein subunits of the signal recognition particle responsible for cotranslational targeting of proteins to the ER. Crystal structures and biochemical characterization of several SRP54 mutants provide insights into how SRP54 mutations affect its function. However, no scalable, flexible platform exists to study the sequence-structure-function relationships of SRP54 mutations. Here, we engineered a Saccharomyces cerevisiae strain whose growth depends on hormone-inducible SRP54 expression. We demonstrate the model's suitability for studying SRP54 and the suspected dominant-negative phenotypes associated with orthologous SRP54 mutants. The most common yeast orthologue, S125del (T117del human orthologue), displayed the least severe growth defect; in contrast, the G234E mutant (G226E human orthologue) displayed the most severe growth defect, in line with clinical presentations in SCN. Lastly, we characterized four previously uncharacterized SRP54 mutations found in clinical data. We also determined the first X-ray structure of Saccharomyces cerevisiae SRP54. The NG domain of the wild-type protein shows predicted high structural similarity to homologs from across phyla. The structure outlines unique features of the yeast protein, along with providing a structural explanation for mutant phenotypes. The ability of this haploid model to recapitulate these phenotypes while remaining amenable to high-throughput screening approaches makes it a powerful tool for studying SRP54. Furthermore, the methodology used to create this model may also be used to study other human diseases involving essential and quasi-essential genes.