Divergent Morphologies and Common Signaling Features of Active and Inactive Oncogenic RHOA Mutants in Yeast

<i>RHOA</i>, a member of the Rho family of small GTPases, harbors recurrent mutations in diverse cancers, but how these mutations cause their cellular effects remains poorly understood. To investigate their cellular consequences, we expressed oncogenic <i>RHOA</i> variants (R5Q, G17V, C16R, and A161P) in <i>Saccharomyces cerevisiae</i>, substituting for the essential yeast homologue <i>RHO1</i>. While the E40Q variant failed to complement <i>RHO1</i> deletion, other mutants supported viability and enabled phenotypic characterization. All four variants conferred myriocin resistance, suggesting activation of the membrane stress response pathway, but induced no major changes in growth or caspofungin sensitivity. Using high-dimensional image analysis, we quantified 501 morphological parameters and applied principal component analysis and linear discriminant analysis to determine distinct phenotypic profiles. Gain-of-function (C16R and A161P) and loss-of-function (R5Q and G17V) mutants formed separate morphological clusters, indicating functional divergence. Our yeast model enabled systematic dissection of the functions of <i>RHOA</i> mutants and highlighted the utility of morphology-based approaches to characterize context-dependent mechanisms of oncogenesis.