The PH domain of the yeast GEF Rom2p serves an essential function in vivo

In a screen designed to identify new upstream components of the Pkc1p-MAP k inase signal transduction pathway that responds to cell wall damage in yeas t, we identified a new mutant allele of the ROM2 gene, which encodes a GDP/ GTP exchange factor that acts on Rho1p. This allele, originally termed ubk1 (upstream of Bck1p) encodes a truncated protein that lacks the putative PH domain. Complementation experiments showed that genes coding for several k nown components of the pathway are able to suppress the ubk1 mutation to va rious degrees when introduced on low- or high-copy-number vectors. Analysis of several rom2 mutants showed that mutants in which the PH domain is dele ted result in a phenotype indistinguishable from that of a strain deleted f or the entire gene, indicating that this domain fulfills an essential funct ion in vivo. Furthermore, we found that the growth phenotype of rom2 mutant s is highly dependent on the strain background. Surprisingly, analysis of t he phosphorylation status of Mpk1p in these mutants showed an elevated leve l of doubly phosphorylated Mpk1 protein. indicating that the growth defect of rom2 mutants is not due to an inability to activate the MAP kinase modul e, but rather to lack of a function of the Rom2 protein that has yet to be identified precisely.