IDENTIFICATION OF GUANINE EXCHANGE FACTOR KEY RESIDUES INVOLVED IN EXCHANGE ACTIVITY AND RAS INTERACTION

We have carried out a functional analysis of the human HGRF55 exchange factor in the yeast Saccharomyces cerevisiae. Twelve residues conserv ed among most of all known guanine exchange factors (GEFs) have been i ndependently changed to alanine. Taking advantage of the ability of Hg rf55p to replace the yeast Cdc25p exchange factor, and using the two-h ybrid system with RAS2(ala22) allele, we have identified key residues for the interaction with Pas and/or its activation. Substitution of ar ginine 392 to alanine leads to a complete loss of interaction with Ras , though the protein remains stable. Substitution of Asp266 or Arg359 to alanine results in inactive proteins at 39 degrees C, still able ho wever to interact with Ras. The other charged-to-alanine substitutions led to no detectable phenotype when present alone but most of them dr amatically increased the temperature sensitive phenotype observed with [Asp266Ala] substitution. Surprisingly, the cysteine to alanine subst itution in the highly conserved PCVPF/Y motif proved to be without eff ect, suggesting that the sulfhydryl group is not essential for stabili ty or interaction with Ras.