MUTAGENIC ALTERATION OF THE DISTAL SWITCH-II REGION OF RAS BLOCKS CDC25-DEPENDENT SIGNALING FUNCTIONS

We have explored the role of the distal switch II region of the yeast RAS2 protein in determining the response to the nucleotide exchange fa ctor CDC25. We first constructed yeast tester strains in which the del etion of the chromosomal CDC25, RAS1, and RAS2 genes, in combination w ith the chromosomal suppressor CRI4, resulted in detectable phenotypes in vivo and in vitro. Phenotypes included impaired growth at 37 degre es C, defective glucose-induced cyclic AMP signaling, and low adenylyl cyclase activity of membrane preparations. Tester strains were subseq uently used for the reintroduction of various combinations of wild-typ e and mutated RAS2 and CDC25 genes by genetic techniques, as well as f or in vitro reconstitution assays with the corresponding proteins. CDC 25 restored both growth and glucose-induced cyclic AMP signaling in th e presence, but not in the absence of wild-type RAS2. A gene encoding a RAS2 protein with a mutationally altered switch II region was expres sed but was ineffective in reintegrating exchange factor-dependent res ponses in vivo. Wild-type, but not mutagenically altered, RAS2 protein s were stimulated by exchange factors in vitro. We conclude that the c onserved distal switch II region is required for CDC25-dependent activ ation of RAS.