Transient palmitoylation supports H-Ras membrane binding but only partial biological activity

H-Ras is >95% membrane-bound when modified by farnesyl and palmitate, but < 10% membrane-bound if only farnesyl is present, implying that palmitate pro vides major support for membrane interaction. However the direct contributi on of palmitate to H-Ras membrane interaction or the extent of its cooperat ion with farnesyl is unknown, because in the native protein the isoprenoid must be present before palmitate can be attached. To examine if palmitates can maintain H-Ras membrane association despite multiple cycles of turnover , a nonfarnesylated H-Ras(Cys186Ser) was constructed, with an N-terminal pa lmitoylation signal, derived from the GAP-43 protein. Although 40% of the G AP43:Ras-(61Leu,186Ser) protein (G43:Ras61L) partitioned with membranes, th e chimera had less than 10% of the transforming activity of fully lipidated H-Ras(61Leu) in NIH 3T3 cells. Poor focus formation was not due to incorre ct targeting or gross structural changes, because G43:Ras61L localized spec ifically to plasma membranes and triggered differentiation of PC12 cells as potently as native H-Ras61L. Proteolytic digestion indicated that in G43:R as61L both the N-terminal and the two remaining C-terminal cysteines of G43 :Ras61L were palmitoylated. A mutant lacking all three C-terminal Cys resid ues had decreased membrane binding and differentiating activity. Therefore, even with correct targeting and palmitates at the C-terminus, G43:Ras61L w as only partially active. These results indicate that although farnesyl and palmitate share responsibility for H-Ras membrane binding, each lipid also has distinct functions. Farnesyl may be important for signaling, especiall y transformation, while palmitates may provide potentially dynamic regulati on of membrane binding.