Newly synthesized Rho A, not Ras, is isoprenylated and translocated to membranes coincident with progression of the G1 to S phase of growth-stimulated rat FRTL-5 cells.

Ras and Rho are involved in the regulation of signal transduction events governing cell growth and cell proliferation. Protein prenylation is essential for the activation and/or the translocation of these small GTPases; however, protein geranylgeranylation rather than farnesylation is required for G1/S transition. We studied prenylation and translocation of Ras and Rho A during G1/S progression in growth-stimulated rat thyroid FRTL-5 cells. Immunoblot analysis revealed that both Ras and Rho A were detected in membrane fractions at G0. Rho A was eliminated from the membrane fraction during G1 and was not detected on the membrane at mid-G1. Translocation of Rho A from the cytoplasm back to the membranes was observed during late G1 phase. In contrast, Ras remains in the membrane fraction through the cell cycle progression from G1 to S phase. The immunoprecipitation of Rho A from the membrane fraction demonstrated that newly synthesized Rho A, labeled by pulsing cells with [35S]methionine and [35S]cysteine, was geranylgeranylated and associated with the membrane in late G1. These results indicate that Rho A, not Ras, was eliminated from membrane fraction during G1 progression and that newly synthesized Rho A is geranylgeranylated and translocated to membranes during G1/S progression in growth-stimulated FRTL-5 cells.