H-Ras Dynamically Interacts with Recycling Endosomes in CHO-K1 Cells

H-, N-, and K-Ras are isoforms of Ras proteins, which undergo different lipid modifications at the C terminus. These post-translational events make possible the association of Ras proteins both with the inner plasma membrane and to the cytosolic surface of endoplasmic reticulum and Golgi complex, which is also required for the proper function of these proteins. To better characterize the intracellular distribution and sorting of Ras proteins, constructs were engineered to express the C-terminal domain of H- and K-Ras fused to variants of green fluorescent protein. Using confocal microscopy, we found in CHO-K1 cells that H-Ras, which is palmitoylated and farnesylated, localized at the recycling endosome in addition to the inner leaflet of the plasma membrane. In contrast, K-Ras, which is farnesylated and nonpalmitoylated, mainly localized at the plasma membrane. Moreover, we demonstrate that sorting signals of H- and K-Ras are contained within the C-terminal domain of these proteins and that palmitoylation on this region of H-Ras might operate as a dominant sorting signal for proper subcellular localization of this protein in CHO-K1 cells. Using selective photobleaching techniques, we demonstrate the dynamic nature of H-Ras trafficking to the recycling endosome from plasma membrane. We also provide evidence that Rab5 and Rab11 activities are required for proper delivery of H-Ras to the endocytic recycling compartment. Using a chimera containing the Ras binding domain of c-Raf-1 fused to a fluorescent protein, we found that a pool of GTP-bound H-Ras localized on membranes from Rab11-positive recycling endosome after serum stimulation. These results suggest that H-Ras present in membranes of the recycling endosome might be activating signal cascades essential for the dynamic and function of the organelle.