Ras proteins were described localizing at the plasma membrane (PM) where they activate diverse signal transduction pathways. There are three isoforms of Ras proteins, H-, N- and K-Ras, which undergo different lipid modifications at the C-terminus. These lipid modifications confer them the capacity to associate with PM.
To better characterize the intracellular distribution and sorting of Ras proteins, two constructs were engineered to express the C-terminal domain of H- and K-Ras fused to GFP (GFP-H-RasC20 and GFP-K-RasC14). By using confocal microscopy, we found that GFP-H-RasC20 (palmitoylated and farnesylated) localized mainly at the PM, recycling endosomes (RE) and in minor extent at late endosomes. In contrast, GFP-K-RasC14 (farnesylated and nonpalmitoylated) localized at the PM and cytoplasm. Interestingly, the subcellular distribution of GFP-H-RasC20 and GFP-K-RasC14 completely colocalized with the full length version of these proteins. We also demonstrate that another unrelated palmitoylated protein, N13GAP43-GFP, also localized, like H-ras, both at PM and RE In conclusion, we demonstrate for the first time that H-Ras localizes, in addition to PM, at RE. Furthermore, our results suggest that palmitoylation at the C-terminus region of H-Ras might be a dominant sorting signal for proper subcellular localization of this protein in CHO-K1 cells.
