It's easy to group all isoforms of a certain protein under the same umbrella. But a closer look at the Ras GTPases H-ras, N-ras and K-ras reminds us that they generate isoform-specific biological outputs, and that this is influenced by their differential localization at the plasma membrane (PM) and subcellular membranes. This, in turn, reflects their C-terminal lipid modifications: after farnesylation, AAX proteolysis and methylation of the farnesylated cysteine, H-ras and N-ras are palmitoylated, which enables them to localize to the PM and Golgi membranes; by contrast, K-ras bypasses the secretory pathway and localizes to the PM.

Both the PM and the Golgi are sites of active Ras signalling, but how this compartmentalized localization and activity of palmitoylated Ras isoforms is accomplished has remained unclear. Work by Rocks et al., reported in Science, now shows that a constitutive de- and re-palmitoylation cycle maintains the specific compartmentalization.

Golgi-localized Ras was thought to comprise nascent proteins trafficking to the PM. But by inhibiting protein synthesis, and thereby removing nascent proteins, the authors showed that H-ras and N-ras still localized at the Golgi. Moreover, photobleaching and photoactivation studies showed that palmitoylated Ras cycled between the PM and the Golgi, and that Golgi Ras was replenished by retrograde transport of PM-localized Ras. Using a hexadecylated version of N-ras — HDFar — that could not undergo de- and re-palmitoylation, the authors showed that this localization wasn't mediated by clathrin, caveolae or cholesterol, but that de- and re-palmitoylation events were required. Microinjected HDFar localized nonspecifically throughout the membrane system.

The authors also noticed that the kinetics of H-ras and N-ras trafficking were different. H-ras is palmitoylated on two cysteines, whereas palmitate moieties are added to only one cysteine in N-ras. N-ras trafficked faster than H-ras, so Rock et al. studied monopalmitoylated H-ras mutants. C181S and C184S H-ras had an increased preference for Golgi localization relative to wild-type H-ras, and showed faster PM–Golgi exchange. By developing a new assay to compare the dynamics of Ras activation at the PM and Golgi, Rock et al. showed H-ras to be rapidly and transiently activated at the PM in response to growth factor stimulation, and to have a delayed onset but to be sustained at the Golgi. By contrast, active H-ras C184S and N-ras were detectable much sooner at the Golgi. Total inhibition of palmitoylation blocked trafficking from the PM to the Golgi and therefore blocked Ras activation.

So, rapid exchange of palmitoylated Ras isoforms at the PM and Golgi is driven by de- and re-palmitoylation cycles. De-palmitoylation confers an equal distribution between the cytosol and membranes. Re-palmitoylation, which enables stable membrane anchorage, occurs at the Golgi; from here, Ras is redirected to the PM in the exocytic pathway. As the authors' findings of such a cycle extended to other proteins that have been reported to localize to the PM and the Golgi, they propose that this process has a universal role in subcellular distribution.