Although our understanding of the mechanisms of depalmitoylation is limited, the spatiotemporal dynamics of this process were investigated by microinjection of semisynthetic N-Ras containing both farnesyl and palmitoyl chains . Previous analysis of an N-Ras protein in which the palmitoyl group was attached by a noncleavable thioether linkage revealed a dispersed intracellular localization without Golgi enrichment (Rocks et al., 2005) . This localization was suggested to reflect a requirement for active palmitoylation/ depalmitoylation cycling to achieve the correct localization of Ras. The palmitoylated protein with a cleavable thioester bond was therefore not expected to display any initial membranetargeting specificity. Despite this, the construct rapidly accumulated at the Golgi (t/2 = 27 s). This Golgi accumulation was suggested to follow on from binding of the farnesylated/ palmitoylated protein to any membrane, depalmitoylation, cytosolic diffusion, and subsequent repalmitoylation at the Golgi. There were two main interpretations made from this behavior of farnesylated and palmitoylated N-Ras: (1) depalmitoylation must be very rapid to account for the speed of Golgi accumulation and (2) depalmitoylation must occur throughout the cell because if it was confined to a specific location, association of the farnesylated and palmitoylated protein with some membranes would be irreversible.