We show that the specific subcellular distribution of H- and Nras guanosine triphosphate-binding proteins is generated by a constitutive de/reacylation cycle that operates on palmitoylated proteins, driving their rapid exchange between the plasma membrane (PM) and the Golgi apparatus. Depalmitoylation redistributes farnesylated Ras in all membranes, followed by repalmitoylation and trapping of Ras at the Golgi, from where it is redirected to the PM via the secretory pathway. This continuous cycle prevents Ras from nonspecific residence on endomembranes, thereby maintaining the specific intracellular compartmentalization. The de/reacylation cycle also initiates Ras activation at the Golgi by transport of PM-localized Ras guanosine triphosphate. Different de/repalmitoylation kinetics account for isoform-specific activation responses to growth factors.

No abstract

Fluorescence resonance energy transfer (FRET) microscopy approaches have been used to study protein interactions in living cells. Up to now, due to the spectral requirements for FRET detection, this has been limited to the measurement of single protein interactions. Here we present a novel time-resolved fluorescence imaging method for simultaneously monitoring the activation state of two proteins in a single cell. A Ras sensor, consisting of fluorescently labelled Ras and a fluorescently labelled Ras binding domain (RBD) of Raf, which reads out Ras activation by its interaction with RBD as a FRET signal, has been adapted for this purpose. By using yellow (YFP) and cyan (CFP) versions of the green fluorescent protein from Aquorea victoria as donors and a tandem construct of Heteractis crispa Red (tHcRed) as acceptor for both donors, two independent FRET signals can be measured at the same time. Measuring the YFP and CFP donor lifetimes by fluorescence-lifetime imaging microscopy (FLIM) allows us to distinguish the two different FRET signals in a single cell. Using this approach, we show that different Ras isoforms and mutants that localize to the plasma membrane, to the Golgi or to both compartments display distinct activation profiles upon growth-factor stimulation; this indicates that there is a differential regulation in cellular compartments. The method presented here is especially useful when studying spatiotemporal aspects of protein regulation as part of larger cellular signalling networks.