SOS is a key activator of the small GTPase Ras. In cells, SOS-Ras signaling is thought to be initiated predominantly by membrane-recruitment of SOS via the adaptor Grb2 and balanced by rapidly reversible Grb2:SOS binding kinetics. However, SOS has multiple protein and lipid interactions that provide linkage to the membrane. In reconstituted membrane experiments, these Grb2-independent interactions are sufficient to retain SOS on the membrane for many minutes, during which a single SOS molecule can processively activate thousands of Ras molecules. These observations raise questions concerning how receptors maintain control of SOS in cells and how membrane-recruited SOS is ultimately released. We addressed these questions in quantitative reconstituted SOS-deficient chicken B cell signaling systems combined with single molecule measurements in supported membranes. These studies reveal an essentially one-way trafficking process in which membrane-recruited SOS remains trapped on the membrane and continuously activates Ras until it is actively removed via endocytosis.

Activation of small GTPases of the Ras superfamily by guanine nucleotide exchange factors (GEFs) is a key step in numerous cell signaling processes. Unveiling the detailed molecular mechanisms of GEF-GTPase signaling interactions is of great importance due to their central roles in cell biology, including critical disease states, and their potential as therapeutic targets. Here we present an assay to monitor individual Ras activation events catalyzed by single molecules of the GEF Son of Sevenless (SOS) in the natural membrane environment. The assay employs zero-mode waveguide (ZMW) nanostructures containing a single Ras-functionalized liposome. The ZMWs facilitate highly localized excitation of fluorophores in the vicinity of the liposome membrane, allowing direct observation of individual Ras activation events as single SOS enzymes catalyze exchange of unlabeled nucleotides bound to Ras with fluorescently labeled nucleotides from solution. The system is compatible with continuous recording of long sequences of individual enzymatic turnover events over hour-long time scales. The single turnover waiting time sequence is a molecular footprint that details the temporal characteristics of the system. Data reported here reveal long-lived activity states that correspond to well-defined conformers of SOS at the membrane. Liposome functionalized ZMWs allow for studies of nucleotide exchange reactions at single GTPase resolution, providing a platform to gauge the mechanisms of these processes.