Abstract
The monomeric GTPase RalB controls crucial physiological processes, including autophagy and invasion, but it still remains unclear how this multi-functionality is achieved. Previously, we reported that the RalGEF (Guanine nucleotide Exchange Factor) RGL2 binds and activates RalB to promote invasion. Here we show that RGL2, a major activator of RalB, is also required for autophagy. Using a novel automated image analysis method, Endomapper, we quantified the endogenous localization of the RGL2 activator and its substrate RalB at different endomembrane compartments, in an isogenic normal and Ras-transformed cell model. In both normal and Ras-transformed cells, we observed that RGL2 and RalB substantially localize at early and recycling endosomes, and to lesser extent at autophagosomes, but not at trans-Golgi. Interestingly the use of a FRET-based RalB biosensor indicated that RalB signaling is active at these endomembrane compartments at basal level in rich medium. Furthermore, induction of autophagy by nutrient starvation led to a considerable reduction of early and recycling endosomes, in contrast to the expected increase of autophagosomes, in both normal and Ras-transformed cells. However, autophagy mildly affected relative abundances of both RGL2 and RalB at early and recycling endosomes, and at autophagosomes. Interestingly, RalB activity increased at autophagosomes upon starvation in normal cells. These results suggest that the contribution of endosome membranes (carrying RGL2 and RalB molecules) increases total pool of RGL2-RalB at autophagosome forming compartments and might contribute to amplify RalB signaling to support autophagy.