Activated G Protein Gαs Samples Multiple Endomembrane Compartments

Heterotrimeric G proteins are localized to the plasma membrane where they transduce extracellular signals to intracellular effectors. G proteins also act at intracellular locations, and can translocate between cellular compartments. For example, G(s) can leave the plasma membrane and move to the cell interior after activation. However, the mechanism of G(s) translocation and its intracellular destination are not known. Here we use bioluminescence resonance energy transfer (BRET) to show that after activation, G(s) rapidly associates with the endoplasmic reticulum, mitochondria, and endosomes, consistent with indiscriminate sampling of intracellular membranes from the cytosol rather than transport via a specific vesicular pathway. The primary source of G(s) for endosomal compartments is constitutive endocytosis rather than activity-dependent internalization. Recycling of G(s) to the plasma membrane is complete 25 min after stimulation is discontinued. We also show that an acylation-deacylation cycle is important for the steady-state localization of G(s) at the plasma membrane, but our results do not support a role for deacylation in activity-dependent G(s) internalization.