Activated G protein Gα_s 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, Gα_s can leave the plasma membrane and move to the cell interior after activation. However, the mechanism of Gα_s translocation and its intracellular destination are not known. Here we use bioluminescence resonance energy transfer (BRET) to show that after activation, Gα_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 Gα_s for endosomal compartments is constitutive endocytosis rather than activity-dependent internalization. Recycling of Gα_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 Gα_s at the plasma membrane, but our results do not support a role for deacylation in activity-dependent Gα_s internalization.