Arf5-mediated regulation of mTORC1 at the plasma membrane

Abstract

The mechanistic target of rapamycin (mTOR) kinase regulates a major signalling pathway in eukaryotic cells. In addition to regulation of mTORC1 at lysosomes, mTORC1 is also localised at other locations. However, little is known about the recruitment and activation of mTORC1 at non-lysosomal sites. To identify regulators of mTORC1 recruitment to non-lysosomal compartments, novel interacting partners with the mTORC1 subunit, Raptor, were identified using immunoprecipitation and mass spectrometry. We show that one of the interacting partners, Arf5, is a novel regulator of mTORC1 signalling at plasma membrane ruffles. Arf5-GFP localizes with endogenous mTOR at PI3,4P2 enriched membrane ruffles together the GTPase required for mTORC1 activation, Rheb. Knockdown of Arf5 reduced the recruitment of mTOR to membrane ruffles. The activation of mTORC1 at membrane ruffles was directly demonstrated using a plasma membrane-targeted mTORC1 biosensor and Arf5 shown to enhance the phosphorylation of the mTORC1 biosensor substrate. In addition, endogenous Arf5 was shown to be required for rapid activation of mTORC1-mediated S6 phosphorylation following nutrient starvation and re-feeding. Our findings reveal a novel Arf5-dependent pathway for recruitment and activation of mTORC1 at plasma membrane ruffles, a process relevant for spatial and temporal regulation of mTORC1 by receptor and nutrient stimuli.