Bunker, E. N.; Fischer, T. D.; Zhu, P.-P.; Le Guerroue, F.; Youle, R. J.

Activation of the cGAS-STING pathway stimulates innate immune signaling as well as LC3B lipidation and ubiquitylation at Golgi-related vesicles upon STING trafficking. Although ubiquitylation at these subcellular sites has been associated with regulating NFkB-related innate immune signaling, the mechanisms of Golgi-localized polyubiquitin chain regulation of immune signaling is not well understood. We report here that the ubiquitin- and LC3B-binding proteins, TNIP1 and autophagy receptors p62, NBR1, NDP52, TAX1BP1, and OPTN associate with STING-induced ubiquitin and LC3B-labeled vesicles, and that p62 and NBR1 act redundantly in spatial clustering of the LC3B-labeled vesicles in the perinuclear region. We also find that while TBK1 kinase activity is not required for the recruitment of TNIP1 and the autophagy receptors, it also plays a role in sequestration of the LC3B-labeled vesicles. The ubiquitin binding domains, rather than the LC3B-interacting regions, of TNIP1 and OPTN are specifically important for their recruitment to Ub/LC3B-associated perinuclear vesicles, while OPTN is also recruited through a TBK1-dependent mechanism. Functionally, we find that TNIP1 and OPTN play a role in STING-mediated innate immune signaling, with TNIP1 acting as a significant negative regulator of both NFkB- and Interferon-mediated gene expression. Together, these results highlight autophagy-independent mechanisms of autophagy receptors and TNIP1 with unanticipated roles in regulating STING-mediated innate immunity.