Ubiquitin and the ubiquitin-like protein ATG8 are covalently attached to their respective targets via a coordinated cascade involving E1 activating, E2 conjugating and E3 ligating enzymes.Whereas ubiquitin is conferred to proteins as mono-and/or polymer(s) to alter their stability,localization and/or activity, ATG8 is conjugated to the phospholipid phosphatidylethanolamine (PE).The best understood function of ATG8 is during autophagy.Here, ATG8-PE conjugates are incorporated into both layers of incipient autophagosomes and serve as multipurpose docking sites for autophagosomal cargo receptors as well as regulatory factors that drive formation and maturation of autophagosomes.Mammalian cells harbor six ATG8 family members that can all be lipidated.However, it is currently unclear to what extent these proteins are functionally redundant or play unique roles.Here, we report an unexpected autophagy-independent link between the GABARAP subfamily of mammalian ATG8 proteins and the ubiquitin proteasome system.We identified an E3 ubiquitin ligase complex composed of the Cullin-3 scaffold, the RING finger protein RBX1 and the dimerizing substrate adaptors KBTBD6 and KBTBD7.Furthermore, we found that this ligase mediates ubiquitylation and subsequent proteasomal degradation of TIAM1, a guanine nucleotide exchange factors for the Rho GTPase Rac1.Conversely, stabilization of TIAM1 upon KBTBD6 and/or KBTBD7 depletion resulted in decreased membrane-bound Rac1 activity concomitant with altered actin morphology and migration.Spatial restricted signaling is a key element of Rac1 regulation.Intriguingly, we uncovered that engagement of TIAM1 by CUL3KBTBD6/KBTBD7 required interaction with members of the GABARAP subfamily of ATG8 proteins and that this binding was mediated by ATG8 interaction regions present adjacent to the KELCH domain in both substrate adaptors.Importantly, neither the CRL3 complex nor its substrate was found to be subject to autophagosomal degradation.Rather, ATG8-binding seems to mediate targeting of CUL3KBTBD6/KBTBD7 to TIAM1-containing membrane compartments.Concurrently,conditions that lead to increased ATG8 lipidation such as mTOR inhibition yielded increased TIAM1 ubiquitylation and decreased Rac1 activity.Together, we identified an autophagy-independent role of a subgroup of mammalian ATG8 proteins as binding platform that facilitate spatial restriction of Rac1 signaling by an ubiquitin E3 ligase complex.