TBC1D4 antagonizes RAB2A-mediated autophagic and endocytic pathways.

ABSTRACT

Macroautophagic/autophagic and endocytic pathways play essential roles in maintaining homeostasis at different levels. It remains poorly understood how both pathways are coordinated and fine-tuned for proper lysosomal degradation of diverse cargoes. We and others recently identified a Golgi-resident RAB GTPase, RAB2A, as a positive regulator that controls both autophagic and endocytic pathways. In the current study, we report that TBC1D4 (TBC1 domain family member 4), a TBC domain-containing protein that plays essential roles in glucose homeostasis, suppresses RAB2A-mediated autophagic and endocytic pathways. TBC1D4 bound to RAB2A through its N-terminal PTB2 domain, which impaired RAB2A-mediated autophagy at the early stage by preventing ULK1 complex activation. During the late stage of autophagy, TBC1D4 impeded the association of RUBCNL/PACER and RAB2A with STX17 on autophagosomes by direct interaction with RUBCNL via its N-terminal PTB1 domain. Disruption of the autophagosomal trimeric complex containing RAB2A, RUBCNL and STX17 resulted in defective HOPS recruitment and eventually abortive autophagosome-lysosome fusion. Furthermore, TBC1D4 inhibited RAB2A-mediated endocytic degradation independent of RUBCNL. Therefore, TBC1D4 and RAB2A form a dual molecular switch to modulate autophagic and endocytic pathways. Importantly, hepatocyte- or adipocyte-specific tbc1d4 knockout in mice led to elevated autophagic flux and endocytic degradation and tissue damage. Together, this work establishes TBC1D4 as a critical molecular brake in autophagic and endocytic pathways, providing further mechanistic insights into how these pathways are intertwined both in vitro and in vivo.Abbreviations ACTB actin beta; ATG9 autophagy related 9; ATG14 autophagy related 14; ATG16L1 autophagy related 16 like 1; CLEM correlative light electron microscopy; Ctrl control; DMSO dimethyl sulfoxide; EGF epidermal growth factor; EGFR epidermal growth factor receptor; FL full length; GAP GTPase-activating protein; GFP green fluorescent protein; HOPS homotypic fusion and protein sorting; IP immunoprecipitation; KD knockdown; KO knockout; LAMP1 lysosomal associated membrane protein 1; MAP1LC3B/LC3B microtubule associated protein 1 light chain 3 beta; OE overexpression; PG phagophore; PtdIns3K class III phosphatidylinositol 3-kinase; SLC2A4/GLUT4 solute carrier family 2 member 4; SQSTM1/p62 sequestosome 1; RUBCNL/PACER rubicon like autophagy enhancer; STX17 syntaxin 17; TAP tandem affinity purification; TBA total bile acid; TBC1D4 TBC1 domain family member 4; TUBA1B tubulin alpha 1b; ULK1 unc-51 like autophagy activating kinase 1; VPS39 VPS39 subunit of HOPS complex; WB western blot; WT wild type.