Macroautophagy is a degradative pathway that sequesters and transports cytosolic cargo in autophagosomes to lysosomes, and its deterioration affects intracellular proteostasis. Membrane dynamics accompanying autophagy are mostly elusive and depend on trafficking processes. RAB GTPase activating proteins (RABGAPs) are important factors for the coordination of cellular vesicle transport systems, and several TBC (TRE2-BUB2-CDC16) domain-containing RABGAPs are associated with autophagy. Employing C. elegans and human primary fibroblasts, we show that RAB3GAP1 and RAB3GAP2, which are components of the TBC domain-free RAB3GAP complex, influence protein aggregation and affect autophagy at basal and rapamycin-induced conditions. Correlating the activity of RAB3GAP1/2 with ATG3 and ATG16L1 and analyzing ATG5 punctate structures, we illustrate that the RAB3GAPs modulate autophagosomal biogenesis. Significant levels of RAB3GAP1/2 colocalize with members of the Atg8 family at lipid droplets, and their autophagy modulatory activity depends on the GTPase-activating activity of RAB3GAP1 but is independent of the RAB GTPase RAB3. Moreover, we analyzed RAB3GAP1/2 in relation to the previously reported suppressive autophagy modulators FEZ1 and FEZ2 and demonstrate that both reciprocally regulate autophagy. In conclusion, we identify RAB3GAP1/2 as novel conserved factors of the autophagy and proteostasis network.
Keywords: ATG, autophagy-related; ATG16L1; ATG3; BSA, bovine serum albumin; Bafi, bafilomycin A1; C. elegans, Caenorhabditis elegans; CALCOCO2, calcium binding and coiled-coil domain 2; DAPI, 4’, 6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; DPH, 1, 6-diphenyl-1, 3, 5-hexatriene; FEZ, fasciculation and elongation protein zeta; FEZ1; FEZ2; GABARAP, GABA(A) receptor-associated protein; GEF, guanine nucleotide exchange factor; GFP, green fluorescent protein; MAP1LC3, microtubule-associated protein 1 light chain 3; NBR1, neighbor of BRCA1 gene 1; PBS, phosphate-buffered saline; PE, phosphatidylethanolamine; RAB3GAP1; RAB3GAP2; RABGAP, RAB GTPase activating protein; SQSTM1, sequestosome 1; TBC domain, TRE2-BUB2-CDC16 domain; autophagy; eV, empty vector; lipid droplets; proteostasis; siRNA, small interfering RNA.