RAB3GAP1 and RAB3GAP2 modulate basal and rapamycin-induced autophagy

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 <i>C. elegans</i> 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.

巨自噬（Macroautophagy）是一类将胞质货物通过自噬体（autophagosomes）隔离并转运至溶酶体（lysosomes）的降解通路，其功能异常会破坏细胞内蛋白质稳态（proteostasis）。伴随自噬发生的膜动态变化大多尚未明确，且依赖于细胞囊泡转运通路。RAB GTP酶激活蛋白（RAB GTPase activating proteins, RABGAPs）是协调细胞囊泡转运系统的关键调控因子，多种含TBC（TRE2-BUB2-CDC16）结构域的RABGAPs已被证实与自噬相关。
本研究以秀丽隐杆线虫（*C. elegans*）与人原代成纤维细胞为实验模型，证实不含TBC结构域的RAB3GAP复合物组分RAB3GAP1与RAB3GAP2，可在基础状态及雷帕霉素（rapamycin）诱导条件下影响蛋白质聚集与自噬进程。通过关联RAB3GAP1/2与自噬相关蛋白ATG3、ATG16L1的活性，并分析ATG5点状聚集结构，我们阐明RAB3GAPs可调控自噬体的生物发生过程。大量RAB3GAP1/2可与Atg8家族蛋白在脂滴（lipid droplets）处发生共定位，其调控自噬的活性依赖于RAB3GAP1的GTP酶激活活性，但不依赖于RAB GTP酶RAB3。此外，我们结合此前报道的自噬负调控因子FEZ1与FEZ2分析了RAB3GAP1/2的功能，证实二者可相互调控自噬。综上，本研究鉴定RAB3GAP1/2为自噬与蛋白质稳态网络中全新的保守调控因子。