Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, 37077, Göttingen, Germany.
Institute for Biosafety in Plant Biotechnology, Julius Kuehn-Institute, 06484, Quedlinburg, Germany.
Sci Rep. 2020 Jul 31;10(1):12956. doi: 10.1038/s41598-020-69637-0.
The Atg12-Atg5/Atg16L1 complex is recruited by WIPI2b to the site of autophagosome formation. Atg16L1 is an effector of the Golgi resident GTPase Rab33B. Here we identified a minimal stable complex of murine Rab33B(30-202) Q92L and Atg16L1(153-210). Atg16L1(153-210) comprises the C-terminal part of the Atg16L1 coiled-coil domain. We have determined the crystal structure of the Rab33B Q92L/Atg16L1(153-210) effector complex at 3.47 Å resolution. This structure reveals that two Rab33B molecules bind to the diverging α-helices of the dimeric Atg16L1 coiled-coil domain. We mutated Atg16L1 and Rab33B interface residues and found that they disrupt complex formation in pull-down assays and cellular co-localization studies. The Rab33B binding site of Atg16L1 comprises 20 residues and immediately precedes the WIPI2b binding site. Rab33B mutations that abolish Atg16L binding also abrogate Rab33B association with the Golgi stacks. Atg16L1 mutants that are defective in Rab33B binding still co-localize with WIPI2b in vivo. The close proximity of the Rab33B and WIPI2b binding sites might facilitate the recruitment of Rab33B containing vesicles to provide a source of lipids during autophagosome biogenesis.
Atg12-Atg5/Atg16L1 复合物通过 WIPI2b 招募到自噬体形成部位。Atg16L1 是高尔基体驻留 GTPase Rab33B 的效应物。在这里，我们鉴定了一种最小稳定的鼠 Rab33B(30-202) Q92L 和 Atg16L1(153-210)复合物。Atg16L1(153-210)包含 Atg16L1 卷曲螺旋域的 C 端部分。我们已经确定了 Rab33B Q92L/Atg16L1(153-210)效应复合物的晶体结构，分辨率为 3.47 Å。该结构揭示了两个 Rab33B 分子结合到二聚体 Atg16L1 卷曲螺旋域的发散α-螺旋上。我们突变了 Atg16L1 和 Rab33B 界面残基，发现它们在下拉实验和细胞共定位研究中破坏复合物的形成。Atg16L1 的 Rab33B 结合位点包含 20 个残基，并且紧邻 WIPI2b 结合位点。使 Atg16L 结合丧失的 Rab33B 突变也使 Rab33B 与高尔基体堆叠的关联丧失。在体内，在 Rab33B 结合中存在缺陷的 Atg16L1 突变体仍然与 WIPI2b 共定位。Rab33B 和 WIPI2b 结合位点的接近可能促进了含有 Rab33B 的囊泡的募集，为自噬体生物发生过程中提供脂质来源。
