Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720.
California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720.
Proc Natl Acad Sci U S A. 2022 Aug 30;119(35):e2205590119. doi: 10.1073/pnas.2205590119. Epub 2022 Aug 22.
The endosomal sorting complex required for transport (ESCRT) machinery is centrally involved in the repair of damage to both the plasma and lysosome membranes. ESCRT recruitment to sites of damage occurs on a fast time scale, and Ca has been proposed to play a key signaling role in the process. Here, we show that the Ca-binding regulatory protein ALG-2 binds directly to negatively charged membranes in a Ca-dependent manner. Next, by monitoring the colocalization of ALIX with ALG-2 on negatively charged membranes, we show that ALG-2 recruits ALIX to the membrane. Furthermore, we show that ALIX recruitment to the membrane orchestrates the downstream assembly of late-acting CHMP4B, CHMP3, and CHMP2A subunits along with the AAA ATPase VPS4B. Finally, we show that ALG-2 can also recruit the ESCRT-III machinery to the membrane via the canonical ESCRT-I/II pathway. Our reconstitution experiments delineate the minimal sets of components needed to assemble the entire membrane repair machinery and open an avenue for the mechanistic understanding of endolysosomal membrane repair.
内体分选复合物需要运输 (ESCRT) 机制在修复质膜和溶酶体膜损伤中起着核心作用。ESCRT 在损伤部位的募集发生在快速的时间尺度上,并且 Ca 被提议在该过程中发挥关键信号作用。在这里,我们表明 Ca 结合调节蛋白 ALG-2 以 Ca 依赖性的方式直接结合带负电荷的膜。接下来,通过监测带负电荷的膜上 ALIX 与 ALG-2 的共定位,我们表明 ALG-2 将 ALIX 募集到膜上。此外,我们表明 ALIX 募集到膜上协调了晚期作用的 CHMP4B、CHMP3 和 CHMP2A 亚基以及 AAA ATPase VPS4B 的下游组装。最后,我们表明 ALG-2 还可以通过规范的 ESCRT-I/II 途径将 ESCRT-III 机械募集到膜上。我们的重建实验描绘了组装整个膜修复机械所需的最小组件集,并为内体溶酶体膜修复的机制理解开辟了途径。