期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2022
卷号：119
期号：35
DOI：10.1073/pnas.2205590119
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance
One of the ways by which protein aggregates can propagate and lead to the progression of a neurodegenerative disease is by damaging the membrane that is destined to degrade these misfolded aggregates. The ESCRT machinery has been implicated in sealing these damaged membranes, and the nature of the membrane recruitment trigger signal for this machinery is a major open question. Here, we show in vitro that ALG-2 alone is sufficient to bring the ESCRT machinery to membranes in a Ca
2+-dependent manner.
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
2+ has been proposed to play a key signaling role in the process. Here, we show that the Ca
2+-binding regulatory protein ALG-2 binds directly to negatively charged membranes in a Ca
2+-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.
