Mechanism and cellular function of direct membrane binding by the ESCRT and ERES-associated Ca 2+ -sensor ALG-2

• Published in: Proceedings of the National Academy of Sciences - PNAS 2024-02, Vol.121 (9), p.e2318046121
• Main Authors: Shukla, Sankalp, Chen, Wei, Rao, Shanlin, Yang, Serim, Ou, Chenxi, Larsen, Kevin P, Hummer, Gerhard, Hanson, Phyllis I, Hurley, James H
• Format: Article
• Language: English
• Subjects: Cell Division; Cell Physiological Phenomena; Endosomal Sorting Complexes Required for Transport - genetics; Intracellular Membranes; Membranes
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.2318046121

Apoptosis linked Gene-2 (ALG-2) is a multifunctional intracellular Ca sensor and the archetypal member of the penta-EF hand protein family. ALG-2 functions in the repair of damage to both the plasma and lysosome membranes and in COPII-dependent budding at ndoplasmic eticulum xit ites (ERES). In the presence of Ca , ALG-2 binds to ESCRT-I and ALIX in membrane repair and to SEC31A at ERES. ALG-2 also binds directly to acidic membranes in the presence of Ca by a combination of electrostatic and hydrophobic interactions. By combining giant unilamellar vesicle-based experiments and molecular dynamics simulations, we show that charge-reversed mutants of ALG-2 at these locations disrupt membrane recruitment. ALG-2 membrane binding mutants have reduced or abrogated ERES localization in response to Thapsigargin-induced Ca release but still localize to lysosomes following lysosomal Ca release. In vitro reconstitution shows that the ALG-2 membrane-binding defect can be rescued by binding to ESCRT-I. These data thus reveal the nature of direct Ca -dependent membrane binding and its interplay with Ca -dependent protein binding in the cellular functions of ALG-2.