Mechanism and cellular function of direct membrane binding by the ESCRT and ERES-associated Ca2+-sensor ALG-2

Sankalp Shukla; Wei Chen; Shanlin Rao; Serim Yang; Chenxi Ou; Kevin P Larsen; Gerhard Hummer; Phyllis I Hanson; James H Hurley

doi:10.1101/2023.10.17.562764

Mechanism and cellular function of direct membrane binding by the ESCRT and ERES-associated Ca²⁺-sensor ALG-2

bioRxiv [Preprint]. 2023 Oct 19:2023.10.17.562764. doi: 10.1101/2023.10.17.562764.

Authors

Sankalp Shukla^{1

2}, Wei Chen³, Shanlin Rao⁴, Serim Yang¹, Chenxi Ou^{1

2}, Kevin P Larsen^{1

2}, Gerhard Hummer^{4

5}, Phyllis I Hanson³, James H Hurley^{1

2

6}

Affiliations

¹ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
² California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA.
³ Department of Biological Chemistry, University of Michigan School of Medicine, 1150 W. Medical Center Drive, Ann Arbor, MI, 48109, USA.
⁴ Department of Theoretical Biophysics, Max Planck Institute of Biophysics, Frankfurt am Main, Germany.
⁵ Institute of Biophysics, Goethe University Frankfurt, Frankfurt am Main 60438, Germany.
⁶ Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, CA 94720, USA.

Abstract

Apoptosis Linked Gene-2 (ALG-2) is a multifunctional intracellular Ca²⁺ 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 endoplasmic reticulum exit sites (ERES). In the presence of Ca²⁺, 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 Ca²⁺ by a combination of electrostatic and hydrophobic interactions. By combining GUV-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 Ca²⁺ release but still localize to lysosomes following lysosomal Ca²⁺ 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 Ca²⁺-dependent membrane binding and its interplay with Ca²⁺-dependent protein binding in the cellular functions of ALG-2.

Keywords: ALG-2; ERES; ESCRTs; lysosome; molecular dynamics; reconstitution.

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Preprint

Abstract

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