Significance

Apoptosis linked Gene-2 (ALG-2) transduces Ca2+ signals to recruit proteins to the plasma membrane, lysosomes, and endoplasmic reticulum (ER) exit sites in many biological processes. The mechanism whereby ALG-2 interprets the Ca2+ signal for protein–protein interactions is well understood, but Ca2+ modulation of membrane binding is not. Here, we show how Ca2+ controls ALG-2 membrane binding. The presence of Ca2+ neutralizes electrostatic repulsion between apo-ALG-2 and acidic phospholipid membranes to allow membrane engagement by a surface that we characterize in the study.

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 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 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.

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Data, Materials, and Software Availability

All study data are included in the article and/or SI Appendix.

Acknowledgments

We thank Liv Jensen for help with data analysis script. This research was supported by Hoffmann-La Roche as part of the Alliance for Therapies in Neuroscience (J.H.H.) and the NIH grants R01 GM122434 (P.I.H.) and F32 AI155226 (K.P.L.). Other unrelated work in the Hurley lab is funded by Genentech.

Author contributions

S.S., G.H., P.I.H., and J.H.H. designed research; S.S., W.C., S.R., and S.Y. performed research; C.O. and K.P.L. contributed new reagents/analytic tools; S.S., W.C., S.R., and C.O. analyzed data; and S.S., S.R., G.H., P.I.H., and J.H.H. wrote the paper.

Competing interests

J.H.H. is a founder and consultant for Casma Therapeutics.

Supporting Information

Appendix 01 (PDF)

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Information & Authors

Information

Published in

Go to Proceedings of the National Academy of Sciences
Go to Proceedings of the National Academy of Sciences
Proceedings of the National Academy of Sciences
Vol. 121 | No. 9
February 27, 2024
PubMed: 38386713

Classifications

Data, Materials, and Software Availability

All study data are included in the article and/or SI Appendix.

Submission history

Received: October 17, 2023
Accepted: January 17, 2024
Published online: February 22, 2024
Published in issue: February 27, 2024

Keywords

  1. ESCRT
  2. membrane repair
  3. calcium-binding protein
  4. reconstitution
  5. lysosome

Acknowledgments

We thank Liv Jensen for help with data analysis script. This research was supported by Hoffmann-La Roche as part of the Alliance for Therapies in Neuroscience (J.H.H.) and the NIH grants R01 GM122434 (P.I.H.) and F32 AI155226 (K.P.L.). Other unrelated work in the Hurley lab is funded by Genentech.
Author Contributions
S.S., G.H., P.I.H., and J.H.H. designed research; S.S., W.C., S.R., and S.Y. performed research; C.O. and K.P.L. contributed new reagents/analytic tools; S.S., W.C., S.R., and C.O. analyzed data; and S.S., S.R., G.H., P.I.H., and J.H.H. wrote the paper.
Competing Interests
J.H.H. is a founder and consultant for Casma Therapeutics.

Notes

This article is a PNAS Direct Submission.

Authors

Affiliations

Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720
California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720
Department of Biological Chemistry, University of Michigan School of Medicine, Ann Arbor, MI 48109
Shanlin Rao
Department of Theoretical Biophysics, Max Planck Institute of Biophysics, Frankfurt am Main 60438, Germany
Serim Yang
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720
Chenxi Ou
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720
California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720
California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720
Department of Theoretical Biophysics, Max Planck Institute of Biophysics, Frankfurt am Main 60438, Germany
Institute of Biophysics, Goethe University Frankfurt, Frankfurt am Main 60438, Germany
Department of Biological Chemistry, University of Michigan School of Medicine, Ann Arbor, MI 48109
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720
California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720
Helen Wills Neuroscience Institute, University of California, Berkeley, CA 94720

Notes

1
To whom correspondence may be addressed. Email: [email protected].

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