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Originally published In Press as doi:10.1074/jbc.M201718200 on April 1, 2002

J. Biol. Chem., Vol. 277, Issue 24, 21947-21954, June 14, 2002
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Biochemical Characterization of Two Analogues of the Apoptosis-linked Gene 2 Protein in Dictyostelium discoideum and Interaction with a Physiological Partner in Mammals, Murine Alix*

Laurence AubryDagger , Sara MatteiDagger , Béatrice Blot§, Rémy Sadoul§, Michel SatreDagger , and Gérard KleinDagger

From the Dagger  Laboratoire de Biochimie et Biophysique des Systèmes Intégrés (Unité Mixte de Recherche 5092 CNRS-Commissariat à l'Energie Atomique-Université Joseph-Fourier), Département Réponse et Dynamique Cellulaires/Biochimie et Biophysique des Systèmes Intégres, Commissariat à l'Energie Atomique-Grenoble, 17 rue des Martyrs, 38054 Grenoble Cedex 9 and the § Equipe Mixte EMI 0108 INSERM-Université Joseph-Fourier, Pavillon de Neurologie, Centre Hospitalier Universitaire de Grenoble, Boite Postale 217, 38043 Grenoble Cedex 9, France

Two homologues, Dd-ALG-2a and Dd-ALG-2b, of the mammalian calcium-binding protein ALG-2 (apoptosis-linked gene 2) have been characterized in the cellular slime mold Dictyostelium discoideum. Fluorescence titrations showed that both proteins bind calcium ions with affinities (Ca2+)0.5 of 30 and 450 µM, respectively, at sites specific to calcium. Calcium ion binding resulted in changes of conformation associated with the unmasking of hydrophobic regions of the proteins. Surface plasmon resonance analysis showed that Dd-ALG-2a homodimers formed (KD of 1 µM) at calcium ion concentrations similar to those necessary for Ca2+-induced conformational changes. Deletion of the hydrophobic N-terminal sequence or EF-hand 5 of Dd-ALG-2a prevented dimerization. The Dd-ALG-2b homodimer was not detected, and the Dd-ALG-2a/2b heterodimer formed only when Dd-ALG-2b was the immobilized partner. Murine Alix formed a heterodimer (KD = 0.6 µM) with Dd-ALG-2a but not with Dd-ALG-2b, and the interaction strictly depended upon calcium ions. The Delta Nter construct of Dd-ALG-2a lost its interaction capacity with mouse Alix. The genes encoding both proteins, Dd-alg-2a and -2b, were expressed in growing cells. The levels of mRNA were at a maximum during aggregation (4-8 h) and decreased rapidly thereafter. In contrast, the levels of proteins remained fairly stable. Dd-ALG-2a and Dd-ALG-2b were found to be dispensable for growth and development, based on the finding that single Dd-alg2a- or Dd-alg-2b- and double Dd-alg2a-/Dd-alg-2b- mutant cell lines showed normal growth in axenic medium or on bacterial lawns and exhibited unaltered development.

* This work was supported in part by the Commissariat à l'Energie Atomique, CNRS, INSERM, the University Joseph Fourier Grenoble, and grants from the Association pour la Recherche sur le Cancer. A preliminary report of this work was presented at the Dictyostelium International Meetings in Dundee, United Kingdom, July 30-August 4, 2000 and San Diego, CA, July 22-26, 2001.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF358913 and AF358911 for Dd-alg-2a and AF358914 and AF358912 for Dd-alg-2b.

To whom correspondence should be addressed. Tel.: 33-438-784-661; Fax: 33-438-786-107; E-mail: gklein@cea.fr.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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