Biochemical Characterization of Two Analogues of the Apoptosis-linked Gene 2 Protein in Dictyostelium discoideumand Interaction with a Physiological Partner in Mammals, Murine Alix

Abstract

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 microm, 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 (K(D) of 1 microm) 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 (K(D) = 0.6 microm) with Dd-ALG-2a but not with Dd-ALG-2b, and the interaction strictly depended upon calcium ions. The DeltaNter 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.