American Society for Biochemistry and Molecular Biology
Journal of Biological Chemistry vol:287 issue:4 pages:2544-2557
Bax Inhibitor-1 (BI-1) is a multi-transmembrane domain-spanning endoplasmic reticulum (ER)-located protein that is evolutionarily conserved and protects against apoptosis and ER stress. Furthermore, BI-1 is proposed to modulate ER Ca2+ homeostasis by acting as a Ca2+-leak channel. Based on experimental determination of the BI-1 topology, we propose that its C-terminus forms a Ca2+ pore responsible for its Ca2+-leak properties. We utilized a set of C-terminal peptides to screen for Ca2+-leak activity in unidirectional 45Ca2+-flux experiments and identified an α-helical 20-amino-acid peptide causing Ca2+ leak from the ER. The Ca2+ leak was independent of endogenous ER Ca2+-release channels or other Ca2+-leak mechanisms, namely translocons and presenilins. The Ca2+-permeating property of the peptide was confirmed in lipid-bilayer experiments. Using mutant peptides, we identified critical residues responsible for the Ca2+-leak properties of this BI-1 peptide, including a series of critical negatively charged aspartate residues. Using peptides corresponding to the equivalent BI-1 domain from various organisms, we found that the Ca2+ -leak properties were conserved among animal, but not plant and yeast orthologs. By mutating one of the critical aspartate residues in the proposed Ca2+-channel pore in full-length BI-1, we found that D213 was essential for BI-1 dependent ER Ca2+-leak. Thus, we elucidated residues critically important for BI-1-mediated Ca2+ leak and its potential channel pore. Remarkably, one of these residues was not conserved among plant and yeast BI-1 orthologs, indicating that the ER Ca2+-leak properties of BI-1 are an added function during evolution.