21Human Bax Inhibitor-1 (HsBI-1/TMBIM6) is the founding member of the evolutionary 22 conserved TMBIM superfamily of proteins that share sequence homology within the 23 transmembrane Bax inhibitor-containing motif (TMBIM). Mechanistically, BI-1/TMBIM6 and 24 all the other mammalian TMBIM proteins appear to be involved in the maintenance of calcium 25 homeostasis, and the crystal structure of a bacterial TMBIM protein, BsYetJ, suggests that the 26 protein is a pH-sensitive calcium leak. The budding yeast, Saccharomyces cerevisiae, has a single 27 TMBIM family member (YNL305C) named Bxi1p/Ybh3p. To determine the function of 28Bxi1p/Ybh3p, we overexpressed Bxi1p-EGFP in E. coli to determine if it is a calcium channel. We 29show that bacterial cells expressing Bxi1p-EGFP are more permeable to calcium than controls. 30Thus, our data suggests that yeast Bax inhibitor (Bxi1p) is a calcium channel in E. coli, lending 31 support to our proposal that Bxi1p is a bona fide member of the TMBIM family of proteins. 32Further, we use our bacterial system to show that gadolinium is an inhibitor of Bxi1p in vivo, 33 suggesting a path forward to identifying other small-molecular inhibitors of this clinically-34 important and highly conserved superfamily of proteins. Finally, parallel experiments revealed 35 that the human Bax Inhibitor-1 (HsBI-1/TMBIM6) is also a calcium channel in bacteria that can 36 be inhibited by gadolinium. 37

Human Bax Inhibitor‐1 (HsBI‐1/TMBIM6) is the founding member of the evolutionary conserved TMBIM superfamily of proteins that share sequence homology within the transmembrane Bax inhibitor‐containing motif (TMBIM). Mechanistically, BI‐1/TMBIM6 and all the other mammalian TMBIM proteins appear to be involved in the maintenance of calcium homeostasis, and the crystal structure of a bacterial TMBIM protein, BsYetJ, suggests that the protein is a pH‐sensitive calcium leak. The budding yeast, Saccharomyces cerevisiae, has a single TMBIM family member (YNL305C) named Bxi1p/Ybh3p. To determine the function of Bxi1p/Ybh3p, we overexpressed Bxi1p‐EGFP in E. coli to determine if it is a calcium channel. We show that bacterial cells expressing Bxi1p‐EGFP are more permeable to calcium than controls. Thus, our data suggests that yeast Bax inhibitor (Bxi1p) is a calcium channel in E. coli, lending support to our proposal that Bxi1p is a bona fide member of the TMBIM family of proteins. Further, we use our bacterial system to show that gadolinium is an inhibitor of Bxi1p in vivo, suggesting a path forward to identifying other small‐molecular inhibitors of this clinically‐important and highly conserved superfamily of proteins. Finally, parallel experiments revealed that the human Bax Inhibitor‐1 (HsBI‐1/TMBIM6) is also a calcium channel in bacteria that can be inhibited by gadolinium. Support or Funding Information Our laboratory is supported by grant NIGMS R15 GM110578, awarded to N. Austriaco.

Human Bax Inhibitor‐1 (HsBI‐1/TMBIM6) is the founding member of the evolutionary conserved TMBIM superfamily of proteins that share sequence homology within the transmembrane Bax inhibitor‐containing motif (TMBIM). Mechanistically, BI‐1/TMBIM6 and all the other mammalian TMBIM proteins appear to be involved in the maintenance of calcium homeostasis, and the crystal structure of a bacterial TMBIM protein, BsYetJ, suggests that the protein is a pH‐sensitive calcium leak. The budding yeast, Saccharomyces cerevisiae, has a single TMBIM family member (YNL305C) named Bxi1p/Ybh3p. To determine the function Bxi1p/Ybh3p, we overexpressed Bxi1p‐GFP in E. coli to interrogate its putative calcium channel function. We show that bacterial cells expressing Bxi1p‐GFP are more permeable to calcium than controls. Our data suggests that yeast Bax inhibitor (Bxi1p) is a calcium channel in E. coli, lending support to our proposal that Bxi1p is a bona fide member of the TMBIM family of proteins. We use our bacterial system to show that gadolinium is an inhibitor of Bxi1p in vivo, suggesting a path forward to identifying other small‐molecular inhibitors of this clinically‐important and highly conserved superfamily of proteins. Finally, parallel experiments revealed that the human Bax Inhibitor‐1 (HsBI‐1/TMBIM6) is also a calcium channel in bacteria that can be inhibited by gadolinium.