CISD2 counteracts the inhibition of ER-mitochondrial calcium transfer by anti-apoptotic BCL-2

CISD2, a 2Fe2S cluster domain-containing protein, is implicated in Wolfram syndrome type 2, longevity and cancer. CISD2 is part of a ternary complex with IP₃ receptors (IP₃Rs) and anti-apoptotic BCL-2 proteins and enhances BCL-2's anti-autophagic function. Here, we examined how CISD2 impacted the function of BCL-2 in apoptosis and in controlling IP₃R-mediated Ca²⁺ signaling. Using purified proteins, we found a direct interaction between the cytosolic region of CISD2 and BCL-2's BH4 domain with a submicromolar affinity. At the functional level, the cytosolic region of CISD2, as a purified protein, did not affect the ability of BCL-2 to inhibit BAX-pore formation. In a cellular context, loss of CISD2 did not impede the suppression of apoptosis by BCL-2. Also, in Ca²⁺-signaling assays, absence of CISD2 did not affect the inhibition of IP₃R-mediated Ca²⁺ release by BCL-2. Combined, these experiments indicate that CISD2 is not essential for BCL-2 function in apoptosis and cytosolic Ca²⁺ signaling. Instead, CISD2 overexpression enhanced BCL-2-mediated suppression of cytosolic IP₃R-mediated Ca²⁺ release. However, consistent with the presence of CISD2 and BCL-2 at mitochondria-associated ER membranes (MAMs), the most striking effect was observed at the level of ER-mitochondrial Ca²⁺ transfer. While BCL-2 overexpression inhibited ER-mitochondrial Ca²⁺ transfer, overexpression of CISD2 together with BCL-2 abrogated the effect of BCL-2. The underlying mechanism is linked to ER-mitochondrial contact sites, since BCL-2 reduced ER-mitochondrial contact sites while co-expression of CISD2 together with BCL-2 abolished this effect. These findings reveal a unique interplay between BCL-2 and CISD2 at Ca²⁺-signaling nanodomains between ER and mitochondria.