After T cell receptor (TCR) engagement, the CARD11-Bcl10-Malt1 (CBM) complex oligomerizes to transduce NF-κB activating signals. Bcl10 is then degraded to limit NF-κB activation. The cDNA AK057716 (BinCARD-1) was reported to encode a novel CARD protein that interacts with Bcl10 and modestly inhibits NF-κB activation. In a later study, a second isoform, BinCARD-2, was identified. Here, we report that the cDNA AK057716 (BinCARD-1) is an incompletely spliced derivative of the gene product of C9orf89, whereas CARD19 (BinCARD-2) represents the properly spliced isoform, with conservation across diverse species. Immunoblotting revealed expression of CARD19 in T cells, but no evidence of BinCARD-1 expression, and microscopy demonstrated that endogenous CARD19 localizes to mitochondria. Although we confirmed that both BinCARD-1 and CARD19 can inhibit NF-κB activation and promote Bcl10 degradation when transiently overexpressed in HEK293T cells, loss of endogenous CARD19 expression had little effect on Bcl10-dependent NF-κB activation, activation of Malt1 protease function, or Bcl10

CARD19 is a mitochondrial protein of unknown function. While CARD19 was originally reported to regulate TCR-dependent NF-κB activation via interaction with BCL10, this function is not recapitulated ex vivo in primary murine CD8+ T cells. Here, we employ a combination of SIM, TEM, and confocal microscopy, along with proteinase K protection assays and proteomics approaches, to identify interacting partners of CARD19 in macrophages. Our data show that CARD19 is specifically localized to the outer mitochondrial membrane. Through deletion of functional domains, we demonstrate that both the distal C-terminus and transmembrane domain are required for mitochondrial targeting, whereas the CARD is not. Importantly, mass spectrometry analysis of 3×Myc-CARD19 immunoprecipitates reveals that CARD19 interacts with the components of the mitochondrial intermembrane bridge (MIB), consisting of mitochondrial contact site and cristae organizing system (MICOS) components MIC19, MIC25, and MIC60, and MICOS-interacting proteins SAMM50 and MTX2. These CARD19 interactions are in part dependent on a properly folded CARD. Consistent with previously reported phenotypes upon siRNA silencing of MICOS subunits, absence of CARD19 correlates with irregular cristae morphology. Based on these data, we propose that CARD19 is a previously unknown interacting partner of the MIB and the MIC19–MIC25–MIC60 MICOS subcomplex that regulates cristae morphology.

CARD19 is a mitochondrial protein of unknown function; gene expression databases indicate that CARD19 is highly expressed in myeloid cells. We have observed that Card19 −/− mice injected with LPS display elevated levels of some pro-inflammatory cytokines relative to Card19 +/+ mice. Because CARD19 is a mitochondrial protein and mitochondrial dysfunction is frequently associated with immune dysregulation and chronic inflammation, we sought to identify the function of CARD19 in macrophages. We demonstrated via super resolution microscopy that endogenous CARD19 colocalizes with mitochondrial markers in bone marrow derived macrophages (BMDMs) and has a punctate distribution. Through immunoprecipitation and mass spectrometry analyses, we determined that CARD19 interacts with MIC19 and other components of the mitochondrial contact site and cristae organizing system (MICOS) complex. We further demonstrated that CARD19 colocalizes with MIC19 in mitochondrial sub-domains via super resolution microscopy. We then measured outcomes of mitochondrial stress in BMDMs. Consistent with previous reports of MICOS deficiencies, we found that Card19 −/− BMDMs have a modestly decreased oxygen consumption rate (OCR) measured by a Seahorse extracellular flux analyzer relative to Card19 +/+ BMDMs. Additionally, Card19 −/− BMDMs display elevated levels of mitochondrial reaction oxygen species (mROS) and a population of mitochondria with a decreased mitochondrial membrane potential, as measured by flow cytometry. Based on these data, we propose that CARD19 may be a previously unknown regulator of MICOS function which potentially links this complex to other mitochondrial and/or non-mitochondrial CARD proteins in macrophages.