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Altered levels of intracellular calcium (Ca2+) are a highly prevalent feature in different forms of cardiac injury, producing changes in contractility, arrhythmias, and mitochondrial dysfunction. In cardiac ischemia-reperfusion injury, mitochondrial Ca2+ overload leads to pathological production of reactive oxygen species (ROS), activates the permeability transition, and cardiomyocyte death. Here we investigated the cardiac phenotype caused by deletion of EF-hand domain-containing protein D1 (Efhd1 −/−), a Ca2+-binding mitochondrial protein whose function is poorly understood. Efhd1 −/− mice are viable and have no adverse cardiac phenotypes. They feature reductions in basal ROS levels and mitoflash events, both important precursors for mitochondrial injury, though cardiac mitochondria have normal susceptibility to Ca2+ overload. Notably, we also find that Efhd1 −/− mice and their cardiomyocytes are resistant to hypoxic injury. [Display omitted] • EFHD1 localizes to the mitochondrial outer membrane and intermembrane space. • Efhd1 −/− mice exhibit normal cardiac function. • Efhd1 −/− mice have reduced mitochondrial Ca2+, ΔΨ, ROS, and mitoflash levels. • Efhd1 −/− mouse cardiomyocytes are resistant to ischemia-reperfusion injury. [ABSTRACT FROM AUTHOR]

Mitochondrial outer membrane ⍺-helical proteins play critical roles in mitochondrial-cytoplasmic communication, but the rules governing the targeting and insertion of these biophysically diverse proteins remain unknown. Here, we first defined the complement of required mammalian biogenesis machinery through genome-wide CRISPRi screens using topologically distinct membrane proteins. Systematic analysis of nine identified factors across 21 diverse ⍺-helical substrates reveals that these components are organized into distinct targeting pathways that act on substrates based on their topology. NAC is required for the efficient targeting of polytopic proteins, whereas signal-anchored proteins require TTC1, a cytosolic chaperone that physically engages substrates. Biochemical and mutational studies reveal that TTC1 employs a conserved TPR domain and a hydrophobic groove in its C-terminal domain to support substrate solubilization and insertion into mitochondria. Thus, the targeting of diverse mitochondrial membrane proteins is achieved through topological triaging in the cytosol using principles with similarities to ER membrane protein biogenesis systems. [Display omitted] • Genome-wide screens identify biogenesis factors for diverse ⍺-helical OMM proteins • Parallel pathways mediate cytosolic targeting based on substrate topology • The NAC complex selectively targets polytopic proteins to the outer membrane • TTC1 is a cytosolic chaperone involved in OMM signal-anchored protein biogenesis Muthukumar et al. use genome-wide and systematic arrayed CRISPRi screens to identify ⍺-helical OMM protein biogenesis and quality control factors that triage substrates by topology. They establish NAC as a targeting factor for polytopic proteins and identify TTC1 as a cytosolic chaperone for signal-anchored proteins, using a C-terminal hydrophobic groove. [ABSTRACT FROM AUTHOR]

The mitochondrial outer membrane creates a diffusion barrier between the cytosol and the mitochondrial intermembrane space, allowing the exchange of metabolic products, important for efficient mitochondrial function in neurons. The ganglioside-induced differentiation-associated protein 1 (GDAP1) is a mitochondrial outer membrane protein with a critical role in mitochondrial dynamics and metabolic balance in neurons. Missense mutations in the GDAP1 gene are linked to the most common human peripheral neuropathy, Charcot-Marie-Tooth disease (CMT). GDAP1 is a distant member of the glutathione- S -transferase (GST) superfamily, with unknown enzymatic properties or functions at the molecular level. The structure of the cytosol-facing GST-like domain has been described, but there is no consensus on how the protein interacts with the mitochondrial outer membrane. Here, we describe a model for GDAP1 assembly on the membrane using peptides vicinal to the GDAP1 transmembrane domain. We used oriented circular dichroism spectroscopy (OCD) with synchrotron radiation to study the secondary structure and orientation of GDAP1 segments at the outer and inner surfaces of the outer mitochondrial membrane. These experiments were complemented by small-angle X-ray scattering, providing the first experimental structural models for full-length human GDAP1. The results indicate that GDAP1 is bound into the membrane via a single transmembrane helix, flanked by two peripheral helices interacting with the outer and inner leaflets of the mitochondrial outer membrane in different orientations. Impairment of these interactions could be a mechanism for CMT in the case of missense mutations affecting these segments instead of the GST-like domain. [Display omitted] • The segments before and after the transmembrane domain in GDAP1 bind to membranes. • Peptide orientation can be followed by oriented CD spectroscopy. • Full-length GDAP1 conformation observed at low resolution with X-ray scattering. • First experiment-based full-length model of GDAP1 [ABSTRACT FROM AUTHOR]

Mitochondria, composed of two membranes, play a key role in energy production in eukaryotic cells. The main function of the inner membrane is oxidative phosphorylation, while the mitochondrial outer membrane (MOM) seems to control the energy flux and exchange of various charged metabolites between mitochondria and the cytosol. Metabolites cross MOM via the various isoforms of voltage-dependent anion channel (VDAC). In turn, VDACs interact with some enzymes, other proteins and molecules, including drugs. This work aimed to analyze various literature experimental data related to targeting mitochondrial VDACs and VDAC-kinase complexes on the basis of the hypothesis of generation of the outer membrane potential (OMP) and OMP-dependent reprogramming of cell energy metabolism. Our previous model of the VDAC-hexokinase-linked generation of OMP was further complemented in this study with an additional regulation of the MOM permeability by the OMP-dependent docking of cytosolic proteins like tubulin to VDACs. Computational analysis of the model suggests that OMP changes might be involved in the mechanisms of apoptosis promotion through the so-called transient hyperpolarization of mitochondria. The high concordance of the performed computational estimations with many published experimental data allows concluding that OMP generation under physiological conditions is highly probable and VDAC might function as an OMP-dependent gatekeeper of mitochondria, controlling cell life and death. The proposed model of OMP generation allows understanding in more detail the mechanisms of cancer death resistance and anticancer action of various drugs and treatments influencing VDAC voltage-gating properties, VDAC content, mitochondrial hexokinase activity and VDAC-kinase interactions in MOM. [Display omitted] • Kinase-linked generation of the outer membrane potential (OMP) in mitochondria • Dependence of OMP generation on the VDAC blockage by proteins like tubulin • OMP generation depends on VDAC and hexokinase overexpression or knockdown. • Glucose-dependent OMP generation in the mechanism of cell death resistance • Possible involvement of OMP changes in various diseases targeting VDAC [ABSTRACT FROM AUTHOR]

Recently reported kinase-linked mild depolarization of mitochondria, which prevents the generation of the reactive oxygen species (ROS) and disappears in various organs of the old mice, has been assumed to represent a crucial component of the mitochondrial anti-aging program. To measure mitochondrial inner membrane potential (IMP), the authors used fluorescent probe safranin O+. It is widely accepted that the accumulation of such cationic probes in the mitochondrial matrix depends exclusively on IMP, thus completely ignoring the possibility of the outer membrane potential (OMP) generation. However, computational analysis performed in the presented work suggests that the kinase-linked generation of the positive OMP might take place under the described conditions, because the measured potential includes the algebraic sum of both IMP and OMP. Alternatively to the suggested mild depolarization of mitochondria, the reported experimental data might reflect mainly a change of the positive OMP generated by the VDAC-kinase complexes. We also demonstrate that the reported in the literature mitochondrial hyperpolarization induced by erastin (known to prevent VDAC-tubulin interactions) and the depolarization caused by the mitochondrial VDAC knockdowns in the cancer cells might actually represent a decrease or increase, respectively, of the magnitude of the kinase-linked positive OMP. This is consistent with our hypothesis that VDAC voltage gating by the kinase-linked metabolically-dependent OMP plays a very important physiological role in regulating the cell energy metabolism under normal and pathological conditions, in the maintenance of the cell death resistance and even in the genetic aging program. [Display omitted] • Kinase-linked generation of the positive outer membrane potential in mitochondria • Alternative explanations of an apparent hyperpolarization-depolarization of mitochondria • VDAC knockdowns increase kinase-linked outer membrane potential in mitochondria. • Role of the positive outer membrane potential of mitochondria in cell death protection • Possible aging program at the level of the mitochondrial outer membrane [ABSTRACT FROM AUTHOR]