The terminal enzyme of the mitochondrial respiratory chain, cytochrome oxidase, transfers electrons to molecular oxygen, generating water. Within the inner mitochondrial membrane, cytochrome oxidase assembles into supercomplexes, together with other respiratory chain complexes, forming so-called respirasomes. Little is known about how these higher oligomeric structures are attained. Here we report on Rcf1 and Rcf2 as cytochrome oxidase subunits in S. cerevisiae. While Rcf2 is specific to yeast, Rcf1 is a conserved subunit with two human orthologs, RCF1a and RCF1b. Rcf1 is required for growth in hypoxia and complex assembly of subunits Cox13 and Rcf2, as well as for the oligomerization of a subclass of cytochrome oxidase complexes into respirasomes. Our analyses reveal that the cytochrome oxidase of mitochondria displays intrinsic heterogeneity with regard to its subunit composition and that distinct forms of respirasomes can be formed by complex variants.

MINOS1/Mio10, a conserved mitochondrial protein, is required for mitochondrial inner membrane organization and cristae morphology. MINOS1/Mio10 is a novel constituent of the mitofilin/Fcj1 complex of the inner membrane, linking the morphology phenotype of the mutant to the activity of the mitochondrial inner membrane organizing complex.

Saccharomyces cerevisiae contains three dynamin-related-proteins, Vps1p, Dnm1p and Mgm1p. Previous data from glucose-grown VPS1 and DNM1 null mutants suggested that Vps1p, but not Dnm1p, plays a role in regulating peroxisome abundance. Here we show that deletion of DNM1 also results in reduction of peroxisome numbers. This was not observed in glucose-grown dnm1 cells, but was evident in cells grown in the presence of oleate. Similar observations were made in cells lacking Fis1p, a protein involved in Dnm1p function. Fluorescence microscopy of cells producing Dnm1-GFP or GFP-Fis1p demonstrated that both proteins had a dual localization on mitochondria and peroxisomes. Quantitative analysis revealed a greater reduction in peroxisome number in oleate-induced vps1 cells relative to dnm1 or fis1 cells. A significant fraction of oleate-induced vps1 cells still contained two or more peroxisomes. Conversely, almost all cells of a dnm1 vps1 double-deletion strain contained only one, enlarged peroxisome. This suggests that deletion of DNM1 reinforces the vps1 peroxisome phenotype. Time-lapse imaging indicated that during budding of dnm1 vps1 cells, the single peroxisome present in the mother cell formed long protrusions into the developing bud. This organelle divided at a very late stage of the budding process, possibly during cytokinesis.

Mitochondrial function is critically dependent on the folding of the mitochondrial inner membrane into cristae; indeed, numerous human diseases are associated with aberrant crista morphologies. With the MICOS complex, OPA1 and the F 1 F o ‐ATP synthase, key players of cristae biogenesis have been identified, yet their interplay is poorly understood. Harnessing super‐resolution light and 3D electron microscopy, we dissect the roles of these proteins in the formation of cristae in human mitochondria. We individually disrupted the genes of all seven MICOS subunits in human cells and re‐expressed Mic10 or Mic60 in the respective knockout cell line. We demonstrate that assembly of the MICOS complex triggers remodeling of pre‐existing unstructured cristae and de novo formation of crista junctions (CJs) on existing cristae. We show that the Mic60‐subcomplex is sufficient for CJ formation, whereas the Mic10‐subcomplex controls lamellar cristae biogenesis. OPA1 stabilizes tubular CJs and, along with the F 1 F o ‐ATP synthase, fine‐tunes the positioning of the MICOS complex and CJs. We propose a new model of cristae formation, involving the coordinated remodeling of an unstructured crista precursor into multiple lamellar cristae.

The mitochondrial inner membrane contains a large protein complex that functions in inner membrane organization and formation of membrane contact sites. The complex was variably named the mitochondrial contact site complex, mitochondrial inner membrane organizing system, mitochondrial organizing structure, or Mitofilin/Fcj1 complex. To facilitate future studies, we propose to unify the nomenclature and term the complex “mitochondrial contact site and cristae organizing system” and its subunits Mic10 to Mic60.

The mitochondrial inner membrane organizing system (MINOS) is a conserved large hetero-oligomeric protein complex in the mitochondrial inner membrane, crucial for the maintenance of cristae morphology. MINOS has been suggested to represent the core of an extended protein network that controls mitochondrial function and structure, and has been linked to several human diseases. The spatial arrangement of MINOS within mitochondria is ill-defined, however. Using super-resolution stimulated emission depletion (STED) microscopy and immunogold electron microscopy, we determined the distribution of three known human MINOS subunits (mitofilin, MINOS1, and CHCHD3) in mammalian cells. Super-resolution microscopy revealed that all three subunits form similar clusters within mitochondria, and that MINOS is more abundant in mitochondria around the nucleus than in peripheral mitochondria. At the submitochondrial level, mitofilin, a core MINOS subunit, is preferentially localized at cristae junctions. In primary human fibroblasts, mitofilin labeling uncovered a regularly spaced pattern of clusters arranged in parallel to the cell growth surfaces. We suggest that this array of MINOS complexes might explain the observed phenomenon of largely horizontally arranged cristae junctions that connect the inner boundary membrane to lamellar cristae. The super-resolution images demonstrate an unexpectedly high level of regularity in the nanoscale distribution of the MINOS complex in human mitochondria, supporting an integrating role of MINOS in the structural organization of the organelle.MitOS | MICOS | membrane architecture | nanoscopy

Coa3 and Cox14 form assembly intermediates with newly synthesized Cox1 and are required for association of the Mss51 translational activator with these complexes.

Here we report that Mdm38 and Mba1 display overlapping functions in mitochondrial protein expression. Both Mdm38 and Mba1 interact with mitochondrial ribosomes and are required for translation of COX1 and cytochrome b mRNAs.