Translocation of the majority of mitochondrial proteins from the cytosol into mitochondria requires the cooperation of TOM and TIM23 complexes in the outer and inner mitochondrial membranes. The molecular mechanisms underlying this cooperation remain largely unknown. Here, we present biochemical and genetic evidence that at least two contacts from the side of the TIM23 complex play an important role in TOM-TIM23 cooperation in vivo. Tim50, likely through its very C-terminal segment, interacts with Tom22. This interaction is stimulated by translocating proteins and is independent of any other TOM-TIM23 contact known so far. Furthermore, the exposure of Tim23 on the mitochondrial surface depends not only on its interaction with Tim50 but also on the dynamics of the TOM complex. Destabilization of the individual contacts reduces the efficiency of import of proteins into mitochondria and destabilization of both contacts simultaneously is not tolerated by yeast cells. We conclude that an intricate and coordinated network of protein-protein interactions involving primarily Tim50 and also Tim23 is required for efficient translocation of proteins across both mitochondrial membranes.

Metabolic function and architecture of mitochondria are intimately linked. More than 60 years ago, cristae were discovered as characteristic elements of mitochondria that harbor the protein complexes of oxidative phosphorylation, but how cristae are formed, remained an open question. Here we present experimental results obtained with yeast that support a novel hypothesis on the existence of two molecular pathways that lead to the generation of lamellar and tubular cristae. Formation of lamellar cristae depends on the mitochondrial fusion machinery through a pathway that is required also for homeostasis of mitochondria and mitochondrial DNA. Tubular cristae are formed via invaginations of the inner boundary membrane by a pathway independent of the fusion machinery. Dimerization of the F1FO-ATP synthase and the presence of the MICOS complex are necessary for both pathways. The proposed hypothesis is suggested to apply also to higher eukaryotes, since the key components are conserved in structure and function throughout evolution.

Mitochondria are organelles with a complex architecture. They are bounded by an envelope consisting of the outer membrane and the inner boundary membrane (IBM). Narrow crista junctions (CJs) link the IBM to the cristae. OMs and IBMs are firmly connected by contact sites (CS). The molecular nature of the CS remained unknown. Using quantitative high-resolution mass spectrometry we identified a novel complex, the mitochondrial contact site (MICOS) complex, formed by a set of mitochondrial membrane proteins that is essential for the formation of CS. MICOS is preferentially located at the CJs. Upon loss of one of the MICOS subunits, CJs disappear completely or are impaired, showing that CJs require the presence of CS to form a superstructure that links the IBM to the cristae. Loss of MICOS subunits results in loss of respiratory competence and altered inheritance of mitochondrial DNA.

The vast majority of mitochondrial proteins are synthesized in the cytosol and transported into the organelle in a largely, if not completely, unfolded state. The proper function of mitochondria thus depends on folding of several hundreds of proteins in the various subcompartments of the organelle. Whereas folding of proteins in the mitochondrial matrix is supported by members of several chaperone families, very little is known about folding of proteins in the intermembrane space (IMS). We targeted dihydrofolate reductase (DHFR) as a model substrate to the IMS of yeast mitochondria and analyzed its folding. DHFR can fold in this compartment, and its aggregation upon heat shock can be prevented in an ATP-dependent manner. Yme1, an AAA (ATPases associated with diverse cellular activities) protease of the IMS, prevented aggregation of DHFR. Analysis of protein aggregates in mitochondria lacking Yme1 revealed the presence of a number of proteins involved in the establishment of mitochondrial ultrastructure, lipid metabolism, protein import, and respiratory growth. These findings explain the pleiotropic effects of deletion of YME1 and suggest an important role for Yme1 as a folding assistant, in addition to its proteolytic function, in the protein homeostasis of mitochondria.

Mitochondria are essential organelles of eukaryotic cells and are characterized by their unique and complex membrane system. They are confined from the cytosol by an envelope consisting of two membranes. Signals, metabolites, proteins and lipids have to be transferred across these membranes via proteinaceous contact sites to keep mitochondria functional. In the present study, we identified a novel mitochondrial contact site in Saccharomyces cerevisiae that is formed by the inner membrane protein Cqd1 and the outer membrane proteins Por1 and Om14. Similar to what is found for the mitochondrial porin Por1, Cqd1 is highly conserved, suggesting that this complex is conserved in form and function from yeast to human. Cqd1 is a member of the UbiB protein kinase-like family (also called aarF domain-containing kinases). It was recently shown that Cqd1, in cooperation with Cqd2, controls the cellular distribution of coenzyme Q by a yet unknown mechanism. Our data suggest that Cqd1 is additionally involved in phospholipid homeostasis. Moreover, overexpression of CQD1 and CQD2 causes tethering of mitochondria to the endoplasmic reticulum, which might explain the ability of Cqd2 to rescue ERMES deletion phenotypes.

The mitochondrial calcium uniporter is a highly selective ion channel composed of species- and tissue-specific subunits. However, the functional role of each component still remains unclear. Here, we establish a synthetic biology approach to dissect the interdependence between the pore-forming subunit MCU and the calcium-sensing regulator MICU1. Correlated evolutionary patterns across 247 eukaryotes indicate that their co-occurrence may have conferred a positive fitness advantage. We find that, while the heterologous reconstitution of MCU and EMRE in vivo in yeast enhances manganese stress, this is prevented by co-expression of MICU1. Accordingly, MICU1 deletion sensitizes human cells to manganese-dependent cell death by disinhibiting MCU-mediated manganese uptake. As a result, manganese overload increases oxidative stress, which can be effectively prevented by NAC treatment. Our study identifies a critical contribution of MICU1 to the uniporter selectivity, with important implications for patients with MICU1 deficiency, as well as neurological disorders arising upon chronic manganese exposure.

The TOB-SAM complex is an essential component of the mitochondrial outer membrane that mediates the insertion of β-barrel precursor proteins into the membrane. We report here its isolation and determine its size, composition, and structural organization. The complex from Neurospora crassa was composed of Tob55-Sam50, Tob38-Sam35, and Tob37-Sam37 in a stoichiometry of 1:1:1 and had a molecular mass of 140 kD. A very minor fraction of the purified complex was associated with one Mdm10 protein. Using molecular homology modeling for Tob55 and cryoelectron microscopy reconstructions of the TOB complex, we present a model of the TOB-SAM complex that integrates biochemical and structural data. We discuss our results and the structural model in the context of a possible mechanism of the TOB insertase.

In the intermembrane space (IMS) of mitochondria, the receptor domain of Tim23 has an essential role during translocation of hundreds of different proteins from the cytosol via the TOM and TIM23 complexes in the outer and inner membranes, respectively. This intrinsically disordered domain, which can even extend into the cytosol, was shown, mostly in vitro, to interact with several subunits of the TOM and TIM23 complexes. To obtain molecular understanding of this organizational hub in the IMS, we dissected the IMS domain of Tim23 in vivo. We show that the interaction surface of Tim23 with Tim50 is larger than previously thought and reveal an unexpected interaction of Tim23 with Pam17 in the IMS, impairment of which influences their interaction in the matrix. Furthermore, mutations of two conserved negatively charged residues of Tim23, close to the inner membrane, prevented dimerization of Tim23. The same mutations increased exposure of Tim23 on the mitochondrial surface, whereas dissipation of membrane potential decreased it. Our results reveal an intricate network of Tim23 interactions in the IMS, whose influence is transduced across two mitochondrial membranes, ensuring efficient translocation of proteins into mitochondria.

Mitochondrial morphology and inheritance of mitochondrial DNA in yeast depend on the dynamin-like GTPase Mgm1. It is present in two isoforms in the intermembrane space of mitochondria both of which are required for Mgm1 function. Limited proteolysis of the large isoform by the mitochondrial rhomboid protease Pcp1/Rbd1 generates the short isoform of Mgm1 but how this is regulated is unclear. We show that near its NH2 terminus Mgm1 contains two conserved hydrophobic segments of which the more COOH-terminal one is cleaved by Pcp1. Changing the hydrophobicity of the NH2-terminal segment modulated the ratio of the isoforms and led to fragmentation of mitochondria. Formation of the short isoform of Mgm1 and mitochondrial morphology further depend on a functional protein import motor and on the ATP level in the matrix. Our data show that a novel pathway, to which we refer as alternative topogenesis, represents a key regulatory mechanism ensuring the balanced formation of both Mgm1 isoforms. Through this process the mitochondrial ATP level might control mitochondrial morphology.

The Mdm10, Mdm12, and Mmm1 proteins have been implicated in several mitochondrial functions including mitochondrial distribution and morphology, assembly of beta-barrel proteins such as Tom40 and porin, association of mitochondria and endoplasmic reticulum, and maintaining lipid composition of mitochondrial membranes. Here we show that loss of any of these three proteins in Neurospora crassa results in the formation of large mitochondrial tubules and reduces the assembly of porin and Tom40 into the outer membrane. We have also investigated the relationship of Mdm10 and Tom7 in the biogenesis of beta-barrel proteins. Previous work showed that mitochondria lacking Tom7 assemble Tom40 more efficiently, and porin less efficiently, than wild-type mitochondria. Analysis of mdm10 and tom7 single and double mutants, has demonstrated that the effects of the two mutations are additive. Loss of Tom7 partially compensates for the decrease in Tom40 assembly resulting from loss of Mdm10, whereas porin assembly is more severely reduced in the double mutant than in either single mutant. The additive effects observed in the double mutant suggest that different steps in beta-barrel assembly are affected in the individual mutants. Many aspects of Tom7 and Mdm10 function in N. crassa are different from those of their homologues in Saccharomyces cerevisiae.

Treatment of cardiac arrhythmia remains challenging due to severe side effects of common anti-arrhythmic drugs. We previously demonstrated that mitochondrial Ca2+ uptake in cardiomyocytes represents a promising new candidate structure for safer drug therapy. However, druggable agonists of mitochondrial Ca2+ uptake suitable for preclinical and clinical studies are still missing.

The induction of the mitochondrial unfolded protein response (UPRmt) results in increased transcription of the gene encoding the mitochondrial chaperone HSP70. We systematically screened the C. elegans genome and identified 171 genes that, when knocked down, induce the expression of an hsp-6 HSP70 reporter and encode mitochondrial proteins. These genes represent many, but not all, mitochondrial processes (e.g., mitochondrial calcium homeostasis and mitophagy are not represented). Knockdown of these genes leads to reduced mitochondrial membrane potential and, hence, decreased protein import into mitochondria. In addition, it induces UPRmt in a manner that is dependent on ATFS-1 but that is not antagonized by the kinase GCN-2. We propose that compromised mitochondrial protein import signals the induction of UPRmt and that the mitochondrial targeting sequence of ATFS-1 functions as a sensor for this signal.

The phospholipid cardiolipin mediates the functional interactions of proteins that reside within energy-conserving biological membranes. However, the molecular basis by which this lipid performs this essential cellular role is not well understood. We address this role of cardiolipin using the multisubunit mitochondrial TIM23 protein transport complex as a model system. The early stages of protein import by this complex require specific interactions between the polypeptide substrate receptor, Tim50, and the membrane-bound channel-forming subunit, Tim23. Using analyses performed in vivo, in isolated mitochondria, and in reductionist nanoscale model membrane systems, we show that the soluble receptor domain of Tim50 interacts with membranes and with specific sites on the Tim23 channel in a manner that is directly modulated by cardiolipin. To obtain structural insights into the nature of these interactions, we obtained the first small-angle x-ray scattering-based structure of the soluble Tim50 receptor in its entirety. Using these structural insights, molecular dynamics simulations combined with a range of biophysical measurements confirmed the role of cardiolipin in driving the association of the Tim50 receptor with lipid bilayers with concomitant structural changes, highlighting the role of key structural elements in mediating this interaction. Together, these results show that cardiolipin is required to mediate specific receptor-channel associations in the TIM23 complex. Our results support a new working model for the dynamic structural changes that occur within the complex during transport. More broadly, this work strongly advances our understanding of how cardiolipin mediates interactions among membrane-associated proteins.

Hundreds of mitochondrial proteins with N-terminal presequences are translocated across the outer and inner mitochondrial membranes via the TOM and TIM23 complexes, respectively. How translocation of proteins across two mitochondrial membranes is coordinated is largely unknown. Here, we show that the two domains of Tim50 in the intermembrane space, named core and PBD, both have essential roles in this process. Building upon the surprising observation that the two domains of Tim50 can complement each other in trans, we establish that the core domain contains the main presequence-binding site and serves as the main recruitment point to the TIM23 complex. On the other hand, the PBD plays, directly or indirectly, a critical role in cooperation of the TOM and TIM23 complexes and supports the receptor function of Tim50. Thus, the two domains of Tim50 both have essential but distinct roles and together coordinate translocation of proteins across two mitochondrial membranes.

beta-Barrel proteins constitute a distinct class of mitochondrial outer membrane proteins. For import into mitochondria, their precursor forms engage the TOM complex. They are then relayed to the TOB complex, which mediates their insertion into the outer membrane. We studied the structure-function relationships of the core component of the TOB complex, Tob55. Tob55 precursors with deletions in the N-terminal domain were not affected in their targeting to and insertion into the mitochondrial outer membrane. Replacement of wild-type Tob55 by these deletion variants resulted in reduced growth of cells, and mitochondria isolated from such cells were impaired in their capacity to import beta-barrel precursors. The purified N-terminal domain was able to bind beta-barrel precursors in a specific manner. Collectively, these results demonstrate that the N-terminal domain of Tob55 recognizes precursors of beta-barrel proteins. This recognition may contribute to the coupling of the translocation of beta-barrel precursors across the TOM complex to their interaction with the TOB complex.

The import motor of the mitochondrial translocase of the inner membrane (TIM23) mediates the ATP-dependent translocation of preproteins into the mitochondrial matrix by cycles of binding to and release from mtHsp70. An essential step of this process is the stimulation of the ATPase activity of mtHsp70 performed by the J cochaperone Tim14. Tim14 forms a complex with the J-like protein Tim16. The crystal structure of this complex shows that the conserved domains of the two proteins have virtually identical folds but completely different surfaces enabling them to perform different functions. The Tim14-Tim16 dimer reveals a previously undescribed arrangement of J and J-like domains. Mutations that destroy the complex between Tim14 and Tim16 are lethal demonstrating that complex formation is an essential requirement for the viability of cells. We further demonstrate tight regulation of the cochaperone activity of Tim14 by Tim16. The first crystal structure of a J domain in complex with a regulatory protein provides new insights into the function of the mitochondrial TIM23 translocase and the Hsp70 chaperone system in general.

The mitochondrial calcium uniporter complex is essential for calcium (Ca2+) uptake into mitochondria of all mammalian tissues, where it regulates bioenergetics, cell death, and Ca2+ signal transduction. Despite its involvement in several human diseases, we currently lack pharmacological agents for targeting uniporter activity. Here we introduce a high-throughput assay that selects for human MCU-specific small-molecule modulators in primary drug screens. Using isolated yeast mitochondria, reconstituted with human MCU, its essential regulator EMRE, and aequorin, and exploiting a D-lactate- and mannitol/sucrose-based bioenergetic shunt that greatly minimizes false-positive hits, we identify mitoxantrone out of more than 600 clinically approved drugs as a direct selective inhibitor of human MCU. We validate mitoxantrone in orthogonal mammalian cell-based assays, demonstrating that our screening approach is an effective and robust tool for MCU-specific drug discovery and, more generally, for the identification of compounds that target mitochondrial functions.