Systematic Identification of MCU Modulators by Orthogonal Interspecies Chemical Screening.

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.

How to get to the other side of the mitochondrial inner membrane - the protein import motor.

Biogenesis of mitochondria relies on import of more than 1000 different proteins from the cytosol. Approximately 70% of these proteins follow the presequence pathway - they are synthesized with cleavable N-terminal extensions called presequences and reach the final place of their function within the organelle with the help of the TOM and TIM23 complexes in the outer and inner membranes, respectively. The translocation of proteins along the presequence pathway is powered by the import motor of the TIM23 complex. The import motor of the TIM23 complex is localized at the matrix face of the inner membrane and is likely the most complicated Hsp70-based system identified to date. How it converts the energy of ATP hydrolysis into unidirectional translocation of proteins into mitochondria remains one of the biggest mysteries of this translocation pathway. Here, the knowns and the unknowns of the mitochondrial protein import motor are discussed.

The conformational dynamics of the mitochondrial Hsp70 chaperone.

Heat shock proteins 70 (Hsp70) represent a ubiquitous and conserved family of molecular chaperones involved in a plethora of cellular processes. The dynamics of their ATP hydrolysis-driven and cochaperone-regulated conformational cycle are poorly understood. We used fluorescence spectroscopy to analyze, in real time and at single-molecule resolution, the effects of nucleotides and cochaperones on the conformation of Ssc1, a mitochondrial member of the family. We report that the conformation of its ADP state is unexpectedly heterogeneous, in contrast to a uniform ATP state. Substrates are actively involved in determining the conformation of Ssc1. The J protein Mdj1 does not interact transiently with the chaperone, as generally believed, but rather is released slowly upon ATP hydrolysis. Analysis of the major bacterial Hsp70 revealed important differences between highly homologous members of the family, possibly explaining tuning of Hsp70 chaperones to meet specific functions in different organisms and cellular compartments.

Nucleotides regulate the mechanical hierarchy between subdomains of the nucleotide binding domain of the Hsp70 chaperone DnaK.

The regulation of protein function through ligand-induced conformational changes is crucial for many signal transduction processes. The binding of a ligand alters the delicate energy balance within the protein structure, eventually leading to such conformational changes. In this study, we elucidate the energetic and mechanical changes within the subdomains of the nucleotide binding domain (NBD) of the heat shock protein of 70 kDa (Hsp70) chaperone DnaK upon nucleotide binding. In an integrated approach using single molecule optical tweezer experiments, loop insertions, and steered coarse-grained molecular simulations, we find that the C-terminal helix of the NBD is the major determinant of mechanical stability, acting as a glue between the two lobes. After helix unraveling, the relative stability of the two separated lobes is regulated by ATP/ADP binding. We find that the nucleotide stays strongly bound to lobe II, thus reversing the mechanical hierarchy between the two lobes. Our results offer general insights into the nucleotide-induced signal transduction within members of the actin/sugar kinase superfamily.

Real-time observation of the conformational dynamics of mitochondrial Hsp70 by spFRET.

The numerous functions of the important class of molecular chaperones, heat shock proteins 70 (Hsp70), rely on cycles of intricate conformational changes driven by ATP-hydrolysis and regulated by cochaperones and substrates. Here, we used Förster resonance energy transfer to study the conformational dynamics of individual molecules of Ssc1, a mitochondrial Hsp70, in real time. The intrinsic dynamics of the substrate-binding domain of Ssc1 was observed to be uncoupled from the dynamic interactions between substrate- and nucleotide-binding domains. Analysis of the fluctuations in the interdomain separation revealed frequent transitions to a nucleotide-free state. The nucleotide-exchange factor Mge1 did not induce ADP release, as expected, but rather facilitated binding of ATP. These results indicate that the conformational cycle of Ssc1 is more elaborate than previously thought and provide insight into how the Hsp70s can perform a wide variety of functions.

Functional complementation analyses reveal that the single PRAT family protein of trypanosoma brucei is a divergent homolog of Tim17 in saccharomyces cerevisiae.

Trypanosoma brucei, a parasitic protozoan that causes African trypanosomiasis, possesses a single member of the presequence and amino acid transporter (PRAT) protein family, which is referred to as TbTim17. In contrast, three homologous proteins, ScTim23, ScTim17, and ScTim22, are found in Saccharomyces cerevisiae and higher eukaryotes. Here, we show that TbTim17 cannot rescue Tim17, Tim23, or Tim22 mutants of S. cerevisiae. We expressed S. cerevisiae Tim23, Tim17, and Tim22 in T. brucei. These heterologous proteins were properly imported into mitochondria in the parasite. Further analysis revealed that although ScTim23 and ScTim17 were integrated into the mitochondrial inner membrane and assembled into a protein complex similar in size to TbTim17, only ScTim17 was stably associated with TbTim17. In contrast, ScTim22 existed as a protease-sensitive soluble protein in the T. brucei mitochondrion. In addition, the growth defect caused by TbTim17 knockdown in T. brucei was partially restored by the expression of ScTim17 but not by the expression of either ScTim23 or ScTim22, whereas the expression of TbTim17 fully complemented the growth defect caused by TbTim17 knockdown, as anticipated. Similar to the findings for cell growth, the defect in the import of mitochondrial proteins due to depletion of TbTim17 was in part restored by the expression of ScTim17 but was not complemented by the expression of either ScTim23 or ScTim22. Together, these results suggest that TbTim17 is divergent compared to ScTim23 but that its function is closer to that of ScTim17. In addition, ScTim22 could not be sorted properly in the T. brucei mitochondrion and thus failed to complement the function of TbTim17.

The mitochondrial contact site complex, a determinant of mitochondrial architecture.

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.

J-domain proteins: From molecular mechanisms to diseases.

J-domain proteins (JDPs) are the largest family of chaperones in most organisms, but much of how they function within the network of other chaperones and protein quality control machineries is still an enigma. Here, we report on the latest findings related to JDP functions presented at a dedicated JDP workshop in Gdansk, Poland. The report does not include all (details) of what was shared and discussed at the meeting, because some of these original data have not yet been accepted for publication elsewhere or represented still preliminary observations at the time.

Role of the import motor in insertion of transmembrane segments by the mitochondrial TIM23 complex.

The TIM23 complex mediates translocation of proteins across, and their lateral insertion into, the mitochondrial inner membrane. Translocation of proteins requires both the membrane-embedded core of the complex and its ATP-dependent import motor. Insertion of some proteins, however, occurs in the absence of ATP, questioning the need for the import motor during lateral insertion. We show here that the import motor associates with laterally inserted proteins even when its ATPase activity is not required. Furthermore, our results suggest a role for the import motor in lateral insertion. Thus, the import motor is involved in ATP-dependent translocation and ATP-independent lateral insertion.

Systematic screen for human disease genes in yeast.

High similarity between yeast and human mitochondria allows functional genomic study of Saccharomyces cerevisiae to be used to identify human genes involved in disease. So far, 102 heritable disorders have been attributed to defects in a quarter of the known nuclear-encoded mitochondrial proteins in humans. Many mitochondrial diseases remain unexplained, however, in part because only 40-60% of the presumed 700-1,000 proteins involved in mitochondrial function and biogenesis have been identified. Here we apply a systematic functional screen using the pre-existing whole-genome pool of yeast deletion mutants to identify mitochondrial proteins. Three million measurements of strain fitness identified 466 genes whose deletions impaired mitochondrial respiration, of which 265 were new. Our approach gave higher selection than other systematic approaches, including fivefold greater selection than gene expression analysis. To apply these advantages to human disorders involving mitochondria, human orthologs were identified and linked to heritable diseases using genomic map positions.

Two domains of Tim50 coordinate translocation of proteins across the two mitochondrial membranes.

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.

Direct interaction of mitochondrial targeting presequences with purified components of the TIM23 protein complex.

Precursor proteins that are imported from the cytosol into the matrix of mitochondria carry positively charged amphipathic presequences and cross the inner membrane with the help of vital components of the TIM23 complex. It is currently unclear which subunits of the TIM23 complex recognize and directly bind to presequences. Here we analyzed the binding of presequence peptides to purified components of the TIM23 complex. The interaction of three different presequences with purified soluble domains of yeast Tim50 (Tim50IMS), Tim23 (Tim23IMS), and full-length Tim44 was examined. Using chemical cross-linking and surface plasmon resonance we demonstrate, for the first time, the ability of purified Tim50IMS and Tim44 to interact directly with the yeast Hsp60 presequence. We also analyzed their interaction with presequences derived from precursors of yeast mitochondrial 70-kDa heat shock protein (mHsp70) and of bovine cytochrome P450SCC. Moreover, we characterized the nature of the interactions and determined their KDs. On the basis of our results, we suggest a mechanism of translocation where stronger interactions of the presequences on the trans side of the channel support the import of precursor proteins through TIM23 into the matrix.

Understanding the molecular mechanism of protein translocation across the mitochondrial inner membrane: still a long way to go.

In order to reach the final place of their function, approximately half of the proteins in any eukaryotic cell have to be transported across or into one of the membranes in the cell. In this article, we present an overview of our current knowledge concerning the structural properties of the TIM23 complex and their relationship with the molecular mechanism of protein transport across the mitochondrial inner membrane. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.

Active remodelling of the TIM23 complex during translocation of preproteins into mitochondria.

The TIM23 (translocase of the mitochondrial inner membrane) complex mediates translocation of preproteins across and their insertion into the mitochondrial inner membrane. How the translocase mediates sorting of preproteins into the two different subcompartments is poorly understood. In particular, it is not clear whether association of two operationally defined parts of the translocase, the membrane-integrated part and the import motor, depends on the activity state of the translocase. We established conditions to in vivo trap the TIM23 complex in different translocation modes. Membrane-integrated part of the complex and import motor were always found in one complex irrespective of whether an arrested preprotein was present or not. Instead, we detected different conformations of the complex in response to the presence and, importantly, the type of preprotein being translocated. Two non-essential subunits of the complex, Tim21 and Pam17, modulate its activity in an antagonistic manner. Our data demonstrate that the TIM23 complex acts as a single structural and functional entity that is actively remodelled to sort preproteins into different mitochondrial subcompartments.

Combining MFD and PIE for accurate single-pair Förster resonance energy transfer measurements.

Single-pair Förster resonance energy transfer (spFRET) experiments using single-molecule burst analysis on a confocal microscope are an ideal tool to measure inter- and intramolecular distances and dynamics on the nanoscale. Different techniques have been developed to maximize the amount of information available in spFRET burst analysis experiments. Multiparameter fluorescence detection (MFD) is used to monitor a variety of fluorescence parameters simultaneously and pulsed interleaved excitation (PIE) employs direct excitation of the acceptor to probe its presence and photoactivity. To calculate accurate FRET efficiencies from spFRET experiments with MFD or PIE, several calibration measurements are usually required. Herein, we demonstrate that by combining MFD with PIE information regarding all calibration factors as well as an accurate determination of spFRET histograms can be performed in a single measurement. In addition, the quality of overlap of the different detection volumes as well as the detection of acceptor photophysics can be investigated with MFD-PIE. Bursts containing acceptor photobleaching can be identified and excluded from further investigation while bursts that contain FRET dynamics are unaffected by this analysis. We have employed MFD-PIE to accurately analyze the effects of nucleotides and substrate on the interdomain separation in DnaK, the major bacterial heat shock protein 70 (Hsp70). The interdomain distance increases from 47 Å in the ATP-bound state to 84 Å in the ADP-bound state and slightly contracts to 77 Å when a substrate is bound. This is in contrast to what was observed for the mitochondrial member of the Hsp70s, Ssc1, supporting the notion of evolutionary specialization of Hsp70s for different cellular functions in different organisms and cell organelles.

ATPase domain and interdomain linker play a key role in aggregation of mitochondrial Hsp70 chaperone Ssc1.

The co-chaperone Hep1 is required to prevent the aggregation of mitochondrial Hsp70 proteins. We have analyzed the interaction of Hep1 with mitochondrial Hsp70 (Ssc1) and the determinants in Ssc1 that make it prone to aggregation. The ATPase and peptide binding domain (PBD) of Hsp70 proteins are connected by a linker segment that mediates interdomain communication between the domains. We show here that the minimal Hep1 binding entity of Ssc1 consists of the ATPase domain and the interdomain linker. In the absence of Hep1, the ATPase domain with the interdomain linker had the tendency to aggregate, in contrast to the ATPase domain with the mutated linker segment or without linker, and in contrast to the PBD. The closest homolog of Ssc1, bacterial DnaK, and a Ssc1 chimera, in which a segment of the ATPase domain of Ssc1 was replaced by the corresponding segment from DnaK, did not aggregate in Delta hep1 mitochondria. The propensity to aggregate appears to be a specific property of the mitochondrial Hsp70 proteins. The ATPase domain in combination with the interdomain linker is crucial for aggregation of Ssc1. In conclusion, our results suggest that interdomain communication makes Ssc1 prone to aggregation. Hep1 counteracts aggregation by binding to this aggregation-prone conformer.

The many faces of the mitochondrial TIM23 complex.

The TIM23 complex in the inner membrane of mitochondria mediates import of essentially all matrix proteins and a large number of inner membrane proteins. Here we present an overview on the latest insights into the structure and function of this remarkable molecular machine.

Coordinated Translocation of Presequence-Containing Precursor Proteins Across Two Mitochondrial Membranes: Knowns and Unknowns of How TOM and TIM23 Complexes Cooperate With Each Other.

The vast majority of mitochondrial proteins are encoded in the nuclear genome and synthesized on cytosolic ribosomes as precursor proteins with specific mitochondrial targeting signals. Mitochondrial targeting signals are very diverse, however, about 70% of mitochondrial proteins carry cleavable, N-terminal extensions called presequences. These amphipathic helices with one positively charged and one hydrophobic surface target proteins to the mitochondrial matrix with the help of the TOM and TIM23 complexes in the outer and inner membranes, respectively. Translocation of proteins across the two mitochondrial membranes does not take place independently of each other. Rather, in the intermembrane space, where the two complexes meet, components of the TOM and TIM23 complexes form an intricate network of protein-protein interactions that mediates initially transfer of presequences and then of the entire precursor proteins from the outer to the inner mitochondrial membrane. In this Mini Review, we summarize our current understanding of how the TOM and TIM23 complexes cooperate with each other and highlight some of the future challenges and unresolved questions in the field.

Drug Discovery Assay to Identify Modulators of the Mitochondrial Ca2+ Uniporter.

The mitochondrial calcium uniporter (MCU ) is an essential protein of the inner mitochondrial membrane that mediates the uptake of calcium into mitochondria of virtually all mammalian tissues, regulating cell metabolism, signaling, and death. MCU-mediated calcium uptake has been shown to play a pathophysiological role in diverse human disease contexts, which qualifies this channel as a druggable target for therapeutic intervention.Here, we present a protocol to perform drug screens to identify effective and specific MCU-targeting inhibitors. The methodology is based on the use of cryopreserved mitochondria that are isolated from a yeast strain engineered to express the human MCU and its essential regulator EMRE together with the luminescence calcium sensor aequorin. Yeast mitochondria with a functionally reconstituted MCU-mediated calcium uptake are then employed as a ready-to-use screening reagent. False discovery rate is further minimized by energizing mitochondria with D-lactate in a mannitol/sucrose-based medium, which provides a mean to discriminate between direct and secondary effects of drugs on mitochondrial calcium uptake. This screening assay is sensitive and robust and can be easily implemented in any laboratory.