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.

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.

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.

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.

InVivo Dissection of the Intrinsically Disordered Receptor Domain of Tim23.

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.

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.

Compromised Mitochondrial Protein Import Acts as a Signal for UPRmt.

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.

A mutagenesis analysis of Tim50, the major receptor of the TIM23 complex, identifies regions that affect its interaction with Tim23.

Maintenance of the mitochondrial proteome depends on import of newly made proteins from the cytosol. More than half of mitochondrial proteins are made as precursor proteins with N-terminal extensions called presequences and use the TIM23 complex for translocation into the matrix, the inner mitochondrial membrane and the intermembrane space (IMS). Tim50 is the central receptor of the complex that recognizes precursor proteins in the IMS. Additionally, Tim50 interacts with the IMS domain of the channel forming subunit, Tim23, an interaction that is essential for protein import across the mitochondrial inner membrane. In order to gain deeper insight into the molecular function of Tim50, we used random mutagenesis to determine residues that are important for its function. The temperature-sensitive mutants isolated were defective in import of TIM23-dependent precursor proteins. The residues mutated map to two distinct patches on the surface of Tim50. Notably, mutations in both patches impaired the interaction of Tim50 with Tim23. We propose that two regions of Tim50 play a role in its interaction with Tim23 and thereby affect the import function of the complex.

MICU1 Confers Protection from MCU-Dependent Manganese Toxicity.

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.

Cardiolipin mediates membrane and channel interactions of the mitochondrial TIM23 protein import complex receptor Tim50.

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.

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.

Chemical Crosslinking in Intact Mitochondria.

Many mitochondrial proteins perform their functions as components of large, multimeric complexes. Chemical crosslinking is a powerful method to analyze protein-protein interactions within such complexes. Using membrane-permeable crosslinkers and isolated intact mitochondria, protein-protein interactions that are secluded by two mitochondrial membranes can be readily analyzed in physiologically active, isolated organelles under a variety of physiological and pathophysiological conditions. Here, we describe two methods for chemical crosslinking in intact yeast mitochondria. The first method enables the analysis of ATP-dependent remodeling of mitochondrial protein complexes while the second one allows the identification of crosslinking partners of a protein of interest.

Role of Tim17 in coupling the import motor to the translocation channel of the mitochondrial presequence translocase.

The majority of mitochondrial proteins use N-terminal presequences for targeting to mitochondria and are translocated by the presequence translocase. During translocation, proteins, threaded through the channel in the inner membrane, are handed over to the import motor at the matrix face. Tim17 is an essential, membrane-embedded subunit of the translocase; however, its function is only poorly understood. Here, we functionally dissected its four predicted transmembrane (TM) segments. Mutations in TM1 and TM2 impaired the interaction of Tim17 with Tim23, component of the translocation channel, whereas mutations in TM3 compromised binding of the import motor. We identified residues in the matrix-facing region of Tim17 involved in binding of the import motor. Our results reveal functionally distinct roles of different regions of Tim17 and suggest how they may be involved in handing over the proteins, during their translocation into mitochondria, from the channel to the import motor of the presequence translocase.

Mitochondrial protein import: An unexpected disulfide bond.

Most mitochondrial proteins are imported through the TIM23 translocation channel, the structure and molecular nature of which are still unclear. In this issue, Ramesh et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201602074) show that the TIM23 subunit Tim17 contains a disulfide bond that is crucial for protein translocation and channel gating.

Protein translocation channel of mitochondrial inner membrane and matrix-exposed import motor communicate via two-domain coupling protein.

The majority of mitochondrial proteins are targeted to mitochondria by N-terminal presequences and use the TIM23 complex for their translocation across the mitochondrial inner membrane. During import, translocation through the channel in the inner membrane is coupled to the ATP-dependent action of an Hsp70-based import motor at the matrix face. How these two processes are coordinated remained unclear. We show here that the two domain structure of Tim44 plays a central role in this process. The N-terminal domain of Tim44 interacts with the components of the import motor, whereas its C-terminal domain interacts with the translocation channel and is in contact with translocating proteins. Our data suggest that the translocation channel and the import motor of the TIM23 complex communicate through rearrangements of the two domains of Tim44 that are stimulated by translocating proteins.

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.

GxxxG motifs hold the TIM23 complex together.

Approximately 99% of the mitochondrial proteome is nucleus-encoded, synthesized in the cytosol, and subsequently imported into and sorted to the correct compartment in the organelle. The translocase of the inner mitochondrial membrane 23 (TIM23) complex is the major protein translocase of the inner membrane, and is responsible for translocation of proteins across the inner membrane and their insertion into the inner membrane. Tim23 is the central component of the complex that forms the import channel. A high-resolution structure of the import channel is still missing, and structural elements important for its function are unknown. In the present study, we analyzed the importance of the highly abundant GxxxG motifs in the transmembrane segments of Tim23 for the structural integrity of the TIM23 complex. Of 10 glycines present in the GxxxG motifs in the first, second and third transmembrane segments of Tim23, mutations of three of them in transmembrane segments 1 and 2 resulted in a lethal phenotype, and mutations of three others in a temperature-sensitive phenotype. The remaining four caused no obvious growth phenotype. Importantly, none of the mutations impaired the import and membrane integration of Tim23 precursor into mitochondria. However, the severity of growth impairment correlated with the destabilization of the TIM23 complex. We conclude that the GxxxG motifs found in the first and second transmembrane segments of Tim23 are necessary for the structural integrity of the TIM23 complex.

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.