Homozygous stop mutation in AHR causes autosomal recessive foveal hypoplasia and infantile nystagmus.

Herein we present a consanguineous family with three children affected by foveal hypoplasia with infantile nystagmus, following an autosomal recessive mode of inheritance. The patients showed normal electroretinography responses, no signs of albinism, and no anterior segment or brain abnormalities. Upon whole exome sequencing, we identified a homozygous mutation (c.1861C>T;p.Q621*) in the aryl hydrocarbon receptor (AHR) gene that perfectly co-segregated with the disease in the larger family. AHR is a ligand-activated transcription factor that has been intensively studied in xenobiotic-induced toxicity. Further, it has been shown to play a physiological role under normal cellular conditions, such as in immunity, inflammatory response and neurogenesis. Notably, knockout of the Ahr gene in mouse impairs optic nerve myelin sheath formation and results in oculomotor deficits sharing many features with our patients: the eye movement disorder in Ahr-/- mice appears early in development and presents as conjugate horizontal pendular nystagmus. We therefore propose AHR to be a novel disease gene for a new, recessively inherited disorder in humans, characterized by infantile nystagmus and foveal hypoplasia.

A novel biallelic loss-of-function mutation in TMCO1 gene confirming and expanding the phenotype spectrum of cerebro-facio-thoracic dysplasia.

The main clinical features of cerebro-facio-thoracic dysplasia (CFTD) syndrome, which were described over four decades ago, include facial dysmorphism, multiple malformations of the vertebrae and ribs, and intellectual disability. Recently, a TMCO1 gene mutation was shown to be responsible for an autosomal recessive CFTD syndrome characterized by craniofacial dysmorphism, skeletal anomalies, and intellectual disability. In the current report, we describe two members of a consanguineous family from an Arab community in Israel who were clinically diagnosed as suffering from craniofacial dysmorphism, skeletal anomalies, intellectual disability, and epilepsy. Both affected siblings had behavioral difficulties such as anxiety and emotional instability with impulsive behaviors. Whole-exome sequencing revealed a homozygous stop-gain mutation NM_019026.4: c.616C > T; p.(Arg206*) in exon 6 of the TMCO1 gene. Bioinformatics analysis suggested a structural model for the TMCO1 protein and its homologues. The clinical features of our patients were compared with those of the only other five studies available in the literature. We conclude that this mutation in the TMCO1 gene is responsible for the various clinical manifestations of CFTD syndrome exhibited by the patients studied that expand the phenotypic spectrum of the disease to include epilepsy as a characteristic feature of this syndrome.

Clinical, radiological, and genetic characteristics of 16 patients with ACO2 gene defects: Delineation of an emerging neurometabolic syndrome.

Mitochondrial aconitase is the second enzyme in the tricarboxylic acid (TCA) cycle catalyzing the interconversion of citrate into isocitrate and encoded by the nuclear gene ACO2. A homozygous pathogenic variant in the ACO2 gene was initially described in 2012 resulting in a novel disorder termed "infantile cerebellar retinal degeneration" (ICRD, OMIM#614559). Subsequently, additional studies reported patients with pathogenic ACO2 variants, further expanding the genetic and clinical spectrum of this disorder to include milder and later onset manifestations. Here, we report an international multicenter cohort of 16 patients (of whom 7 are newly diagnosed) with biallelic pathogenic variants in ACO2 gene. Most patients present in early infancy with severe truncal hypotonia, truncal ataxia, variable seizures, evolving microcephaly, and ophthalmological abnormalities of which the most dominant are esotropia and optic atrophy with later development of retinal dystrophy. Most patients remain nonambulatory and do no acquire any language, but a subgroup of patients share a more favorable course. Brain magnetic resonance imaging (MRI) is typically normal within the first months but global atrophy gradually develops affecting predominantly the cerebellum. Ten of our patients were homozygous to the previously reported c.336C>G founder mutation while the other six patients were all compound heterozygotes displaying 10 novel mutations of whom 2 were nonsense predicting a deleterious effect on enzyme function. Structural protein modeling predicted significant impairment in aconitase substrate binding in the additional missense mutations. This study provides the most extensive cohort of patients and further delineates the clinical, radiological, biochemical, and molecular features of ACO2 deficiency.

Crystal structure of the human mitochondrial chaperonin symmetrical football complex.

Human mitochondria harbor a single type I chaperonin system that is generally thought to function via a unique single-ring intermediate. To date, no crystal structure has been published for any mammalian type I chaperonin complex. In this study, we describe the crystal structure of a football-shaped, double-ring human mitochondrial chaperonin complex at 3.15 Å, which is a novel intermediate, likely representing the complex in an early stage of dissociation. Interestingly, the mitochondrial chaperonin was captured in a state that exhibits subunit asymmetry within the rings and nucleotide symmetry between the rings. Moreover, the chaperonin tetradecamers show a different interring subunit arrangement when compared to GroEL. Our findings suggest that the mitochondrial chaperonins use a mechanism that is distinct from the mechanism of the well-studied Escherichia coli system.

The TIM23 mitochondrial protein import complex: function and dysfunction.

Mitochondria acquire the majority of their proteins from the cytosol in a process that is mediated by intricate multimeric machineries designed to allow proteins to cross and/or to insert themselves into the two mitochondrial membranes. Ongoing studies carried out in yeast over the past few decades have led to the discovery of numerous protein components that constitute several mitochondrial translocases. One of these complexes, the mitochondrial TIM23, is the major translocase for matrix proteins and is the focus of this review. The components of the TIM23 complex are categorized into four functional types. The first type plays the role of receptor for preproteins in the intermembrane space. The second type forms the actual channel that allows proteins to cross the inner mitochondrial membrane. The third species functions as part of the motor that mediates the final steps of import across the inner membrane. Additional components play regulatory roles orchestrating the action of this myriad of subunits. Recent studies provide new insights into the function of the mammalian TIM23 complex and the role that it plays under pathological conditions.

Homozygous mutation in PTRH2 gene causes progressive sensorineural deafness and peripheral neuropathy.

PTRH2 is an evolutionarily highly conserved mitochondrial protein that belongs to a family of peptidyl-tRNA hydrolases. Recently, patients from two consanguineous families with mutations in the PTRH2 gene were reported. Global developmental delay associated with microcephaly, growth retardation, progressive ataxia, distal muscle weakness with ankle contractures, demyelinating sensorimotor neuropathy, and sensorineural hearing loss were present in all patients, while facial dysmorphism with widely spaced eyes, exotropia, thin upper lip, proximally placed thumbs, and deformities of the fingers and toes were present in some individuals. Here, we report a new family with three siblings affected by sensorineural hearing loss and peripheral neuropathy. Autozygosity mapping followed by exome sequencing identified a previously reported homozygous missense mutation in PTRH2 (c.254A>C; p.(Gln85Pro)). Sanger sequencing confirmed that the variant segregated with the phenotype. In contrast to the previously reported patient, the affected siblings had normal intelligence, milder microcephaly, delayed puberty, myopia, and moderate insensitivity to pain. Our findings expand the clinical phenotype and further demonstrate the clinical heterogeneity related to PTRH2 variants.

GroEL and CCT are catalytic unfoldases mediating out-of-cage polypeptide refolding without ATP.

Chaperonins are cage-like complexes in which nonnative polypeptides prone to aggregation are thought to reach their native state optimally. However, they also may use ATP to unfold stably bound misfolded polypeptides and mediate the out-of-cage native refolding of large proteins. Here, we show that even without ATP and GroES, both GroEL and the eukaryotic chaperonin containing t-complex polypeptide 1 (CCT/TRiC) can unfold stable misfolded polypeptide conformers and readily release them from the access ways to the cage. Reconciling earlier disparate experimental observations to ours, we present a comprehensive model whereby following unfolding on the upper cavity, in-cage confinement is not needed for the released intermediates to slowly reach their native state in solution. As over-sticky intermediates occasionally stall the catalytic unfoldase sites, GroES mobile loops and ATP are necessary to dissociate the inhibitory species and regenerate the unfolding activity. Thus, chaperonin rings are not obligate confining antiaggregation cages. They are polypeptide unfoldases that can iteratively convert stable off-pathway conformers into functional proteins.

Methylation-controlled J-protein MCJ acts in the import of proteins into human mitochondria.

Loss of expression of the methylation-controlled J gene, MCJ (DNAJC15), is observed in cases of several tumors and plays a crucial role in the chemoresistance of ovarian cancer cells. Aside from the pathophysiological effects, almost nothing is known about the cellular function of MCJ. Here, we provide the first evidence that MCJ acts in the biogenesis of mitochondria. Our results demonstrate that MCJ is located in mitochondria. It is anchored in the mitochondrial inner membrane with the C-terminal J domain facing the matrix space. We show that MCJ forms a stable subcomplex with a component of the mitochondrial import motor, MAGMAS, a protein overexpressed in cells treated with granulocyte-macrophage colony-stimulating factor and in prostate carcinomas. In addition, MCJ and MAGMAS interact with the core components of the TIM23 pre-protein translocase. We demonstrate that the recombinant soluble MCJ domain stimulates the ATPase activity of the human mtHsp70 chaperone, mortalin, the central component of the import motor of the TIM23 translocase. This stimulation is counteracted by MAGMAS. Moreover, pre-protein import into mitochondria is impaired in the absence of MCJ. Interestingly, MCJ is able to take over the function of Tim14, the essential J co-chaperone of the mitochondrial protein import motor in yeast. In summary, our results show that MCJ functions as J co-chaperone of the human TIM23 pre-protein translocase, suggesting a link between mitochondrial pre-protein import and tumorigenesis.

Crystal and solution studies of the "Plus-C" odorant-binding protein 48 from Anopheles gambiae: control of binding specificity through three-dimensional domain swapping.

Much physiological and behavioral evidence has been provided suggesting that insect odorant-binding proteins (OBPs) are indispensable for odorant recognition and thus are appealing targets for structure-based discovery and design of novel host-seeking disruptors. Despite the fact that more than 60 putative OBP-encoding genes have been identified in the malaria vector Anopheles gambiae, the crystal structures of only six of them are known. It is therefore clear that OBP structure determination constitutes the bottleneck for structure-based approaches to mosquito repellent/attractant discovery. Here, we describe the three-dimensional structure of an A. gambiae "Plus-C" group OBP (AgamOBP48), which exhibits the second highest expression levels in female antennae. This structure represents the first example of a three-dimensional domain-swapped dimer in dipteran species. A combined binding site is formed at the dimer interface by equal contribution of each monomer. Structural comparisons with the monomeric AgamOBP47 revealed that the major structural difference between the two Plus-C proteins localizes in their N- and C-terminal regions, and their concerted conformational change may account for monomer-swapped dimer conversion and furthermore the formation of novel binding pockets. Using a combination of gel filtration chromatography, differential scanning calorimetry, and analytical ultracentrifugation, we demonstrate the AgamOBP48 dimerization in solution. Eventually, molecular modeling calculations were used to predict the binding mode of the most potent synthetic ligand of AgamOBP48 known so far, discovered by ligand- and structure-based virtual screening. The structure-aided identification of multiple OBP binders represents a powerful tool to be employed in the effort to control transmission of the vector-borne diseases.

SCAPER-Related Autosomal Recessive Retinitis Pigmentosa with Intellectual Disability: Confirming and Extending the Phenotypic Spectrum and Bioinformatics Analyses.

Mutations in the gene SCAPER (S phase Cyclin A-Associated Protein residing in the Endoplasmic Reticulum) have recently been associated with retinitis pigmentosa (RP) and intellectual disability (ID). In 2011, a possible involvement of SCAPER in human diseases was discovered for the first time due to the identification of a homozygous mutation causing ID in an Iranian family. Later, five studies were published in 2019 that described patients with autosomal recessive syndromic retinitis pigmentosa (arRP) accompanied by ID and attention-deficit/hyperactivity disorder (ADHD). This present study describes three patients from an Arab consanguineous family in Israel with similar clinical features of the SCAPER syndrome. In addition, new manifestations of ocular symptoms, nystagmus, glaucoma, and elevator palsy, were observed. Genetic testing of the patients and both parents via whole-exome sequencing revealed the homozygous mutation c.2023-2A>G in SCAPER. Phenotypic and genotypic descriptions for all available cases described in the literature including our current three cases (37 cases) were carried out, in addition to a bioinformatics analysis for all the genetic variants that was undertaken. Our study confirms and extends the clinical manifestations of SCAPER-related disorders.

Biochemical and neurophysiological effects of deficiency of the mitochondrial import protein TIMM50.

TIMM50, an essential TIM23 complex subunit, is suggested to facilitate the import of ∼60% of the mitochondrial proteome. In this study, we characterized a TIMM50 disease causing mutation in human fibroblasts, and noted significant decreases in TIM23 core protein levels (TIMM50, TIMM17A/B, and TIMM23). Strikingly, TIMM50 deficiency had no impact on the steady state levels of most of its substrates, challenging the currently accepted import dogma of the essential general import role of TIM23 and suggesting that fully functioning TIM23 complex is not essential for maintaining the steady state level of the majority of mitochondrial proteins. As TIMM50 mutations have been linked to severe neurological phenotypes, we aimed to characterize TIMM50 defects in manipulated mammalian neurons. TIMM50 knockdown in mouse neurons had a minor effect on the steady state level of most of the mitochondrial proteome, supporting the results observed in patient fibroblasts. Amongst the few affected TIM23 substrates, a decrease in the steady state level of components of the intricate oxidative phosphorylation and mitochondrial ribosome complexes was evident. This led to declined respiration rates in fibroblasts and neurons, reduced cellular ATP levels and defective mitochondrial trafficking in neuronal processes, possibly contributing to the developmental defects observed in patients with TIMM50 disease. Finally, increased electrical activity was observed in TIMM50 deficient mice neuronal cells, which correlated with reduced levels of KCNJ10 and KCNA2 plasma membrane potassium channels, likely underlying the patients' epileptic phenotype.

Tracking the interplay between bound peptide and the lid domain of DnaK, using molecular dynamics.

Hsp70 chaperones consist of two functional domains: the 44 kDa Nucleotide Binding Domain (NBD), that binds and hydrolyses ATP, and the 26 kDa Substrate Binding Domain (SBD), which binds unfolded proteins and reactivates them, utilizing energy obtained from nucleotide hydrolysis. The structure of the SBD of the bacterial Hsp70, DnaK, consists of two sub-domains: A ß-sandwich part containing the hydrophobic cavity to which the hepta-peptide NRLLLTG (NR) is bound, and a segment made of 5 α-helices, called the "lid" that caps the top of the ß-sandwich domain. In the present study we used the Escherichia coli Hsp70, DnaK, as a model for Hsp70 proteins, focusing on its SBD domain, examining the changes in the lid conformation. We deliberately decoupled the NBD from the SBD, limiting the study to the structure of the SBD section, with an emphasis on the interaction between the charges of the peptide with the residues located in the lid. Molecular dynamics simulations of the complex revealed significant mobility within the lid structure; as the structure was released from the forces operating during the crystallization process, the two terminal helices established a contact with the positive charge at the tip of the peptide. This contact is manifested only in the presence of electrostatic attraction. The observed internal motions within the lid provide a molecular role for the function of this sub-domain during the reaction cycle of Hsp 70 chaperones.

PTRH2 Gene Variants: Recent Review of the Phenotypic Features and Their Bioinformatics Analysis.

Peptidyl-tRNA hydrolase 2 (PTRH2) is an evolutionarily highly conserved mitochondrial protein. The biallelic mutations in the PTRH2 gene have been suggested to cause a rare autosomal recessive disorder characterized by an infantile-onset multisystem neurologic endocrine and pancreatic disease (IMNEPD). Patients with IMNEPD present varying clinical manifestations, including global developmental delay associated with microcephaly, growth retardation, progressive ataxia, distal muscle weakness with ankle contractures, demyelinating sensorimotor neuropathy, sensorineural hearing loss, and abnormalities of thyroid, pancreas, and liver. In the current study, we conducted an extensive literature review with an emphasis on the variable clinical spectrum and genotypes in patients. Additionally, we reported on a new case with a previously documented mutation. A bioinformatics analysis of the various PTRH2 gene variants was also carried out from a structural perspective. It appears that the most common clinical characteristics among all patients include motor delay (92%), neuropathy (90%), distal weakness (86.4%), intellectual disability (84%), hearing impairment (80%), ataxia (79%), and deformity of head and face (~70%). The less common characteristics include hand deformity (64%), cerebellar atrophy/hypoplasia (47%), and pancreatic abnormality (35%), while the least common appear to be diabetes mellitus (~30%), liver abnormality (~22%), and hypothyroidism (16%). Three missense mutations were revealed in the PTRH2 gene, the most common one being Q85P, which was shared by four different Arab communities and was presented in our new case. Moreover, four different nonsense mutations in the PTRH2 gene were detected. It may be concluded that disease severity depends on the PTRH2 gene variant, as most of the clinical features are manifested by nonsense mutations, while only the common features are presented by missense mutations. A bioinformatics analysis of the various PTRH2 gene variants also suggested the mutations to be deleterious, as they seem to disrupt the structural confirmation of the enzyme, leading to loss of stability and functionality.

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.

Cross-linking with bifunctional reagents and its application to the study of the molecular symmetry and the arrangement of subunits in hexameric protein oligomers.

Cross-linking with a bifunctional reagent and subsequent SDS gel electrophoresis is a simple but effective method to study the symmetry and arrangement of subunits in oligomeric proteins. In this study, theoretical expressions for the description of cross-linking patterns were derived for protein homohexamers through extension of the method used for tetramers by Hajdu et al. (1976). The derived equations were used for the analysis of cross-linking by glutardialdehyde of four protein hexamers: beef liver glutamate dehydrogenase (GDH), jack bean urease, hemocyanin from the spiny lobster Panulirus pencillatus (PpHc), Escherichia coli glutamate decarboxylase (GDC) and for analysis of published data on the cross-linking of hexameric E. coli rho by dimethyl suberimidate. Best fit models showed that the subunits in the first four proteins are arranged according to D(3) symmetry in two layers, each subunit able to cross-link to three neighboring subunits for GDH and urease, or to four for PpHc and GDC. The findings indicate a dimer-of-trimers eclipsed arrangement of subunits for GDH and urease and a trimer-of-dimers staggered one for PpHc and GDC. In rho, the subunits are arranged according to D(3) symmetry in a trimer-of-dimers ring. The conclusions from cross-linking of GDH and GDC, PpHc and rho are consistent with results from X-ray crystal structure, those for urease with findings from electron microscopy.

Chloroplast ß chaperonins from A. thaliana function with endogenous cpn10 homologs in vitro.

The involvement of type I chaperonins in bacterial and organellar protein folding has been well-documented. In E. coli and mitochondria, these ubiquitous and highly conserved proteins form chaperonin oligomers of identical 60 kDa subunits (cpn60), while in chloroplasts, two distinct cpn60 α and ß subunit types co-exist together. The primary sequence of α and ß subunits is ~50% identical, similar to their respective homologies to the bacterial GroEL. Moreover, the A. thaliana genome contains two α and four ß genes. The functional significance of this variability in plant chaperonin proteins has not yet been elucidated. In order to gain insight into the functional variety of the chloroplast chaperonin family members, we reconstituted ß homo-oligomers from A. thaliana following their expression in bacteria and subjected them to a structure-function analysis. Our results show for the first time, that A. thaliana ß homo-oligomers can function in vitro with authentic chloroplast co-chaperonins (ch-cpn10 and ch-cpn20). We also show that oligomers made up of different ß subunit types have unique properties and different preferences for co-chaperonin partners. We propose that chloroplasts may contain active ß homo-oligomers in addition to hetero-oligomers, possibly reflecting a variety of cellular roles.

The MitCHAP-60 disease is due to entropic destabilization of the human mitochondrial Hsp60 oligomer.

The 60-kDa heat shock protein (mHsp60) is a vital cellular complex that mediates the folding of many of the mitochondrial proteins. Its function is executed in cooperation with the co-chaperonin, mHsp10, and requires ATP. Recently, the discovery of a new mHsp60-associated neurodegenerative disorder, MitCHAP-60 disease, has been reported. The disease is caused by a point mutation at position 3 (D3G) of the mature mitochondrial Hsp60 protein, which renders it unable to complement the deletion of the homologous bacterial protein in Escherichia coli (Magen, D., Georgopoulos, C., Bross, P., Ang, D., Segev, Y., Goldsher, D., Nemirovski, A., Shahar, E., Ravid, S., Luder, A., Heno, B., Gershoni-Baruch, R., Skorecki, K., and Mandel, H. (2008) Am. J. Hum. Genet. 83, 30-42). The molecular basis of the MitCHAP-60 disease is still unknown. In this study, we present an in vitro structural and functional analysis of the purified wild-type human mHsp60 and the MitCHAP-60 mutant. We show that the D3G mutation leads to destabilization of the mHsp60 oligomer and causes its disassembly at low protein concentrations. We also show that the mutant protein has impaired protein folding and ATPase activities. An additional mutant that lacks the first three amino acids (N-del), including Asp-3, is similarly impaired in refolding activity. Surprisingly, however, this mutant exhibits profound stabilization of its oligomeric structure. These results suggest that the D3G mutation leads to entropic destabilization of the mHsp60 oligomer, which severely impairs its chaperone function, thereby causing the disease.

Gene and Protein Expression in Subjects With a Nystagmus-Associated AHR Mutation.

Recently, a consanguineous family was identified in Israel with three children affected by Infantile Nystagmus and Foveal Hypoplasia, following an autosomal recessive mode of inheritance. A homozygous stop mutation c.1861C > T; p.Q621∗ in the aryl hydrocarbon receptor (AHR) gene (AHR; MIM 600253) was identified that co-segregated with the disease in the larger family. AHR is the first gene to be identified causing an autosomal recessive Infantile Nystagmus-related disease in humans. The goal of this study is to delineate the molecular basis of this newly discovered human genetic disorder associated with a rare AHR gene mutation. The gene and protein expression levels of AHR and selected AHR targets from leukocyte cultures of healthy subjects and the patients were analyzed. We observed significant variation between mRNA and protein expression of CYP1A1, CYP1B1, and TiPARP under rest and AHR-induced conditions. The CYP1A1 enzymatic activity in induced leukocytes also differs significantly between the patients and healthy volunteers. Intriguingly, the heterozygous subjects demonstrate CYP1A1 and TiPARP gene and protein expression similar to homozygous patients. In contrast, CYP1B1 inducibility and expression vary between hetero- and homozygous subjects. Similarity and differences in gene and protein expression between heterozygotes and homozygous patients can give us a hint as to which metabolic pathway/s might be involved in the Nystagmus etiology. Thus, we have a unique human model for AHR deficiency that will allow us the opportunity to study the biochemical basis of this rare human mutation, as well as the involvement of AHR in other physiological processes.

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.