Chemical inhibition of phosphatidylcholine biogenesis reveals its role in mitochondrial division.

Phospholipids are major components of biological membranes and play structural and regulatory roles in various biological processes. To determine the biological significance of phospholipids, the use of chemical inhibitors of phospholipid metabolism offers an effective approach; however, the availability of such compounds is limited. In this study, we performed a chemical-genetic screening using yeast and identified small molecules capable of inhibiting phosphatidylcholine (PC) biogenesis, which we designated PC inhibitors 1, 2, 3, and 4 (PCiB-1, 2, 3, and 4). Biochemical analyses indicated that PCiB-2, 3, and 4 inhibited the phosphatidylethanolamine (PE) methyltransferase activity of Cho2, whereas PCiB-1 may inhibit PE transport from mitochondria to the endoplasmic reticulum (ER). Interestingly, we found that PCiB treatment resulted in mitochondrial fragmentation, which was suppressed by expression of a dominant-negative mutant of the mitochondrial division factor Dnm1. These results provide evidence that normal PC biogenesis is important for the regulation of mitochondrial division.

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.

TIM23 facilitates PINK1 activation by safeguarding against OMA1-mediated degradation in damaged mitochondria.

PINK1 is activated by autophosphorylation and forms a high-molecular-weight complex, thereby initiating the selective removal of damaged mitochondria by autophagy. Other than translocase of the outer mitochondrial membrane complexes, members of PINK1-containing protein complexes remain obscure. By mass spectrometric analysis of PINK1 co-immunoprecipitates, we identify the inner membrane protein TIM23 as a component of the PINK1 complex. TIM23 downregulation decreases PINK1 levels and significantly delays autophosphorylation, indicating that TIM23 promotes PINK1 accumulation in response to depolarization. Moreover, inactivation of the mitochondrial protease OMA1 not only enhances PINK1 accumulation but also represses the reduction in PINK1 levels induced by TIM23 downregulation, suggesting that TIM23 facilitates PINK1 activation by safeguarding against degradation by OMA1. Indeed, deficiencies of pathogenic PINK1 mutants that fail to interact with TIM23 are partially restored by OMA1 inactivation. These findings indicate that TIM23 plays a distinct role in activating mitochondrial autophagy by protecting PINK1.

A multipoint guidance mechanism for ß-barrel folding on the SAM complex.

Mitochondrial ß-barrel proteins are essential for the transport of metabolites, ions and proteins. The sorting and assembly machinery (SAM) mediates their folding and membrane insertion. We report the cryo-electron microscopy structure of the yeast SAM complex carrying an early eukaryotic ß-barrel folding intermediate. The lateral gate of Sam50 is wide open and pairs with the last ß-strand (ß-signal) of the substrate-the 19-ß-stranded Tom40 precursor-to form a hybrid barrel in the membrane plane. The Tom40 barrel grows and curves, guided by an extended bridge with Sam50. Tom40's first ß-segment (ß1) penetrates into the nascent barrel, interacting with its inner wall. The Tom40 amino-terminal segment then displaces ß1 to promote its pairing with Tom40's last ß-strand to complete barrel formation with the assistance of Sam37's dynamic α-protrusion. Our study thus reveals a multipoint guidance mechanism for mitochondrial ß-barrel folding.

Proofreading of protein localization mediated by a mitochondrial AAA-ATPase Msp1.

Normal cellular functions rely on correct protein localization within cells. Protein targeting had been thought to be a precise process, and even if it fails, the mistargeted proteins were supposed to be quickly degraded. However, this view is rapidly changing. Tail-anchored (TA) proteins are a class of membrane proteins that possess a single transmembrane domain (TMD) near the C-terminus and are posttranslationally targeted to the endoplasmic reticulum (ER) membrane, mitochondrial outer membrane (OM), and peroxisomal membrane, yet they can be mistargeted to the mitochondrial OM. The mistargeted TA proteins can be extracted from the OM by a mitochondrial AAA-ATPase Msp1/ATAD1 and transferred to the ER. If they are regarded as aberrant by the ER protein quality control system, they are extracted from the ER membrane for proteasomal degradation in the cytosol. If they are not regarded as aberrant, they are further transported to downstream organelles or original destinations along the secretory pathway. Thus, Msp1 contributes to not only degradation but also "proofreading" of the targeting of mislocalized TA proteins.

Dissociation of ERMES clusters plays a key role in attenuating the endoplasmic reticulum stress.

In yeast, ERMES, which mediates phospholipid transport between the ER and mitochondria, forms a limited number of oligomeric clusters at ER-mitochondria contact sites in a cell. Although the number of the ERMES clusters appears to be regulated to maintain proper inter-organelle phospholipid trafficking, its underlying mechanism and physiological relevance remain poorly understood. Here, we show that mitochondrial dynamics control the number of ERMES clusters. Moreover, we find that ER stress causes dissociation of the ERMES clusters independently of Ire1 and Hac1, canonical ER-stress response pathway components, leading to a delay in the phospholipid transport from the ER to mitochondria. Our biochemical and genetic analyses strongly suggest that the impaired phospholipid transport contributes to phospholipid accumulation in the ER, expanding the ER for ER stress attenuation. We thus propose that the ERMES dissociation constitutes an overlooked pathway of the ER stress response that operates in addition to the canonical Ire1/Hac1-dependent pathway.

Structural overview of the translocase of the mitochondrial outer membrane complex.

Most mitochondrial proteins are synthesized as precursor proteins (preproteins) in the cytosol and imported into mitochondria. The translocator of the outer membrane (TOM) complex functions as a main entry gate for the import of mitochondrial proteins. The TOM complex is a multi-subunit membrane protein complex composed of a ß-barrel channel Tom40 and six single-pass membrane proteins. Recent cryo-EM studies have revealed high-resolution structures of the yeast and human TOM complexes, which enabled us to discuss the mechanism of protein import at an amino-acid residue level. The cryo-EM structures show that two Tom40 ß-barrels are surrounded by two sets of small Tom subunits to form a dimeric structure. The intermembrane space (IMS) domains of Tom40, Tom22, and Tom7 form a binding site for presequence-containing preproteins in the middle of the dimer to achieve their efficient transfer of to the downstream translocase, the TIM23 complex. The N-terminal segment of Tom40 spans the channel from the cytosol to the IMS to interact with Tom5 at the periphery of the dimer, where downstream components of presequence-lacking preproteins are recruited. Structure-based biochemical analyses together with crosslinking experiments revealed that each Tom40 channel possesses two distinct paths and exit sites for protein translocation of different sets of mitochondrial preproteins. Here we summarize the current knowledge on the structural features, protein translocation mechanisms, and remaining questions for the TOM complexes, with particular emphasis on their determined cryo-EM structures. This article is an extended version of the Japanese article, Structural basis for protein translocation by the translocase of the outer mitochondrial membrane, published in SEIBUTSU BUTSURI Vol. 60, p. 280-283 (2020).

GET pathway mediates transfer of mislocalized tail-anchored proteins from mitochondria to the ER.

Tail-anchored (TA) membrane proteins have a potential risk to be mistargeted to the mitochondrial outer membrane (OM). Such mislocalized TA proteins can be extracted by the mitochondrial AAA-ATPase Msp1 from the OM and transferred to the ER for ER protein quality control involving ubiquitination by the ER-resident Doa10 complex. Yet it remains unclear how the extracted TA proteins can move to the ER crossing the aqueous cytosol and whether this transfer to the ER is essential for the clearance of mislocalized TA proteins. Here we show by time-lapse microscopy that mislocalized TA proteins, including an authentic ER-TA protein, indeed move from mitochondria to the ER in a manner strictly dependent on Msp1 expression. The Msp1-dependent mitochondria-to-ER transfer of TA proteins is blocked by defects in the GET system, and this block is not due to impaired Doa10 functions. Thus, the GET pathway facilitates the transfer of mislocalized TA proteins from mitochondria to the ER.

Role of the TOM Complex in Protein Import into Mitochondria: Structural Views.

Mitochondria are central to energy production, metabolism and signaling, and apoptosis. To make new mitochondria from preexisting mitochondria, the cell needs to import mitochondrial proteins from the cytosol into the mitochondria with the aid of translocators in the mitochondrial membranes. The translocase of the outer membrane (TOM) complex, an outer membrane translocator, functions as an entry gate for most mitochondrial proteins. Although high-resolution structures of the receptor subunits of the TOM complex were deposited in the early 2000s, those of entire TOM complexes became available only in 2019. The structural details of these TOM complexes, consisting of the dimer of the ß-barrel import channel Tom40 and four α-helical membrane proteins, revealed the presence of several distinct paths and exits for the translocation of over 1,000 different mitochondrial precursor proteins. High-resolution structures of TOM complexes now open up a new era of studies on the structures, functions, and dynamics of the mitochondrial import system.

Crystal structure of Tam41 cytidine diphosphate diacylglycerol synthase from a Firmicutes bacterium.

Translocator assembly and maintenance 41 (Tam41) catalyses the synthesis of cytidine diphosphate diacylglycerol (CDP-DAG), which is a high-energy intermediate phospholipid critical for generating cardiolipin in mitochondria. Although Tam41 is present almost exclusively in eukaryotic cells, a Firmicutes bacterium contains the gene encoding Tam41-type CDP-DAG synthase (FbTam41). FbTam41 converted phosphatidic acid (PA) to CDP-DAG using a ternary complex mechanism in vitro. Additionally, FbTam41 functionally substituted yeast Tam41 in vivo. These results demonstrate that Tam41-type CDP-DAG synthase functions in some prokaryotic cells. We determined the crystal structure of FbTam41 lacking the C-terminal 18 residues in the cytidine triphosphate (CTP)-Mg2+ bound form at a resolution of 2.6 Å. The crystal structure showed that FbTam41 contained a positively charged pocket that specifically accommodated CTP-Mg2+ and PA in close proximity. By using this structure, we constructed a model for the full-length structure of FbTam41 containing the last a-helix, which was missing in the crystal structure. Based on this model, we propose a molecular mechanism for CDP-DAG synthesis in bacterial cells and mitochondria.

Acellular Cartilage Repair Technique Based on Ultrapurified Alginate Gel Implantation for Advanced Capitellar Osteochondritis Dissecans.

BACKGROUND: One of the most important limitations of osteochondral autograft transplant is the adverse effect on donor sites in the knee. Ultrapurified alginate (UPAL) gel is a novel biomaterial that enhances hyaline-like cartilage repair for articular defects. To avoid the need for knee cartilage autografting when treating osteochondritis dissecans (OCD) of the capitellum, we developed a surgical procedure involving a bone marrow stimulation technique (BMST) augmented by implantation of UPAL gel. HYPOTHESIS: BMST augmented by UPAL gel implantation improves the cartilage repair capacity and provides satisfactory clinical outcomes in OCD of the capitellum. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: A total of 5 athletes with advanced capitellar OCD in the dominant elbow underwent BMST augmented by implantation of UPAL gel. The osteochondral defects were filled with UPAL gel after BMST. At a mean follow-up of 97 weeks, all patients were evaluated clinically and radiographically. RESULTS: At final follow-up, all 5 patients had returned to competitive-level sports, and 4 patients were free from elbow pain. The mean Timmerman-Andrews score significantly improved from 100 to 194 points. Radiographically, all patients exhibited graft incorporation and a normal contour of the subchondral cortex. Magnetic resonance imaging showed that the preoperative heterogeneity of the lesion had disappeared, and the signal intensity had returned to normal. Arthroscopic examinations consistently exhibited improvement in the International Cartilage Regeneration and Joint Preservation Society (ICRS) grade of lesions from 3 or 4 to 1 or 2 in 4 patients at 85 weeks postoperatively. Histologic analysis of biopsy specimens revealed an average total ICRS Visual Assessment Scale II histologic score of 1060. CONCLUSION: The acellular cartilage repair technique using UPAL gel for advanced capitellar OCD provided satisfactory clinical and radiographic results. The present results suggest that this novel technique is a useful, minimally invasive approach for treating cartilaginous lesions in athletes.

Shear bond strength of orthodontic brackets bonded with resin coating material.

The purpose of this study was to determine whether a system using a resin coating material (PRG Barrier Coat) with anticariogenic ability can effectively bond orthodontic brackets to human teeth. Resin-modified glass-ionomer cement system (Fuji Ortho LC, group 1) and resin composite cement systems (BeautyOrtho Bond) combined with a self-etching primer (group 2), with the resin coating material (group 3), and with the resin coating material after an organic acid etching agent (group 4) were used for bracket bonding. The mean shear bond strength (SBS) was significantly higher in group 1 than in groups 2, 3 and 4. Groups 2 and 4 exhibited a significantly higher mean SBS than group 3. The resin composite cement system combined with the resin coating material after the organic acid etching agent can serve as an alternative for orthodontic bracket bonding.

Effects of plastic bracket primer on the shear bond strengths of orthodontic brackets.

BACKGROUND/PURPOSE: To assess the usefulness of plastic bracket primer (PBP) for improving the bond strength of plastic brackets (PBs) using three types of orthodontic brackets, including PBs, metal brackets (MBs), and ceramic brackets (CBs). MATERIALS AND METHODS: A total of 162 premolars were gathered and divided equally into six groups of 27. Three groups were tested with the application of PBP (PB+, MB+, and CB+), and three groups were tested without primer (groups PB-, MB-, and CB-). All the groups were bonded using BeautiOrtho Bond II self-etching adhesive. The shear bond strength (SBS) was measured and the bond failure mode was evaluated using the adhesive remnant index after debonding. RESULTS: There were significant differences in the mean SBS between groups PB-, MB and CB-, between PB+ and CB+, and between MB+ and CB+. Group PB + had a significantly higher mean SBS than group PB-. The occurrence of bond failure at the enamel and adhesive interface was more frequent in groups PB+ and CB- than in group PB-; and in groups PB+ and CB + than in group MB+. CONCLUSION: Plastic bracket primer can increase the bond strength of PBs to the level of metal brackets, but not to the level of ceramic brackets.

Mitochondrial sorting and assembly machinery operates by ß-barrel switching.

The mitochondrial outer membrane contains so-called ß-barrel proteins, which allow communication between the cytosol and the mitochondrial interior1-3. Insertion of ß-barrel proteins into the outer membrane is mediated by the multisubunit mitochondrial sorting and assembly machinery (SAM, also known as TOB)4-6. Here we use cryo-electron microscopy to determine the structures of two different forms of the yeast SAM complex at a resolution of 2.8-3.2 Å. The dimeric complex contains two copies of the ß-barrel channel protein Sam50-Sam50a and Sam50b-with partially open lateral gates. The peripheral membrane proteins Sam35 and Sam37 cap the Sam50 channels from the cytosolic side, and are crucial for the structural and functional integrity of the dimeric complex. In the second complex, Sam50b is replaced by the ß-barrel protein Mdm10. In cooperation with Sam50a, Sam37 recruits and traps Mdm10 by penetrating the interior of its laterally closed ß-barrel from the cytosolic side. The substrate-loaded SAM complex contains one each of Sam50, Sam35 and Sam37, but neither Mdm10 nor a second Sam50, suggesting that Mdm10 and Sam50b function as placeholders for a ß-barrel substrate released from Sam50a. Our proposed mechanism for dynamic switching of ß-barrel subunits and substrate explains how entire precursor proteins can fold in association with the mitochondrial machinery for ß-barrel assembly.

Structural snapshot of the mitochondrial protein import gate.

The translocase of the outer mitochondrial membrane (TOM) complex is the main entry gate for most mitochondrial proteins. The TOM complex is a multisubunit membrane protein complex consisting of a ß-barrel protein Tom40 and six α-helical transmembrane (TM) proteins, receptor subunits Tom20, Tom22, and Tom70, and regulatory subunits Tom5, Tom6, and Tom7. Although nearly 30 years have passed since the main components of the TOM complex were identified and characterized, the structural details of the TOM complex remained poorly understood until recently. Thanks to the rapid development of the cryoelectron microscopy (EM) technology, high-resolution structures of the yeast TOM complex have become available. The identified structures showed a symmetric dimer containing five different subunits including Tom22. Biochemical and mutational analyses based on the TOM complex structure revealed the presence of different translocation paths within the Tom40 import channel for different classes of translocating precursor proteins. Previous studies including our cross-linking analyses indicated that the TOM complex in intact mitochondria is present as a mixture of the trimeric complex containing Tom22. Furthermore, the dimeric complex lacking Tom22, and the trimer and dimer may handle different sets of mitochondrial precursor proteins for translocation across the outer membrane. In this Structural Snapshot, we will discuss possible rearrangement of the subunit interactions upon dynamic conversion of the TOM complex between the different subunit assembly states, the Tom22-containing core dimer and trimer.

Relationships between skeletal morphology and patterns of bilateral agenesis of third molars in Japanese orthodontic patients.

The aim of this study was to reveal the correlations between bilateral agenesis of third molars (M3s) and skeletal morphology in Japanese male and female orthodontic patients. Sixty patients (30 males, 30 females), with bilateral agenesis of maxillary M3s and without agenesis of mandibular M3s (group U), and 60 patients (30 males, 30 females), with bilateral agenesis of mandibular M3s and without agenesis of maxillary M3s (group L), were selected as agenesis groups. Additionally, 60 patients (30 males, 30 females) with all four M3s were selected as the control group (group C). Patients in these three groups had no agenesis of teeth other than M3s. Lateral cephalograms of each patient were used to evaluate skeletal morphology of the maxilla and mandible. Two-way analysis of variance was used for statistical comparisons. Groups U and L had significantly smaller maxillary length and area than group C. Group U exhibited a significantly smaller lower facial height than group C. Males showed significantly larger maxillary length; total mandibular and mandibular body length; mandibular ramus height; SNB angle; maxillary area; and mandibular symphysis, corpus and ramus areas than females. Females had significantly larger lower facial height, gonial angle and ANB angle than males. Smaller maxillary length and area and lower facial height should be considered in planning orthodontic treatment for patients with bilateral agenesis of maxillary and mandibular M3s.

Phosphatidylserine flux into mitochondria unveiled by organelle-targeted Escherichia coli phosphatidylserine synthase PssA.

Most phospholipids are synthesised in the endoplasmic reticulum and distributed to other cellular membranes. Although the vesicle transport contributes to the phospholipid distribution among the endomembrane system, exactly how phospholipids are transported to, from and between mitochondrial membranes remains unclear. To gain insights into phospholipid transport routes into mitochondria, we expressed the Escherichia coli phosphatidylserine (PS) synthase PssA in various membrane compartments with distinct membrane topologies in yeast cells lacking a sole PS synthase (Cho1). Interestingly, PssA could complement loss of Cho1 when targeted to the endoplasmic reticulum (ER), peroxisome, or lipid droplet membranes. Synthesised PS could be converted to phosphatidylethanolamine (PE) by Psd1, the mitochondrial PS decarboxylase, suggesting that phospholipids synthesised in the peroxisomes and low doses (LDs) can efficiently reach mitochondria. Furthermore, we found that PssA which has been integrated into the mitochondrial inner membrane (MIM) from the matrix side could partially complement the loss of Cho1. The PS synthesised in the MIM was also converted to PE, indicating that PS flops across the MIM to become PE. These findings expand our understanding of the intracellular phospholipid transport routes via mitochondria.

Bond strength of indirect bonded brackets in orthodontic adhesives with different viscosities.

The aim of this study was to assess the effects of three adhesives with different viscosities and an adhesion promoter on the shear bond strength (SBS) of orthodontic brackets bonded to human premolars with an indirect bonding system (IDBS). High, medium and low viscosity IDBSs with and without application of the adhesion promoter were used. The mean SBSs of the high and low viscosity IDBSs were significantly higher than that of the medium viscosity IDBS. Application of the adhesion promoter significantly increased the SBSs. The adhesion promoter significantly increased the surface roughness and free-energy of enamel. Irrespective of application or nonapplication of the adhesion promoter, the high and low viscosity IDBSs are effective for bracket bonding. Use of the medium viscosity IDBS in combination with the adhesion promoter is recommended for obtaining a clinically acceptable SBS.