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.

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.

Msp1 Clears Mistargeted Proteins by Facilitating Their Transfer from Mitochondria to the ER.

Normal mitochondrial functions rely on optimized composition of their resident proteins, and proteins mistargeted to mitochondria need to be efficiently removed. Msp1, an AAA-ATPase in the mitochondrial outer membrane (OM), facilitates degradation of tail-anchored (TA) proteins mistargeted to the OM, yet how Msp1 cooperates with other factors to conduct this process was unclear. Here, we show that Msp1 recognizes substrate TA proteins and facilitates their transfer to the endoplasmic reticulum (ER). Doa10 in the ER membrane then ubiquitinates them with Ubc6 and Ubc7. Ubiquitinated substrates are extracted from the ER membrane by another AAA-ATPase in the cytosol, Cdc48, with Ufd1 and Npl4 for proteasomal degradation in the cytosol. Thus, Msp1 functions as an extractase that mediates clearance of mistargeted TA proteins by facilitating their transfer to the ER for protein quality control.

Porin Associates with Tom22 to Regulate the Mitochondrial Protein Gate Assembly.

Mitochondria import nearly all of their resident proteins from the cytosol, and the TOM complex functions as their entry gate. The TOM complex undergoes a dynamic conversion between the majority population of a three-channel gateway ("trimer") and the minor population that lacks Tom22 and has only two Tom40 channels ("dimer"). Here, we found that the porin Por1 acts as a sink to bind newly imported Tom22. This Por1 association thereby modulates Tom22 integration into the TOM complex, guaranteeing formation of the functional trimeric TOM complex. Por1 sequestration of Tom22 dissociated from the trimeric TOM complex also enhances the dimeric TOM complex, which is preferable for the import of TIM40/MIA-dependent proteins into mitochondria. Furthermore, Por1 appears to contribute to cell-cycle-dependent variation of the functional trimeric TOM complex by chaperoning monomeric Tom22, which arises from the cell-cycle-controlled variation of phosphorylated Tom6.

Mitochondrial matrix reloaded with RNA.

Although mitochondrial biogenesis requires the import of specific RNAs, the pathways and cellular machineries involved are only poorly understood. Wang et al. (2010) now find that polynucleotide phosphorylase in the intermembrane space of mammalian mitochondria facilitates import of several RNAs into the mitochondrial matrix.

Structure of the mitochondrial import gate reveals distinct preprotein paths.

The translocase of the outer mitochondrial membrane (TOM) is the main entry gate for proteins1-4. Here we use cryo-electron microscopy to report the structure of the yeast TOM core complex5-9 at 3.8-Å resolution. The structure reveals the high-resolution architecture of the translocator consisting of two Tom40 ß-barrel channels and α-helical transmembrane subunits, providing insight into critical features that are conserved in all eukaryotes1-3. Each Tom40 ß-barrel is surrounded by small TOM subunits, and tethered by two Tom22 subunits and one phospholipid. The N-terminal extension of Tom40 forms a helix inside the channel; mutational analysis reveals its dual role in early and late steps in the biogenesis of intermembrane-space proteins in cooperation with Tom5. Each Tom40 channel possesses two precursor exit sites. Tom22, Tom40 and Tom7 guide presequence-containing preproteins to the exit in the middle of the dimer, whereas Tom5 and the Tom40 N extension guide preproteins lacking a presequence to the exit at the periphery of the dimer.

Organelle membrane-specific chemical labeling and dynamic imaging in living cells.

Lipids play crucial roles as structural elements, signaling molecules and material transporters in cells. However, the functions and dynamics of lipids within cells remain unclear because of a lack of methods to selectively label lipids in specific organelles and trace their movement by live-cell imaging. We describe here a technology for the selective labeling and fluorescence imaging (microscopic or nanoscopic) of phosphatidylcholine in target organelles. This approach involves the metabolic incorporation of azido-choline, followed by a spatially limited bioorthogonal reaction that enables the visualization and quantitative analysis of interorganelle lipid transport in live cells. More importantly, with live-cell imaging, we obtained direct evidence that the autophagosomal membrane originates from the endoplasmic reticulum. This method is simple and robust and is thus powerful for real-time tracing of interorganelle lipid trafficking.

Structural basis for interorganelle phospholipid transport mediated by VAT-1.

Eukaryotic cells are compartmentalized to form organelles, whose functions rely on proper phospholipid and protein transport. Here we determined the crystal structure of human VAT-1, a cytosolic soluble protein that was suggested to transfer phosphatidylserine, at 2.2 Å resolution. We found that VAT-1 transferred not only phosphatidylserine but also other acidic phospholipids between membranes in vitro Structure-based mutational analyses showed the presence of a possible lipid-binding cavity at the interface between the two subdomains, and two tyrosine residues in the flexible loops facilitated phospholipid transfer, likely by functioning as a gate to this lipid-binding cavity. We also found that a basic and hydrophobic loop with two tryptophan residues protruded from the molecule and facilitated binding to the acidic-lipid membranes, thereby achieving efficient phospholipid transfer.

ERdj3B-Mediated Quality Control Maintains Anther Development at High Temperatures.

Pollen development is highly sensitive to heat stress, which impairs cellular proteostasis by causing misfolded proteins to accumulate. Therefore, each cellular compartment possesses a dedicated protein quality control system. An elaborate quality control system involving molecular chaperones, including immunoglobulin-binding protein (BiP), heat shock protein70, and regulatory J domain-containing cochaperones (J proteins), allows the endoplasmic reticulum (ER) to withstand a large influx of proteins. Here, we found that Arabidopsis (Arabidopsis thaliana) mutants of ER-localized DnaJ family 3B (ERdj3B), one of three ER-resident J proteins involved in ER quality control, produced few seeds at high temperatures (29°C) due to defects in anther development. This temperature-sensitive fertility defect is specific to the defective interactions of BiP with ERdj3B but not with the other two J proteins, indicating functional differences between ERdj3B and the other J proteins. RNA sequencing analysis revealed that heat stress affects pollen development in both wild-type and mutant buds, but the erdj3b mutant is more susceptible, possibly due to defects in ER quality control. Our results highlight the importance of a specific ER quality control factor, ERdj3B, for plant reproduction, particularly anther development, at high temperatures.

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.

Fertilization-Coupled Sperm Nuclear Fusion Is Required for Normal Endosperm Nuclear Proliferation.

Angiosperms exhibit double fertilization, a process in which one of the sperm cells released from the pollen tube fertilizes the egg, while the other sperm cell fertilizes the central cell, giving rise to the embryo and endosperm, respectively. We have previously reported two polar nuclear fusion-defective double knockout mutants of Arabidopsis thaliana immunoglobulin binding protein (BiP), a molecular chaperone of the heat shock protein 70 (Hsp70) localized in the endoplasmic reticulum (ER), (bip1 bip2) and its partner ER-resident J-proteins, ERdj3A and P58IPK (erdj3a p58ipk). These mutants are defective in the fusion of outer nuclear membrane and exhibit characteristic seed developmental defects after fertilization with wild-type pollen, which are accompanied by aberrant endosperm nuclear proliferation. In this study, we used time-lapse live-cell imaging analysis to determine the cause of aberrant endosperm nuclear division in these mutant seeds. We found that the central cell of bip1 bip2 or erdj3a p58ipk double mutant female gametophytes was also defective in sperm nuclear fusion at fertilization. Sperm nuclear fusion was achieved after the onset of the first endosperm nuclear division. However, division of the condensed sperm nucleus resulted in aberrant endosperm nuclear divisions and delayed expression of paternally derived genes. By contrast, the other double knockout mutant, erdj3b p58ipk, which is defective in the fusion of inner membrane of polar nuclei but does not show aberrant endosperm nuclear proliferation, was not defective in sperm nuclear fusion at fertilization. We thus propose that premitotic sperm nuclear fusion in the central cell is critical for normal endosperm nuclear proliferation.

Lipid homeostasis in mitochondria.

Mitochondria are surrounded by the two membranes, the outer and inner membranes, whose lipid compositions are optimized for proper functions and structural organizations of mitochondria. Although a part of mitochondrial lipids including their characteristic lipids, phosphatidylethanolamine and cardiolipin, are synthesized within mitochondria, their precursor lipids and other lipids are transported from other organelles, mainly the ER. Mitochondrially synthesized lipids are re-distributed within mitochondria and to other organelles, as well. Recent studies pointed to the important roles of inter-organelle contact sites in lipid trafficking between different organelle membranes. Identification of Ups/PRELI proteins as lipid transfer proteins shuttling between the mitochondrial outer and inner membranes established a part of the molecular and structural basis of the still elusive intra-mitochondrial lipid trafficking.

Ubiquitin is phosphorylated by PINK1 to activate parkin.

PINK1 (PTEN induced putative kinase 1) and PARKIN (also known as PARK2) have been identified as the causal genes responsible for hereditary recessive early-onset Parkinsonism. PINK1 is a Ser/Thr kinase that specifically accumulates on depolarized mitochondria, whereas parkin is an E3 ubiquitin ligase that catalyses ubiquitin transfer to mitochondrial substrates. PINK1 acts as an upstream factor for parkin and is essential both for the activation of latent E3 parkin activity and for recruiting parkin onto depolarized mitochondria. Recently, mechanistic insights into mitochondrial quality control mediated by PINK1 and parkin have been revealed, and PINK1-dependent phosphorylation of parkin has been reported. However, the requirement of PINK1 for parkin activation was not bypassed by phosphomimetic parkin mutation, and how PINK1 accelerates the E3 activity of parkin on damaged mitochondria is still obscure. Here we report that ubiquitin is the genuine substrate of PINK1. PINK1 phosphorylated ubiquitin at Ser 65 both in vitro and in cells, and a Ser 65 phosphopeptide derived from endogenous ubiquitin was only detected in cells in the presence of PINK1 and following a decrease in mitochondrial membrane potential. Unexpectedly, phosphomimetic ubiquitin bypassed PINK1-dependent activation of a phosphomimetic parkin mutant in cells. Furthermore, phosphomimetic ubiquitin accelerates discharge of the thioester conjugate formed by UBCH7 (also known as UBE2L3) and ubiquitin (UBCH7∼ubiquitin) in the presence of parkin in vitro, indicating that it acts allosterically. The phosphorylation-dependent interaction between ubiquitin and parkin suggests that phosphorylated ubiquitin unlocks autoinhibition of the catalytic cysteine. Our results show that PINK1-dependent phosphorylation of both parkin and ubiquitin is sufficient for full activation of parkin E3 activity. These findings demonstrate that phosphorylated ubiquitin is a parkin activator.

Size and bridging of the sella turcica in Japanese orthodontic patients with tooth agenesis.

The purpose of this study was to investigate the association between the size and bridging of the sella turcica and tooth agenesis, and whether the likelihood of second premolar agenesis can be predicted from the sella turcica size and bridging in Japanese orthodontic patients. Patients were divided into four groups of 32: groups 1 and 2 consisted of patients with agenesis of the maxillary and mandibular second premolars, respectively; group 3, patients with severe tooth agenesis; and group 4, patients without tooth agenesis. Each group was divided into two subgroups of 16 each based on the patient's age: patients under 14 years of age (groups 1A through 4A, group A) and patients 14 years of age or older (groups 1B through 4B, group B). Lateral cephalograms were used to evaluate the size and bridging of the sella turcica. The interclinoidal distance (ID) was significantly shorter in groups 1 and 3 than in group 4, and in group 3 than in group 2. Group B exhibited significantly greater depth, diameter, area, and perimeter of the sella turcica than group A. Groups 3 and 1B had a significantly higher prevalence of sella turcica bridging than groups 4 and 4B, respectively. Maxillary second premolar agenesis and severe tooth agenesis were associated with a reduced ID irrespective of age and increased occurrence of sella turcica bridging. The early emergence in life of a short ID might be a predictor of possible second premolar agenesis in later life.

Correction to: Use of the Er,Cr:YSGG laser for removing remnant adhesive from the enamel surface in rebonding of orthodontic brackets.

In the original publication of the article, Fig. 2 was published incorrectly.

Use of the Er,Cr:YSGG laser for removing remnant adhesive from the enamel surface in rebonding of orthodontic brackets.

The aim of this study was to compare the use of the Er,Cr:YSGG laser with the use of adhesive-removing pliers for removing remnant adhesive from the enamel surface. A total of 54 sound premolars were divided into two groups of 27. Each group was assigned one of two adhesive removal systems: the Er,Cr:YSGG laser, and adhesive-removing pliers. Shear bond strength was measured 24 h after bracket bonding, with the bracket bonding/debonding and residual adhesive removal procedures repeated twice after the first adhesive removal. Before the first bonding and after each adhesive removal procedure, the average surface roughness (Ra) of each tooth was measured. Additionally, 14 of the premolars were examined under a scanning electron microscope. There were no significant differences in the shear bond strength between the two removal systems or between the three debonding sequences. There were significant differences in the enamel surface roughness after each removal sequence between the adhesive removal systems, and a significant increase in the enamel surface roughness was noted in each group with successive removal sequences. The scanning electron microscope images revealed evaporation of the primer and adhesive on the enamel surface and laser etching of enamel in the laser removal group, and the remnants of primer and adhesive on the enamel surface after each removal sequence in the plier removal group. The Er,Cr:YSGG laser was useful for removing remnant adhesive from the enamel surface for bracket rebonding.

Maxillary sinus size and posterior tooth inclination in Japanese orthodontic patients with agenesis of maxillary second premolars.

The purpose of this study was to investigate the expansion of the maxillary sinus and the inclinations of posterior teeth in orthodontic patients with maxillary second premolar agenesis. A total of 30 subjects with one or two congenitally missing maxillary second premolars and retained maxillary deciduous second molars (a agenesis group) were selected and divided into a unilateral agenesis group (20 subjects with one maxillary second premolar missing) and a bilateral agenesis group (10 subjects with two maxillary second premolars missing). As controls, 30 sex- and age-matched subjects without agenesis of the maxillary second premolars were selected. Oblique cephalograms were used to investigate the association of maxillary second premolar agenesis and lower maxillary sinus size and posterior tooth inclinations. Agenesis of the maxillary second premolars induced significantly large lower maxillary sinus length, depth, area and mesial inclination of the maxillary first premolar, a significantly small anterior maxillary length, and a significantly more distal position for root apex of the maxillary first premolar. There were no significant differences in any measurements of the lower maxillary sinus and posterior teeth between the non-agenesis side in the unilateral agenesis group and the control group. Maxillary second premolar agenesis caused inferior and anterior expansion of the lower maxillary sinus and the mesial inclination of the maxillary first premolars with a distal position of root apex. Unilateral agenesis of the maxillary second premolar did not affect on the lower maxillary sinus size or posterior tooth inclinations of the unaffected antimere.

Regulation of the protein entry gate assembly by mitochondrial porin.

Mitochondrial biogenesis and functions rely on transport of their resident proteins as well as small molecules/ions across their membranes. The TOM complex functions as a protein entry gate for most mitochondrial proteins and mitochondrial porin facilitates transport of small-molecule metabolites and ions. We recently found a novel role of porin in regulation of the TOM complex assembly, the dynamic exchange between the dimer and trimer, and different substrate specificities of the dimer and trimer. Using distinct assembly forms customized for different client proteins, the TOM complex can handle ~ 1000 different mitochondrial protein for their import into mitochondria.

Multifaceted roles of porin in mitochondrial protein and lipid transport.

Mitochondria are essential eukaryotic organelles responsible for primary cellular energy production. Biogenesis, maintenance, and functions of mitochondria require correct assembly of resident proteins and lipids, which require their transport into and within mitochondria. Mitochondrial normal functions also require an exchange of small metabolites between the cytosol and mitochondria, which is primarily mediated by a metabolite channel of the outer membrane (OM) called porin or voltage-dependent anion channel. Here, we describe recently revealed novel roles of porin in the mitochondrial protein and lipid transport. First, porin regulates the formation of the mitochondrial protein import gate in the OM, the translocase of the outer membrane (TOM) complex, and its dynamic exchange between the major form of a trimer and the minor form of a dimer. The TOM complex dimer lacks a core subunit Tom22 and mediates the import of a subset of mitochondrial proteins while the TOM complex trimer facilitates the import of most other mitochondrial proteins. Second, porin interacts with both a translocating inner membrane (IM) protein like a carrier protein accumulated at the small TIM chaperones in the intermembrane space and the TIM22 complex, a downstream translocator in the IM for the carrier protein import. Porin thereby facilitates the efficient transfer of carrier proteins to the IM during their import. Third, porin facilitates the transfer of lipids between the OM and IM and promotes a back-up pathway for the cardiolipin synthesis in mitochondria. Thus, porin has roles more than the metabolite transport in the protein and lipid transport into and within mitochondria, which is likely conserved from yeast to human.