Angiosperm female gametophytes contain a central cell with two polar nuclei. In many species, including Arabidopsis thaliana, the polar nuclei fuse during female gametogenesis. We previously showed that BiP, an Hsp70 in the endoplasmic reticulum (ER), was essential for membrane fusion during female gametogenesis. Hsp70 function requires partner proteins for full activity. J-domain containing proteins (J-proteins) are the major Hsp70 functional partners. A. thaliana ER contains three soluble J-proteins, AtERdj3A, AtERdj3B, and AtP58(IPK). Here, we analyzed mutants of these proteins and determined that double-mutant ovules lacking AtP58(IPK) and AtERdj3A or AtERdj3B were defective in polar nuclear fusion. Electron microscopy analysis identified that polar nuclei were in close contact, but no membrane fusion occurred in mutant ovules lacking AtP58(IPK) and AtERdj3A. The polar nuclear outer membrane appeared to be connected via the ER remaining at the inner unfused membrane in mutant ovules lacking AtP58(IPK) and AtERdj3B. These results indicate that ER-resident J-proteins, AtP58(IPK)/AtERdj3A and AtP58(IPK)/AtERdj3B, function at distinct steps of polar nuclear-membrane fusion. Similar to the bip1 bip2 double mutant female gametophytes, the aterdj3a atp58(ipk) double mutant female gametophytes defective in fusion of the outer polar nuclear membrane displayed aberrant endosperm proliferation after fertilization with wild-type pollen. However, endosperm proliferated normally after fertilization of the aterdj3b atp58(ipk) double mutant female gametophytes defective in fusion of the inner membrane. Our results indicate that the polar nuclear fusion defect itself does not cause an endosperm proliferation defect.
In eukaryotic cells, complex membrane structures called organelles are highly developed to exert specialized functions. Mitochondria are one of such organelles consisting of the outer and inner membranes (OM and IM) with characteristic protein and phospholipid compositions. Maintaining proper phospholipid compositions of the membranes is crucial for mitochondrial integrity, thereby contributing to normal cell activities. As cellular locations for phospholipid synthesis are restricted to specific compartments such as the endoplasmic reticulum (ER) membrane and the mitochondrial inner membrane, newly synthesized phospholipids have to be transported and distributed properly from the ER or mitochondria to other cellular membranes. Although understanding of molecular mechanisms of phospholipid transport are much behind those of protein transport, recent studies using yeast as a model system began to provide intriguing insights into phospholipid exchange between the ER and mitochondria as well as between the mitochondrial OM and IM. In this review, we summarize the latest findings of phospholipid transport via mitochondria and discuss the implicated molecular mechanisms.
The membrane topology of Om45 in the yeast mitochondrial outer membrane (OM) is under debate. Here, we confirm that Om45 is anchored to the OM from the intermembrane space (IMS) by its N-terminal hydrophobic segment. We show that import of Om45 requires the presequence receptors, Tom20 and Tom22, and the import channel of Tom40. Unlike any of the known OM proteins, Om45 import requires the TIM23 complex in the inner membrane, a translocator for presequence-containing proteins, and the membrane potential (??). Therefore, Om45 is anchored to the OM via the IMS by a novel import pathway involving the TIM23 complex.
Immunoglobulin-binding protein (BiP) is a molecular chaperone of the heat shock protein 70 (Hsp70) family. BiP is localized in the endoplasmic reticulum (ER) and plays key roles in protein translocation, protein folding and quality control in the ER. The genomes of flowering plants contain multiple BiP genes. Arabidopsis thaliana has three BiP genes. BIP1 and BIP2 are ubiquitously expressed. BIP3 encodes a less well conserved BiP paralog, and it is expressed only under ER stress conditions in the majority of organs. Here, we report that all BiP genes are expressed and functional in pollen and pollen tubes. Although the bip1 bip2 double mutation does not affect pollen viability, the bip1 bip2 bip3 triple mutation is lethal in pollen. This result indicates that lethality of the bip1 bip2 double mutation is rescued by BiP3 expression. A decrease in the copy number of the ubiquitously expressed BiP genes correlates well with a decrease in pollen tube growth, which leads to reduced fitness of mutant pollen during fertilization. Because an increased protein secretion activity is expected to increase the protein folding demand in the ER, the multiple BiP genes probably cooperate with each other to ensure ER homeostasis in cells with active secretion such as rapidly growing pollen tubes.
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.
The mitochondrial targeting signal in the presequence of mitochondrial precursor proteins is recognized by Tom20 and subsequently by Tim50 in mitochondria. Yeast Tim50 contains two presequence binding sites in the conserved core domain and in the fungi-specific C-terminal presequence binding domain (PBD). We report the NMR analyses on interactions of a shorter variant of PBD (sPBD), a shorter variant of PBD, with presequences. The presequence is recognized by sPBD in a similar manner to Tom20. sPBD can also bind to the core domain of Tim50 through the presequence binding region, which could promote transfer of the presequence from sPBD to the core domain in Tim50.
Mitochondrial proteins require protein machineries called translocators in the outer and inner membranes for import into and sorting to their destination submitochondrial compartments. Among them, the TIM22 complex mediates insertion of polytopic membrane proteins into the inner membrane, and Tim22 constitutes its central insertion channel. Here we report that the conserved Cys residues of Tim22 form an intramolecular disulfide bond. By comparison of Tim22 Cys ? Ser mutants with wild-type Tim22, we show that the disulfide bond of Tim22 stabilizes Tim22 especially at elevated temperature through interactions with Tim18, which are also important for the stability of the TIM22 complex. We also show that lack of the disulfide bond in Tim22 impairs the assembly of TIM22 pathway substrate proteins into the inner membrane especially when the TIM22 complex handles excess amounts of substrate proteins. Our findings provide a new insight into the mechanism of the maintenance of the structural and functional integrity of the TIM22 complex.
Recent development of methods for genetic incorporation of unnatural amino acids into proteins in live cells enables us to analyze protein interactions by site-specific photocrosslinking. Here we describe a method to incorporate p-benzoyl-L-phenylalanine (pBpa), a photoreactive unnatural amino acid, into defined positions of a target protein in living yeast cells. Photocrosslinking using the pBpa-incorporated proteins has been proven to be a powerful method for analyzing protein-protein interactions at the spatial resolution of amino-acid residues. Since photocrosslinking can be performed for pBpa-incorporated proteins that are properly assembled into a protein complex in living cells, this method will allow us to reveal protein-protein interactions of the target proteins at work.
Orthodontic medical treatment is performed to move a tooth to the optimal position to obtain optimal occlusion. Orthodontic treatment is accompanied by mechanical stress due to orthodontic force and by psychological stress that is experienced as pain or displeasure. The purpose of this study was to identify stress marker proteins during orthodontic treatment. Levels of receptor activator of NF?B (RANKL) and heat shock protein 70 (HSP70) in the gingival crevicular fluid (GCF) were analyzed as markers of mechanical stress, and levels of chromogranin A (CgA) and amylase in whole saliva were analyzed as markers of psychological stress. GCF was collected from control and experimental teeth at initiation of treatment and 24 h after treatment. Whole saliva was collected before treatment, at initiation of treatment and 24 h after treatment. RANKL was expressed at 24 h after treatment in the experimental GCF, but not in the control GCF. HSP70 appeared to be constitutively expressed in GCF, and its levels showed no major change between the control and experimental groups from initiation of treatment to 24 h after treatment. Amylase activity in whole saliva was enhanced at 24 h after treatment compared to control, but CgA levels showed little change between the groups. These results indicated that RANKL and amylase may be the candidate markers for mechanical and psychological stress, respectively, during orthodontic treatment, even though the total protein concentration and amylase activity displayed a large standard deviation among subjects. Further studies are therefore required to establish these markers for clinical use.
CDP-diacylglycerol (CDP-DAG) is central to the phospholipid biosynthesis pathways in cells. A prevailing view is that only one CDP-DAG synthase named Cds1 is present in both the endoplasmic reticulum (ER) and mitochondrial inner membrane (IM) and mediates generation of CDP-DAG from phosphatidic acid (PA) and CTP. However, we demonstrate here by using yeast Saccharomyces cerevisiae as a model organism that Cds1 resides in the ER but not in mitochondria, and that Tam41, a highly conserved mitochondrial maintenance protein, directly catalyzes the formation of CDP-DAG from PA in the mitochondrial IM. We also find that inositol depletion by overexpressing an arrestin-related protein Art5 partially restores the defects of cell growth and CL synthesis in the absence of Tam41. The present findings unveil the missing step of the cardiolipin synthesis pathway in mitochondria as well as the flexibile regulation of phospholipid biosynthesis to respond to compromised CDP-DAG synthesis in mitochondria.
The purpose of this study was to investigate the patterns of third molar agenesis and incidence of agenesis of other permanent teeth in a Japanese orthodontic patient group. A total of 417 Japanese subjects (134 males and 283 females) with agenesis of one or more third molars were divided into four groups according to the agenesis pattern, and 874 other Japanese subjects (302 males and 572 females) without third molar agenesis were assigned to a control group. Panoramic radiographs and medical and dental records were used to examine for tooth agenesis. The Chi-square test and odds ratio were used to make statistical comparisons. The prevalence of third molar agenesis worked out at 32.3 % with no statistically significant gender difference. A high prevalence rate of agenesis of third molars, unilateral or bilateral, could be considered characteristic of the Japanese orthodontic population. Significant increases in occurrence of oligodontia, and unilateral or bilateral agenesis of other teeth, including maxillary lateral incisors and maxillary and mandibular second premolars, were observed in all or almost all of the third molar agenesis groups, compared with the controls. A significantly increased prevalence rate of mandibular lateral incisor agenesis was observed in almost all of the third molar agenesis groups. The Japanese patients with third molar agenesis had a significantly increased occurrence of oligodontia, and unilateral or bilateral agenesis of maxillary and mandibular lateral incisors and second premolars, except for bilateral agenesis of mandibular lateral incisors.
Mitochondrial protein import requires cooperation of the machineries called translocators in the outer and inner mitochondrial membranes. Here we analyze the interactions of Tom22, a multifunctional subunit of the outer membrane translocator TOM40 complex, with other translocator subunits such as Tom20, Tom40, and Tim50 and with substrate precursor proteins at a spatial resolution of the amino acid residue by in vivo and in organello site-specific photocross-linking. Changes in cross-linking patterns caused by excess substrate precursor proteins or presequence peptides indicate how the cytosolic receptor domain of Tom22 accepts substrate proteins and how the intermembrane space domain of Tom22 transfers them to Tim50 of the inner-membrane translocator.
A part of eukaryotic tRNA genes harbor an intron at one nucleotide 3 to the anticodon, so that removal of the intron is an essential processing step for tRNA maturation. While some tRNA introns have important roles in modification of certain nucleotides, essentiality of the tRNA intron in eukaryotes has not been tested extensively. This is partly because most of the eukaryotic genomes have multiple genes encoding an isoacceptor tRNA. Here, we examined whether the intron of tRNA-Trp(CCA) genes, six copies of which are scattered on the genome of yeast, Saccharomyces cerevisiae, is essential for growth or translation of the yeast in vivo. We devised a procedure to remove all of the tRNA introns from the yeast genome iteratively with marker cassettes containing both positive and negative markers. Using this procedure, we removed all the introns from the six tRNA-Trp(CCA) genes, and found that the intronless strain grew normally and expressed tRNA-Trp(CCA) in an amount similar to that of the wild-type genes. Neither incorporation of (35)S-labeled amino acids into a TCA-insoluble fraction nor the major protein pattern on SDS-PAGE/2D gel were affected by complete removal of the intron, while expression levels of some proteins were marginally affected. Therefore, the tRNA-Trp(CCA) intron is dispensable for growth and bulk translation of the yeast. This raises the possibility that some mechanism other than selective pressure from translational efficiency maintains the tRNA intron on the yeast genome.
This study was conducted to examine the bond strength of rebonded orthodontic brackets after adhesive residuals on the surface of the bracket bases were removed by Er,Cr:YSGG lasers. Seventy-six brackets bonded to premolars with a self-etching primer adhesive system were equally divided into four groups after the first debonding with the bracket bases (Group 1) untreated, and treated by (Group 2) Er,Cr:YSGG laser, (Group 3) sandblaster, and (Group 4) Er,Cr:YSGG laser/sandblaster. The treated brackets were rebonded to the new premolars in the same manner as the first-stage experiment. The shear bond strengths were measured, with the bonding/debonding procedures repeated once after the first debonding, and the bracket/adhesive failure modes were evaluated after each debonding. The treated bracket base surfaces were observed under a scanning electron microscopy (SEM). The mean rebond strengths were significantly lower in group 1 than in other groups, and there were no significant differences between the other groups. The mean initial bond strength was significantly higher than the mean rebond strength in group 1 but there was no significant difference between the two in the other three groups. Failures at the bracket-adhesive interface occurred frequently at second debonding in group 1. Under the SEM, residual adhesive was removed from the bracket bases by Er,Cr:YSGG laser, while adhesive remnant was seen underneath the meshwork of the bracket bases and microroughness appeared on the meshwork after sandblasting. Er,Cr:YSGG laser certainly could serve the purpose of promoting the use of recycled orthodontic brackets.
The purpose of this study was to ascertain the effects of tooth bleaching on the shear bond strength of orthodontic brackets rebonded with a self-etching adhesive system. A total of 39 premolars were collected and divided into three equal groups: in group 1 bracket bonding was performed without bleaching treatment; specimens in group 2 were bonded immediately after bleaching; and group 3 teeth were bleached, then immersed in artificial saliva and left for 7 days before bonding. The shear bond strength was measured, with the bonding/debonding procedures repeated once after the first debonding, and the bracket/adhesive failure modes were evaluated by the adhesive remnant index after each debonding. Excepting the mean shear bond strength for group 2 after the first debonding, the overall mean values reached the minimum clinical requirement of 6 MPa. The mean values at the first and second debondings were significantly higher in groups 1 and 3 than in group 2. Between groups 1 and 3, significant differences were noted at the first debonding, but not at the second debonding. Group 2 showed significant differences in mean shear bond strength between the first and second debondings. Bond failure at the enamel-adhesive interface occurred more frequently in group 2 than in groups 1 or 3 after the first debonding. The bracket-rebonding procedure can recover the reduced shear bond strength caused by immediate bonding after bleaching to a clinically acceptable level, but not to the prebleaching level.
The purpose of this study was to evaluate the effects of the maxillary arch expansion on maxillomandibular arch widths in patients treated with the quad-helix versus untreated controls. The treatment group consisted of 50 consecutive patients treated for maxillary incisor crowding with a quad-helix appliance in the early mixed dentition. Lateral cephalograms and dental casts taken at the start (T0) and end (T1) of the quad-helix treatment were obtained. The control group consisted of 50 untreated patients with the same type of malocclusion. Two consecutive lateral cephalograms and dental casts of each untreated patient were taken at about the same time as T0 and T1. All these study materials were analyzed for comparison between the two groups. The mean ages at T0 and T1 in the two groups were about the same. The maxillary first molars moved and tipped distally in the treatment group and mesially in the control group. The quad-helix treatment actually expanded the mandibular and maxillary arches concurrently. The more the maxillary arch widths were expanded and the less the maxillary first molars were inclined distally, the more the mandibular arch widths were expanded. The quad-helix activation caused lingual tipping and mesiobuccal rotation of the maxillary first molars. The mesiobuccal rotation of the maxillary first molars could turn molar occlusal relationships for the better from Class II to Class I. The quad-helix treatment gives rise to spontaneous expansion of the mandibular arch concurrent with maxillary expansion in the early mixed dentition patients with maxillary incisor crowding.
While overall hydrophobicity is generally recognized as the main characteristic of transmembrane (TM) ?-helices, the only membrane system for which there are detailed quantitative data on how different amino acids contribute to the overall efficiency of membrane insertion is the endoplasmic reticulum (ER) of eukaryotic cells. Here, we provide comparable data for TIM23-mediated membrane protein insertion into the inner mitochondrial membrane of yeast cells. We find that hydrophobicity and the location of polar and aromatic residues are strong determinants of membrane insertion. These results parallel what has been found previously for the ER. However, we see striking differences between the effects elicited by charged residues flanking the TM segments when comparing the mitochondrial inner membrane and the ER, pointing to an unanticipated difference between the two insertion systems.
Mitochondria import most of their resident proteins from the cytosol, and the import receptor Tom20 of the outer-membrane translocator TOM40 complex plays an essential role in specificity of mitochondrial protein import. Here we analyzed the effects of Tom20 binding on NMR spectra of a long mitochondrial presequence and found that it contains two distinct Tom20-binding elements. In vitro import and cross-linking experiments revealed that, although the N-terminal Tom20-binding element is essential for targeting to mitochondria, the C-terminal element increases efficiency of protein import in the step prior to translocation across the inner membrane. Therefore Tom20 has a dual role in protein import into mitochondria: recognition of the targeting signal in the presequence and tethering the presequence to the TOM40 complex to increase import efficiency.
?-barrel membrane proteins in the mitochondrial outer membrane use the TOM40 complex to enter mitochondria and then the TOB/SAM complex to be assembled into the outer membrane. Tom7, a subunit of the TOM40 complex, regulates association of Mdm10 with the TOB complex. Here, we analyzed the role of Tom7 in assembly of ?-barrel proteins, including Tom40, a central channel subunit of the TOM40 complex, and porin. Depletion of Tom7 decreased transient accumulation of Tom40 at the level of the TOB complex and retarded assembly of porin in vitro. On the other hand, overexpression of Tom7 resulted in enhanced accumulation of in vitro imported Tom40 in the TOB complex, yet it did not affect the in vitro assembly of porin. Site-specific photocross-linking in vivo revealed that Tom7 directly interacts with Tom40 through its transmembrane segment and with Mdm10. These results collectively show that Tom7 recruits Mdm10, enhancing its association with the MMM1 complex, to regulate timing of the release of Tom40 from the TOB complex for subsequent assembly into the TOM40 complex.
The unfolded protein response (UPR) is an essential signal transduction to cope with protein-folding stress in the endoplasmic reticulum. In the yeast UPR, the unconventional splicing of HAC1 mRNA is a key step. Translation of HAC1 pre-mRNA (HAC1(u) mRNA) is attenuated on polysomes and restarted only after splicing upon the UPR. However, the precise mechanism of this restart remained unclear. Here we show that yeast tRNA ligase (Rlg1p/Trl1p) acting on HAC1 ligation has an unexpected role in HAC1 translation. An RLG1 homologue from Arabidopsis thaliana (AtRLG1) substitutes for yeast RLG1 in tRNA splicing but not in the UPR. Surprisingly, AtRlg1p ligates HAC1 exons, but the spliced mRNA (HAC1(i) mRNA) is not translated efficiently. In the AtRLG1 cells, the HAC1 intron is circularized after splicing and remains associated on polysomes, impairing relief of the translational repression of HAC1(i) mRNA. Furthermore, the HAC1 5 UTR itself enables yeast Rlg1p to regulate translation of the following ORF. RNA IP revealed that yeast Rlg1p is integrated in HAC1 mRNP, before Ire1p cleaves HAC1(u) mRNA. These results indicate that the splicing and the release of translational attenuation of HAC1 mRNA are separable steps and that Rlg1p has pivotal roles in both of these steps.
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.
The purposes of this study were to: (1) examine the shear bond strengths (SBSs) of orthodontic brackets bonded to primary teeth with conventional, self-etching, and moisture-insensitive primers; and (2) evaluate the mode of bracket failure.
Mitochondrial functions rely on precise and efficient transport of 1000-1500 different mitochondrial proteins from the cytosol to appropriate mitochondrial subcompartments. Those mitochondrial protein transport processes are mediated by the dedicated mitochondrial protein import system comprised of translocators in the outer and inner mitochondrial membranes and soluble factors in the cytosol, intermembrane space, and matrix. In the last decade, high-resolution structures of many of the components of the mitochondrial protein import machineries have become available, which has significantly advanced our understanding of the molecular mechanisms of mitochondrial protein transport. Here we review the currently available high-resolution structures of the components of the mitochondrial protein import machineries that afford structural and mechanistic insight into how the mitochondrial import system works. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.
We found few studies on the association between maxillary sinus size and malocclusion in an electronic search using PubMed. The purpose of this study was to investigate maxillary sinus size in different malocclusion groups and the association between maxillary sinus size and dentofacial morphology by the use of lateral cephalometric radiographs. A total of 120 lateral cephalograms were used. These radiographs were derived from subjects with skeletal Class I, Class II, and Class III malocclusions, classified on the basis of the A-N-B angle. Each malocclusion group consisted of 20 boys and 20 girls ranging in age from 12 to 16 years. Two linear measurements and three area measurements were made to evaluate maxillary sinus size, and four angular and eight linear measurements were made to evaluate dentofacial morphology. Analysis of variance and Pearsons correlation analysis were performed for statistical comparison. The maxillary sinuses showed no significant differences in size between the different classes of skeletal malocclusion or between sexes. However, the maxillary sinus measurements were significantly correlated with several dentofacial morphological measurements. When formulating an orthodontic treatment plan, orthodontists should take into consideration the fact that the patients 12 to 16 years old with large cranial bases and nasomaxillary complexes tend to have larger maxillary sinuses, but there is no significant association between maxillary sinus size and the A-N-B angle denoting the sagittal skeletal jaw relationship.
Mitochondria contain two biological membranes. Although reducing agents can diffuse from the cytosol into the intermembrane space (IMS) between the outer and inner mitochondrial membranes, the IMS has a dedicated disulfide relay system to introduce disulfide bonds into mainly small and soluble proteins. This system consists of two essential proteins, a disulfide carrier Tim40/Mia40 and a flavin-dependent sulfhydryl oxidase Erv1, high-resolution structures that have recently become available. Tim40/Mia40 transfers disulfide bonds to newly imported IMS proteins by dithiol/disulfide exchange reactions involving mixed disulfide intermediates. Tight folding by introduction of disulfide bonds prevents egress of these small IMS proteins, resulting in their selective retention in the compartment. After disulfide transfer from Tim40/Mia40 to substrate proteins, Tim40/Mia40 is reoxidized again by Erv1, which is then oxidized by electron transfer to either cytochrome c or molecular oxygen. Here we review the recent advancement of the knowledge on the mechanism of the disulfide relay system in the mitochondrial IMS, especially shedding light on the structural aspects of its components.
Protein translocation across membranes is a fundamental cellular process. The majority of the proteins of organelles such as mitochondria and chloroplasts is synthesized in the cytosol and subsequently imported in a post-translational manner. The precursor proteins have to be unfolded at least for translocation, but it has also been assumed that they are unfolded during transport to the organelle in the cytosol. Unfolding is governed by chaperones and the translocon itself. At the same time, chaperones provide the energy for the import process. The energetic properties of the chloroplast translocon were studied by import of the Ig-like module of the muscle protein titin fused to the transit peptide of the chloroplast targeted oxygen evolving complex subunit of 33 kDa (OE33). Our results suggest that p(OE33)titin is folded prior to import and that translocation is initiated by unfolding after having bound to the translocon at the chloroplast surface. Using a set of stabilizing and destabilizing mutants of titin previously analyzed by atomic force microscopy and as passenger for mitochondrial translocation, we studied the unfolding force provided by the chloroplast translocon. Based on these results, a model for translocation is discussed.
The mitochondrial outer membrane contains two protein translocators: the TOM40 and TOB/SAM complexes. Mdm10 is distributed in the TOB complex for beta-barrel protein assembly and in the MMM1 complex for tethering of the endoplasmic reticulum and mitochondria. Here, we establish a system in which the Mdm10 level in the TOB complex--but not in the MMM1 complex--is altered to analyse its part in beta-barrel protein assembly. A decrease in the Mdm10 level results in accumulation of in vitro imported Tom40, which is a beta-barrel protein, at the level of the TOB complex. An increase in the Mdm10 level inhibits association not only of Tom40 but also of other beta-barrel proteins with the TOB complex. These results show that Mdm10 regulates the timing of release of unassembled Tom40 from the TOB complex, to facilitate its coordinated assembly into the TOM40 complex.
Point mutations in proteins can have different effects on protein stability depending on the mechanism of unfolding. In the most interesting case of I27, the Ig-like module of the muscle protein titin, one point mutation (Y9P) yields opposite effects on protein stability during denaturant-induced "global unfolding" versus "vectorial unfolding" by mechanical pulling force or cellular unfolding systems. Here, we assessed the reason for the different effects of the Y9P mutation of I27 on the overall molecular stability and N-terminal unraveling by NMR. We found that the Y9P mutation causes a conformational change that is transmitted through beta-sheet structures to reach the central hydrophobic core in the interior and alters its accessibility to bulk solvent, which leads to destabilization of the hydrophobic core. On the other hand, the Y9P mutation causes a bend in the backbone structure, which leads to the formation of a more stable N-terminal structure probably through enhanced hydrophobic interactions.
Misfolded proteins produced in the endoplasmic reticulum (ER) are degraded by a mechanism, the ER-associated degradation (ERAD). Here we report establishment of the experimental system to analyze the ERAD in plant cells. Carboxypeptidase Y (CPY) is a vacuolar enzyme and its mutant CPY* is degraded by the ERAD in yeast. Since Arabidopsis thaliana has AtCPY, an ortholog of yeast CPY, we constructed and expressed fusion proteins consisting of AtCPY and GFP and of AtCPY*, which carries a mutation homologous to yeast CPY*, and GFP in A. thaliana cells. While AtCPY-GFP was efficiently transported to the vacuole, AtCPY*-GFP was retained in the ER to be degraded in proteasome- and Cdc48-dependent manners. We also found that AtCPY*-GFP was degraded by the ERAD in yeast cells, but that its single N-glycan did not function as a degradation signal in yeast or plant cells. Therefore, AtCPY*-GFP can be used as a marker protein to analyze the ERAD pathway, likely for nonglycosylated substrates, in plant cells.
Normal mitochondrial protein import requires multiple translocator complexes in the outer and inner mitochondrial membrane. Tam41 is a peripheral inner membrane protein that is involved in the structural maintenance of the inner membrane translocator the TIM23 complex. Here we identified an arrestin-related protein Art5 as a multicopy suppressor for the Tam41-deficient yeast mutant, which exhibited the deteriorated TIM23 complex and temperature-sensitive growth defects. Overexpression of Art5 suppressed growth defects of tam41Delta cells and partially restored the destabilized TIM23 complex structure in tam41Delta mitochondria, so that the defects in mitochondrial protein import via the TIM23 complex were partially recovered. Deletion of the ART5 gene in turn exhibited synthetic growth defects with the TAM41 deletion. Art5 as a functional partner for Tam41 will provide a starting point to reveal the precise function of Tam41 in the maintenance of the TIM23 complex.
Nuclear fusion is an essential process in the sexual reproduction of animals and plants. In flowering plants, nuclear fusion occurs three times: once during female gametogenesis, when the two polar nuclei fuse to produce the diploid central cell nucleus, and twice during double fertilization. The yeast Ig binding protein (BiP) is a molecular chaperone Hsp70 in the endoplasmic reticulum that regulates nuclear membrane fusion during mating. Here we report that in Arabidopsis thaliana, BiP is involved in the fusion of polar nuclei during female gametophyte development. BiP-deficient mature female gametophytes contain two unfused polar nuclei, in spite of their close contact. This indicates a surprising conservation of BiP function in nuclear fusion between plants and yeasts. We also found that endosperm nuclear division becomes aberrant after fertilization of the BiP-deficient female gametophytes with wild-type pollen. This is experimental evidence for the importance of fusion of the polar nuclei in the proliferation of endosperm nuclei.
XB-S is a protein with an amino-terminal-truncated form of tenascin-X (TNXB). However, the precise roles of XB-S in vivo are unknown. In this study, to determine the role of XB-S in vivo, we screened XB-S-binding proteins. FLAG-tagged XB-S was transiently introduced into 293T cells. Then its associated proteins were purified by immunoprecipitation using an anti-FLAG antibody and its components were identified by mass spectrometric analyses. Mitotic motor kinesin Eg5 was identified in the immunoprecipitates. XB-S and Eg5 proteins were co-localized in the cytoplasm in interphase and mitosis, but XB-S did not localize on mitotic spindle microtubules, on which Eg5 prominently localized in mitosis. As for Eg5 binding to XB-S, glutathione S-transferase-fused XB-S expressed in vitro directly bound to full-length Eg5 translated in reticulocyte lysate, and the XB-S-binding region was located in the motor domain of Eg5. Furthermore, during cell cycle progression XB-S showed a similar expression profile to that of Eg5. These results suggest possible involvement of XB-S in the function of Eg5.
Mitochondria are surrounded by two biological membranes. The outer mitochondrial membrane contains two major translocators, the TOM40 (TOM) and TOB/SAM complexes for protein translocation across and/or insertion into the outer membrane. The TOM40 complex functions as an entry gate for most mitochondrial proteins, and the TOB/SAM complex as a specialized insertion machinery for beta-barrel membrane proteins. In order to handle loosely folded or unfolded precursor polypeptides, those translocators cooperate with chaperones in the cytosol and intermembrane space, and also exhibit chaperone-like functions on their own. Several alpha-helical membrane proteins take non-standard routes to be inserted into the outer membrane. Here we review the current view on a remarkable variety of mechanisms of protein transport taking place at the mitochondrial outer membrane.
Mitochondrial protein traffic requires precise recognition of the mitochondrial targeting signals by the import receptors on the mitochondrial surface including a general import receptor Tom20 and a receptor for presequence-less proteins, Tom70. Here we took a proteome-wide approach of mitochondrial protein import in vitro to find a set of presequence-containing precursor proteins for recognition by Tom70. The presequences of the Tom70-dependent precursor proteins were recognized by Tom20, whereas their mature parts exhibited Tom70-dependent import when attached to the presequence of Tom70-independent precursor proteins. The mature parts of the Tom70-dependent precursor proteins have the propensity to aggregate, and the presence of the receptor domain of Tom70 prevents their aggregate formation. Therefore Tom70 plays the role of a docking site for not only cytosolic chaperones but also aggregate-prone substrates to maintain their solubility for efficient transfer to downstream components of the mitochondrial import machineries.
The mitochondrial intermembrane space (IMS) contains many small cysteine-bearing proteins, and their passage across the outer membrane and subsequent folding require recognition and disulfide bond transfer by an oxidative translocator Tim40/Mia40 in the inner membrane facing the IMS. Here we determined the crystal structure of the core domain of yeast Mia40 (Mia40C4) as a fusion protein with maltose-binding protein at a resolution of 3 A. The overall structure of Mia40C4 is a fruit-dish-like shape with a hydrophobic concave region, which accommodates a linker segment of the fusion protein in a helical conformation, likely mimicking a bound substrate. Replacement of the hydrophobic residues in this region resulted in growth defects and impaired assembly of a substrate protein. The Cys296-Cys298 disulfide bond is close to the hydrophobic concave region or possible substrate-binding site, so that it can mediate disulfide bond transfer to substrate proteins. These results are consistent with the growth phenotypes of Mia40 mutant cells containing Ser replacement of the conserved cysteine residues.
Cardiolipin, a unique phospholipid composed of four fatty acid chains, is located mainly in the mitochondrial inner membrane (IM). Cardiolipin is required for the integrity of several protein complexes in the IM, including the TIM23 translocase, a dynamic complex which mediates protein import into the mitochondria through interactions with the import motor presequence translocase-associated motor (PAM). In this study, we report that two homologous intermembrane space proteins, Ups1p and Ups2p, control cardiolipin metabolism and affect the assembly state of TIM23 and its association with PAM in an opposing manner. In ups1Delta mitochondria, cardiolipin levels were decreased, and the TIM23 translocase showed altered conformation and decreased association with PAM, leading to defects in mitochondrial protein import. Strikingly, loss of Ups2p restored normal cardiolipin levels and rescued TIM23 defects in ups1Delta mitochondria. Furthermore, we observed synthetic growth defects in ups mutants in combination with loss of Pam17p, which controls the integrity of PAM. Our findings provide a novel molecular mechanism for the regulation of cardiolipin metabolism.
Mitochondria are two-membrane bounded organelles consisting of 1000-2000 different proteins, most of which are synthesized in the cytosol and subsequently imported into mitochondria. The imported proteins are further sorted to one of the four compartments, the outer membrane, intermembrane space, inner membrane, and matrix, mostly following one of the five major pathways. Mitochondrial protein import and sorting are mediated by the translocator complexes in the membranes and chaperones in the aqueous compartments operating along the import pathways. Here, we summarize the expanding knowledge on the roles of translocators, chaperones, and related components in the multiple pathways for mitochondrial protein trafficking.
To ascertain the effects of repeated bonding on the shear bond strength of orthodontic brackets bonded with a fluoride-releasing and -recharging adhesive system with a self-etching primer in comparison with two other types of adhesive system.
The endoplasmic reticulum (ER) has a strict protein quality control system. Misfolded proteins generated in the ER are degraded by the ER-associated degradation (ERAD). Yeast Mnl1p consists of an N-terminal mannosidase homology domain and a less conserved C-terminal domain and facilitates the ERAD of glycoproteins. We found that Mnl1p is an ER luminal protein with a cleavable signal sequence and stably interacts with a protein-disulfide isomerase (PDI). Analyses of a series of Mnl1p mutants revealed that interactions between the C-terminal domain of Mnl1p and PDI, which include an intermolecular disulfide bond, are essential for subsequent introduction of a disulfide bond into the mannosidase homology domain of Mnl1p by PDI. This disulfide bond is essential for the ERAD activity of Mnl1p and in turn stabilizes the prolonged association of PDI with Mnl1p. Close interdependence between Mnl1p and PDI suggests that these two proteins form a functional unit in the ERAD pathway.
An arg120gly (R120G) missense mutation in HSPB5 (alpha-beta-crystallin ), which belongs to the small heat shock protein (HSP) family, causes desmin-related cardiomyopathy (DRM), a muscle disease that is characterized by the formation of inclusion bodies, which can contain pre-amyloid oligomer intermediates (amyloid oligomer). While we have shown that small HSPs can directly interrupt amyloid oligomer formation, the in vivo protective effects of the small HSPs on the development of DRM is still uncertain.
Protein import into mitochondria requires unfolding of the folded mature domain of precursor proteins. Here we compared the effects of amino-acid replacement between the core region and the N-terminal region of the titin I27 domain (the 27th Ig domain of human titin) on its import into isolated mitochondria when attached to a short presequence (pb(2)(35)). We found that several mutations in the core region around Trp34 of the I27 domain enhanced the import rates of the fusion proteins, while the N-terminal K6P mutation, which increases mechanical stability around the N-terminal region, decreases the import rate. When the K6P mutation is combined with core-destabilizing mutations, the import rates of the fusion proteins still decrease, unless a long segment is deleted. These results suggest that mutations in the core region could destabilize the transition state for unfolding from the intermediate with the detached N-terminal segment during import, leading to enhanced unfolding rates, although stabilization of the N-terminal region masks these effects. In other words, the rate-limiting step of the global unfolding upon import into mitochondria switches, depending on the balance between the stability of the N-terminal structure and the stability of the core region of the I27 domain.
The purpose of the present study was to probe into the effects of maxillary second molar extraction on dentofacial morphology by the use of cephalometric radiographs taken before and after anterior open-bite treatment. The subjects were 30 Japanese patients who had received multiloop edgewise archwire therapy without premolar extraction. They were divided into two groups. Group 1 consisted of 15 patients who had their maxillary second molars removed. Group 2 comprised 15 patients who had been treated without extraction of maxillary second molars. Lateral cephalograms were taken before and after treatment. Seventeen angular and 31 linear measurements were obtained from the lateral cephalograms. Paired and unpaired t tests were used to determine the significance of differences in measurements. The retrusion and extrusion of the maxillary incisors, the distal movement of the maxillary first molars, and the uprighting of the maxillary first premolars and the mandibular first molars were greater in group 1 than in group 2. Moreover, the maxillary first molars intruded significantly in group 1 but extruded insignificantly in group 2. Extraction of maxillary second molars, as well as nonextraction, can be an effective operating procedure for open-bite correction of the permanent dentition.
Mitochondrial protein traffic requires coordinated operation of protein translocator complexes in the mitochondrial membrane. The TIM23 complex translocates and inserts proteins into the mitochondrial inner membrane. Here we analyze the intermembrane space (IMS) domains of Tim23 and Tim50, which are essential subunits of the TIM23 complex, in these functions. We find that interactions of Tim23 and Tim50 in the IMS facilitate transfer of precursor proteins from the TOM40 complex, a general protein translocator in the outer membrane, to the TIM23 complex. Tim23-Tim50 interactions also facilitate a late step of protein translocation across the inner membrane by promoting motor functions of mitochondrial Hsp70 in the matrix. Therefore, the Tim23-Tim50 pair coordinates the actions of the TOM40 and TIM23 complexes together with motor proteins for mitochondrial protein import.
Phosphatidylethanolamine (PE) plays important roles for the structure and function of mitochondria and other intracellular organelles. In yeast, the majority of PE is produced from phosphatidylserine (PS) by a mitochondrion-located PS decarboxylase, Psd1p. Because PS is synthesized in the endoplasmic reticulum (ER), PS is transported from the ER to mitochondria and converted to PE. After its synthesis, a portion of PE moves back to the ER. Two mitochondrial proteins located in the intermembrane space, Ups1p and Ups2p, have been shown to regulate PE metabolism by controlling the export of PE. It remains to be determined where PS is decarboxylated in mitochondria and whether decarboxylation is coupled to trafficking of PS. Here, using fluorescent PS as a substrate in an in vitro assay for Psd1p-dependent PE production in isolated mitochondria, we show that PS is transferred from the mitochondrial outer membrane to the inner membrane independently of Psd1p, Ups1p, and Ups2p and decarboxylated to PE by Psd1p in the inner membrane. Interestingly, Ups1p is required for the maintenance of Psd1p and therefore PE production. Restoration of Psd1p levels rescued PE production defects in ups1? mitochondria. Our data provide novel mechanistic insight into PE biogenesis in mitochondria.
This study was conducted to compare subjective and objective assessment methods of a two-colored chewing gum test and to find out whether these methods are capable of discriminating masticatory performances between sexes. 31 adults, 16 males and 15 females participated in this study. Each subject chewed five samples of two-colored chewing gum sticks for 5, 10, 20, 30 and 50 chewing strokes, respectively. The subjective color-mixing and shape indices for the gum bolus (SCMI-B, SSI-B) and the subjective color-mixing index and objective color-mixing ratio for the gum wafer (SCMI-W, OCMR-W) were evaluated by two independent examiners and, on a different day, re-evaluated by one of the examiners. The SCMI-B and SCMI-W assessments had inter- and intra-examiner reliable agreement at 20 or more chewing strokes. The OCMR-W measurement demonstrated high accuracy and low reproducibility between and within the examiners. There were significant gender differences in the distribution of SCMI-W scores (P = 0.044) and in the mean OCMI-W (P = 0.007). The SCMI-B and SCMI-W assessments and the OCMR-W measurement were reliable and valid at the 20 and 30 chewing strokes in this two-colored chewing gum test. The subjective color-mixing index (SCMI-W) and objective color-mixing ratio (OCMR-W) for the chewing gum wafer are capable of discriminating masticatory performance between sexes in this two-colored chewing gum test and that the OCMR-W measurement is discriminating better than the SCMI-W assessment.
Because messenger RNAs without a stop codon (nonstop mRNAs) generate stalled ribosomes, cells have developed a mechanism allowing degradation of nonstop mRNAs and their translation products (nonstop proteins) in the cytosol. Here, we observe the fate of nonstop proteins destined for organelles such as the endoplasmic reticulum (ER) and mitochondria. Nonstop mRNAs for secretory-pathway proteins in yeast generate nonstop proteins that become stuck in the translocator, the Sec61 complex, in the ER membrane. These stuck nonstop secretory proteins avoid proteasomal degradation in the cytosol, but are instead released into the ER lumen through stalled ribosome and translocator channels by Dom34:Hbs1. We also found that nonstop mitochondrial proteins are cleared from the mitochondrial translocator, the TOM40 complex, by Dom34:Hbs1. Clearance of stuck nonstop proteins from organellar translocator channels is crucial for normal protein influx into organelles and for normal cell growth, especially when nonstop mRNA decay does not function efficiently.
The TOM40 complex is a protein translocator in the mitochondrial outer membrane and consists of several different subunits. Among them, Tom40 is a central subunit that constitutes a protein-conducting channel by forming a ?-barrel structure. To probe the nature of the assembly process of Tom40 in the outer membrane, we attached various mitochondrial presequences to Tom40 that possess sorting information for the intermembrane space (IMS), inner membrane, and matrix and would compete with the inherent Tom40 assembly process. We analyzed the mitochondrial import of those fusion proteins in vitro. Tom40 crossed the outer membrane and/or inner membrane even in the presence of various sorting signals. N-terminal anchorage of the attached presequence to the inner membrane did not prevent Tom40 from associating with the TOB/SAM complex, although it impaired its efficient release from the TOB complex in vitro but not in vivo. The IMS or matrix-targeting presequence attached to Tom40 was effective in substituting for the requirement for small Tim proteins in the IMS for the translocation of Tom40 across the outer membrane. These results provide insight into the mechanism responsible for the precise delivery of ?-barrel proteins to the outer mitochondrial membrane.
The aim of this study was to explore the patterns of tooth agenesis in Japanese orthodontic patients with bilateral agenesis of mandibular second premolars. A total of 80 Japanese orthodontic patients with 2 congenitally missing mandibular second premolars were selected as the subjects of this study (experimental group). Another 80 individuals without bilateral agenesis of mandibular second premolars were collected for comparison (control group). The 2 groups were matched with regard to sex. Radiographs, dental casts and records of medical and/or dental treatment were used to identify tooth agenesis. The Chi-square test, odds ratio, and test for equality were used to make statistical comparisons. The prevalence rates of other types of tooth agenesis were significantly higher in the experimental group than in the control group. The occurrence of agenesis of maxillary and mandibular lateral incisors and third molars, and maxillary second premolars was also significantly higher in the experimental group than in the control group. Significantly increased prevalence rates of symmetrical tooth agenesis, with third molars included, and oligodontia were observed in the experimental group. Japanese subjects with bilateral agenesis of mandibular second premolars are at significantly high risk of agenesis of other types of permanent teeth, symmetrical tooth agenesis and oligodontia.
To examine the initial shear bond strength of orthodontic brackets bonded to bleached teeth with a self-etching adhesive system, as well as the effect of the length of time after bleaching on the initial bond strength.
The endoplasmic reticulum (ER) has an elaborate quality control system, which retains misfolded proteins and targets them to ER-associated protein degradation (ERAD). To analyze sorting between ER retention and ER exit to the secretory pathway, we constructed fusion proteins containing both folded carboxypeptidase Y (CPY) and misfolded mutant CPY (CPY*) units. Although the luminal Hsp70 chaperone BiP interacts with the fusion proteins containing CPY* with similar efficiency, a lectin-like ERAD factor Yos9p binds to them with different efficiency. Correlation between efficiency of Yos9p interactions and ERAD of these fusion proteins indicates that Yos9p but not BiP functions in the retention of misfolded proteins for ERAD. Yos9p targets a CPY*-containing ERAD substrate to Hrd1p E3 ligase, thereby causing ER retention of the misfolded protein. This ER retention is independent of the glycan degradation signal on the misfolded protein and operates even when proteasomal degradation is inhibited. These results collectively indicate that Yos9p and Hrd1p mediate ER retention of misfolded proteins in the early stage of ERAD, which constitutes a process separable from the later degradation step.
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