p32/gC1qR is indispensable for fetal development and mitochondrial translation: importance of its RNA-binding ability.

p32 is an evolutionarily conserved and ubiquitously expressed multifunctional protein. Although p32 exists at diverse intra and extracellular sites, it is predominantly localized to the mitochondrial matrix near the nucleoid associated with mitochondrial transcription factor A. Nonetheless, its function in the matrix is poorly understood. Here, we determined p32 function via generation of p32-knockout mice. p32-deficient mice exhibited mid-gestation lethality associated with a severe developmental defect of the embryo. Primary embryonic fibroblasts isolated from p32-knockout embryos showed severe dysfunction of the mitochondrial respiratory chain, because of severely impaired mitochondrial protein synthesis. Recombinant p32 binds RNA, not DNA, and endogenous p32 interacts with all mitochondrial messenger RNA species in vivo. The RNA-binding ability of p32 is well correlated with the mitochondrial translation. Co-immunoprecipitation revealed the close association of p32 with the mitoribosome. We propose that p32 is required for functional mitoribosome formation to synthesize proteins within mitochondria.

Arg 901 in the AE1 C-terminal tail is involved in conformational change but not in substrate binding.

In our previous paper, we demonstrated that Arg 901 in the C-terminal tail of human AE1 (band 3, anion exchanger 1) had a functional role in conformational change during anion exchange. To further examine how Arg 901 is involved in conformational change, we expressed various Arg 901 mutants and alanine mutants of the C-terminal tail (from Leu 886 to Val 911) on the plasma membrane of Saccharomyces cerevisiae and evaluated the kinetic parameters of sulfate ion transport. As a result, Vmax decreased as the hydrophobicities of the 901st and peripheral hydrophilic residues increased, indicating that the hydrophobicity of the C-terminal residue is involved in the conformational change. We also found the alkali and protease resistance of the C-terminal region after Arg 901 modification with hydroxyphenylglyoxal (HPG) or phenylglyoxal (PG), a hydrophobic reagent. These results suggested that the increased hydrophobicity of the C-terminal region around Arg 901 leads to inefficient conformational change by the newly produced hydrophobic interaction.

Ribonucleoprotein Y-box-binding protein-1 regulates mitochondrial oxidative phosphorylation (OXPHOS) protein expression after serum stimulation through binding to OXPHOS mRNA.

Mitochondria play key roles in essential cellular functions, such as energy production, metabolic pathways and aging. Growth factor-mediated expression of the mitochondrial OXPHOS (oxidative phosphorylation) complex proteins has been proposed to play a fundamental role in metabolic homoeostasis. Although protein translation is affected by general RNA-binding proteins, very little is known about the mechanism involved in mitochondrial OXPHOS protein translation. In the present study, serum stimulation induced nuclear-encoded OXPHOS protein expression, such as NDUFA9 [NADH dehydrogenase (ubiquinone) 1α subcomplex, 9, 39 kDa], NDUFB8 [NADH dehydrogenase (ubiquinone) 1ß subcomplex, 8, 19 kDa], SDHB [succinate dehydrogenase complex, subunit B, iron sulfur (Ip)] and UQCRFS1 (ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1), and mitochondrial ATP production, in a translation-dependent manner. We also observed that the major ribonucleoprotein YB-1 (Y-box-binding protein-1) preferentially bound to these OXPHOS mRNAs and regulated the recruitment of mRNAs from inactive mRNPs (messenger ribonucleoprotein particles) to active polysomes. YB-1 depletion led to up-regulation of mitochondrial function through induction of OXPHOS protein translation from inactive mRNP release. In contrast, YB-1 overexpression suppressed the translation of these OXPHOS mRNAs through reduced polysome formation, suggesting that YB-1 regulated the translation of mitochondrial OXPHOS mRNAs through mRNA binding. Taken together, our findings suggest that YB-1 is a critical factor for translation that may control OXPHOS activity.

Mutation of His 834 in human anion exchanger 1 affects substrate binding.

Anion exchanger 1 (AE1 or band 3) is responsible for Cl(-)-HCO3(-) exchange on erythrocyte membrane. Previously, we showed that band 3 is fixed in an inward-facing conformation by specific modification of His 834 with DEPC, resulting in a strong inhibition of its anion transport activity. To clarify the physiological role of His 834, we evaluated the sulfate transport activities of various band 3 mutants: different mutants at His 834 and alanine mutants of peripheral residues around 834 (Lys 829-Phe 836) in yeast cell membranes. The K(m) values of the His 834 mutants were 4-10 times higher than that of the wild type, while their V(max) values were barely lower than that of wild type. Meanwhile, the K(m) values of the peripheral alanine mutants were only slightly increased. These data suggest that His 834 is critically important for the efficient binding of sulfate anion, but not for the conformational change induced by substrate binding.

The functional role of arginine 901 at the C-terminus of the human anion transporter band 3 protein.

To determine which arginine residues are responsible for band 3-mediated anion transport, we analyzed hydroxyphenylglyoxal (HPG)-modified band 3 protein in native erythrocyte membranes. HPG-modification leads to inhibition of the transport of phosphoenolpyruvate, a substrate for band 3-mediated transport. We analyzed the HPG-modified membranes by reverse phase-HPLC, and determined that arginine 901 was modified by HPG. To determine the role of Arg 901 in the conformational change induced by anion exchange, we analyzed HPG-modification of the membranes when 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) or diethypyrocarbonate (DEPC) was present. DNDS and DEPC fix band 3 in the outward and inward conformations, respectively. HPG-modification was unaffected in the presence of DEPC but decreased in the presence of DNDS. In addition to that, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), which specifically reacts with the outward conformation of band 3, did not react with HPG-modified membranes. Furthermore, we expressed a band 3 mutant in which Arg 901 was replaced by alanine (R901A) on yeast membranes. The kinetic parameters indicated that the R901A mutation affected the rate of conformational change of the band 3 protein. From these results, we conclude that the most C-terminal arginine, Arg 901, has a functional role in the conformational change that is necessary for anion transport.

A simple search of TM segments in polytopic membrane protein using matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Using both high performance liquid chromatography (HPLC) and amino acid sequencing (AAS), we previously analyzed band 3 TM peptide-segments that make up the transmembrane protein structure. However, the HPLC/AAS combination method was highly time-consuming. Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) mass spectrometry is used to obtain accurate molecular weight information for proteins/peptides simply and sensitively. We applied the MALDI-TOF mass spectrometry technique to search for TM segments in membrane proteins. In combination with trypsin cleavages after alkali treatments (pH12 or 13) and sample preparation using organic solvents for MALDI-TOF mass spectrometry, we determined the TM segments of band 3 and glycophorin A in erythrocyte membrane. The method can be applied to other polytopic membrane proteins in erythrocyte membrane.

Effect on catalysis by replacement of catalytic residue from hen egg white lysozyme to Venerupis philippinarum lysozyme.

Asn46Asp/Asp52Ser or Asn46Glu/Asp52Ser hen egg white lysozyme (HEL) mutant was designed by introducing the substituted catalytic residue Asp46 or Glu46, respectively, based on Venerupis philippinarum (Vp) lysozyme structure as a representative of invertebrate-type (i-type) lyzozyme. These mutations restored the bell-shaped pH-dependency of the enzyme activity from the sigmoidal pH-dependency observed for the Asp52Ser mutant. Furthermore both lysozyme mutants possessed retaining mechanisms like Vp lysozyme and HEL. The Asn46Glu/Asp52Ser mutant, which has a shorter distance between two catalytic residues, formed a glycosyl adduct in the reaction with the N-acetylglucosamine oligomer. Furthermore, we found the accelerated turnover through its glycosyl adduct formation and decomposition. The turnover rate estimated from the glycosyl formation and decomposition rates was only 20% of the observed hydrolysis rate of the substrate. Based on these results, we discussed the catalytic mechanism of lysozymes.

Dihydro-orotate dehydrogenase is physically associated with the respiratory complex and its loss leads to mitochondrial dysfunction.

Some mutations of the DHODH (dihydro-orotate dehydrogenase) gene lead to postaxial acrofacial dysostosis or Miller syndrome. Only DHODH is localized at mitochondria among enzymes of the de novo pyrimidine biosynthesis pathway. Since the pyrimidine biosynthesis pathway is coupled to the mitochondrial RC (respiratory chain) via DHODH, impairment of DHODH should affect the RC function. To investigate this, we used siRNA (small interfering RNA)-mediated knockdown and observed that DHODH knockdown induced cell growth retardation because of G2/M cell-cycle arrest, whereas pyrimidine deficiency usually causes G1/S arrest. Inconsistent with this, the cell retardation was not rescued by exogenous uridine, which should bypass the DHODH reaction for pyrimidine synthesis. DHODH depletion partially inhibited the RC complex III, decreased the mitochondrial membrane potential, and increased the generation of ROS (reactive oxygen species). We observed that DHODH physically interacts with respiratory complexes II and III by IP (immunoprecipitation) and BN (blue native)/SDS/PAGE analysis. Considering that pyrimidine deficiency alone does not induce craniofacial dysmorphism, the DHODH mutations may contribute to the Miller syndrome in part through somehow altered mitochondrial function.

Localization of mRNAs encoding human mitochondrial oxidative phosphorylation proteins.

The mitochondrial oxidative phosphorylation (OXPHOS) proteins are encoded by both nuclear and mitochondrial DNA. The nuclear-encoded OXPHOS mRNAs have specific subcellular localizations, but little is known about which localize near mitochondria. Here, we compared mRNAs in mitochondria-bound polysome fractions with those in cytosolic, free polysome fractions. mRNAs encoding hydrophobic OXPHOS proteins, which insert into the inner membrane, were localized near mitochondria. Conversely, OXPHOS gene which mRNAs were predominantly localized in cytosol had less than one transmembrane domain. The RNA-binding protein Y-box binding protein-1 is localized at the mitochondrial outer membrane and bound to the OXPHOS mRNAs. Our findings offer new insight into mitochondrial co-translational import in human cells.

Comparative analysis of cells and proteins of pumpkin plants for the control of fruit size.

Common pumpkin plants (Cucurbita maxima) produce fruits of 1-2 kg size on the average, while special varieties of the same species called Atlantic Giant are known to produce a huge fruit up to several hundred kilograms. As an approach to determine the factors controlling the fruit size in C. maxima, we cultivated both AG and control common plants, and found that both the cell number and cell sizes were increased in a large fruit while DNA content of the cell did not change significantly. We also compared protein patterns in the leaves, stems, ripe and young fruits by two-dimensional (2D) gel electrophoresis, and identified those differentially expressed between them with mass spectroscopy. Based on these results, we suggest that factors in photosynthesis such as ribulose-bisphosphate carboxylase, glycolysis pathway enzymes, heat-shock proteins and ATP synthase play positive or negative roles in the growth of a pumpkin fruit. These results provide a step toward the development of plant biotechnology to control fruit size in the future.

Protein instability and functional defects caused by mutations of dihydro-orotate dehydrogenase in Miller syndrome patients.

Miller syndrome is a recessive inherited disorder characterized by postaxial acrofacial dysostosis. It is caused by dysfunction of the DHODH (dihydroorotate dehydrogenase) gene, which encodes a key enzyme in the pyrimidine de novo biosynthesis pathway and is localized at mitochondria intermembrane space. We investigated the consequence of three missense mutations, G202A, R346W and R135C of DHODH, which were previously identified in patients with Miller syndrome. First, we established HeLa cell lines stably expressing DHODH with Miller syndrome-causative mutations: G202A, R346W and R135C. These three mutant proteins retained the proper mitochondrial localization based on immunohistochemistry and mitochondrial subfractionation studies. The G202A, R346W DHODH proteins showed reduced protein stability. On the other hand, the third one R135C, in which the mutation lies at the ubiquinone-binding site, was stable but possessed no enzymatic activity. In conclusion, the G202A and R346W mutation causes deficient protein stability, and the R135C mutation does not affect stability but impairs the substrate-induced enzymatic activity, suggesting that impairment of DHODH activity is linked to the Miller syndrome phenotype.

Recombinant mitochondrial transcription factor A protein inhibits nuclear factor of activated T cells signaling and attenuates pathological hypertrophy of cardiac myocytes.

The overexpression of mitochondrial transcription factor A (TFAM) attenuates the decrease in mtDNA copy number after myocardial infarction, ameliorates pathological hypertrophy, and markedly improves survival. However, non-transgenic strategy to increase mtDNA for the treatment of pathological hypertrophy remains unknown. We produced recombinant human TFAM protein (rhTFAM). rhTFAM rapidly entered into mitochondria of cultured cardiac myocytes. rhTFAM increased mtDNA and abolished the activation of nuclear factor of activated T cells (NFAT), which is well known to activate pathological hypertrophy. rhTFAM attenuated subsequent morphological hypertrophy of myocytes as well. rhTFAM would be an attractive molecule in attenuating cardiac pathological hypertrophy.

Island specific expression of a novel [Lys(49)]phospholipase A(2) (BPIII) in Protobothrops flavoviridis venom in Amami-Oshima, Japan.

In search of the transcripts expressed in Protobothrops flavoviridis venom gland, 466 expressed sequence tags (ESTs) were generated from the venom gland cDNA library of P. flavoviridis in Amami-Oshima, Japan. The sequencing of randomly selected cDNA clones followed by identification in similarity search against existing databases led to the finding of a novel lysine-49-phospholipase A(2) ([Lys(49)]PLA(2)) clone. It coded for one amino acid-substituted BPII homologue or two amino acids-substituted BPI homologue in which BPII and BPI are [Lys(49)]PLA(2)s contained in Amami-Oshima and Tokunoshima P. flavoviridis venoms. This isozyme, named BPIII, was isolated from Amami-Oshima P. flavoviridis venom. BPIII gave a specific [M+2H](2+) peak of m/z 736.3 on mass spectrometry (MS) analysis after S-carboxamidomethylation and trypsin digestion when compared with BPII. It became evident from MS analysis after S-carboxamidomethylation and trypsin digestion of the mixed protein peaks ranging from BPI to BPII obtained by fractionation on a carboxymethyl cellulose column of Amami-Oshima and Tokunoshima P. flavoviridis venoms that BPIII protein is contained in Amami-Oshima P. flavoviridis venom but not in Tokunoshima P. flavoviridis venom. It is for the first time that a protein present in Amami-Oshima P. flavoviridis venom is not found in Tokunoshima P. flavoviridis venom.

Identification of oxidized methionine sites in erythrocyte membrane protein by liquid chromatography/electrospray ionization mass spectrometry peptide mapping.

In this study, we used peptide mapping combined with liquid chromatography/electrospray ionization mass spectrometry (LC/ESI MS) to examine the methionine oxidation of band 3 of erythrocyte membrane protein. Initially, we identified the methionine sites oxidized by chloramine T (N-chloro-p-toluenesulfoamide), a hydrophilic reagent. There were three oxidized methionines (Met 559, Met 741, and Met 909) in band 3, and these methionines were located in a hydrophilic region determined by previous topological studies of band 3. In addition, we found that C12E8, a polyoxyethylene detergent, leads to the oxidation of methionines in a transmembrane segment in band 3, and this oxidation occurs in a C12E8 preincubation time-dependent manner. In a previous study, it was found that peroxides accumulate in a polyoxyethylene detergent. Thus, our method enabled the direct and quantitative detection of protein damage due to detergent peroxides. Furthermore, we examined methionine oxidation in the presence of 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) or diethyl pyrocarbonate (DEPC), which induced either an outward or an inward conformation in band 3, respectively. Our results indicated that the location of Met 741 was associated with the band 3 conformation induced by band 3-mediated anion transport. In conclusion, we found that methionine oxidation can be applied to examine membrane protein structures as follows: (1) for topological studies of membrane proteins, (2) for assessing the quality of proteins in detergent solubilization studies, and (3) for the detection of conformational changes in membrane proteins.