Biomedical subjects

Toshihiko Oka

Publications and source records attributed to Toshihiko Oka.

At least 19 recordsLinked to original sources

PubMed2006

Identification of a novel protein that regulates mitochondrial fusion by modulating mitofusin (Mfn) protein function.

Mitofusin proteins 1 and 2 (Mfn1 and Mfn2, respectively) of the mammalian mitochondrial outer membrane are homologues of Drosophila FZO and yeast Fzo1, and both are essential for GTP-dependent mitochondrial fusion. We identified a 55-kDa Mfn-binding protein named MIB. It is a member of the medium-chain dehydrogenase/reductase protein superfamily, and has a conserved coenzyme-binding domain (CBD). The majority of MIB is localized in the cytoplasm but a small amount is associated with mitochondria. Exogenous expression of MIB in HeLa cells induced mitochondrial fragmentation, which was prevented by coexpression of Mfn1, suggesting a functional interaction of MIB with Mfn proteins; the GGVG sequence in the CBD of MIB is essential for its function. By contrast, MIB knockdown resulted in growth arrest of the cells, although apoptotic sensitivity was not affected by either its knockdown or its overexpression. Furthermore, MIB knockdown induced a large extension of mitochondrial network structures. By contrast, a double knockdown of MIB and Mfn1 resulted in mitochondrial fragmentation and reversal of the growth arrest, the morphology and growth phenotype induced by knockdown of Mfn1 alone, again suggesting that MIB modulates Mfn1 function. Together, these findings suggest that MIB is essential for cellular function by regulating mitochondrial membrane dynamics in cooperation with Mfn proteins.

Acyl-CoA Dehydrogenase↗

PubMed2006

IntraGolgi distribution of the Conserved Oligomeric Golgi (COG) complex.

The Conserved Oligomeric Golgi (COG) complex is an eight-subunit (Cog1-8) peripheral Golgi protein involved in membrane trafficking and glycoconjugate synthesis. COG appears to participate in retrograde vesicular transport and is required to maintain normal Golgi structure and function. COG mutations interfere with normal transport, distribution, and/or stability of Golgi proteins associated with glycoconjugate synthesis and trafficking, and lead to failure of spermatogenesis in Drosophila melanogaster, misdirected migration of gonadal distal tip cells in Caenorhabditis elegans, and type II congenital disorders of glycosylation in humans. The mechanism by which COG influences Golgi structure and function is unclear. Immunogold electron microscopy was used to visualize the intraGolgi distribution of a functional, hemagglutinin epitope-labeled COG subunit, Cog1-HA, that complements the Cog1-deficiency in Cog1-null Chinese hamster ovary cells. COG was found to be localized primarily on or in close proximity to the tips and rims of the Golgi's cisternae and their associated vesicles and on vesicles and vesiculo-tubular structures seen on both the cis and trans-Golgi Network faces of the cisternal stacks, in some cases on COPI containing vesicles. These findings support the proposal that COG is directly involved in controlling vesicular retrograde transport of Golgi resident proteins throughout the Golgi apparatus.

Adaptor Proteins, Vesicular Transport↗

PubMed2006

Regulation of mitochondrial morphology through proteolytic cleavage of OPA1.

The dynamin-like GTPase OPA1, a causal gene product of human dominant optic atrophy, functions in mitochondrial fusion and inner membrane remodeling. It has several splice variants and even a single variant is found as several processed forms, although their functional significance is unknown. In yeast, mitochondrial rhomboid protease regulates mitochondrial function and morphology through proteolytic cleavage of Mgm1, the yeast homolog of OPA1. We demonstrate that OPA1 variants are synthesized with a bipartite-type mitochondrial targeting sequence. During import, the matrix-targeting signal is removed and processed forms (L-isoforms) are anchored to the inner membrane in type I topology. L-isoforms undergo further processing in the matrix to produce S-isoforms. Knockdown of OPA1 induced mitochondrial fragmentation, whose network morphology was recovered by expression of L-isoform but not S-isoform, indicating that only L-isoform is fusion-competent. Dissipation of membrane potential, expression of m-AAA protease paraplegin, or induction of apoptosis stimulated this processing along with the mitochondrial fragmentation. Thus, mammalian mitochondrial function and morphology is regulated through processing of OPA1 in a DeltaPsi-dependent manner.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2006

High-resolution powder diffraction study of purple membrane with a large Guinier-type camera.

X-ray diffraction patterns from a film of oriented purple membranes, which comprise two-dimensional crystals of bacteriorhodopsin (BR) trimers, were recorded with a 1 m-pathlength Guinier-type camera at SPring-8 BL40B2. A well focused X-ray beam and a camera with a high angular resolution of 0.024 degrees enabled a powder diffraction profile with very sharp and well separated peaks to be obtained up to a resolution of 2.3 A. Using integrated diffraction intensities up to a Bragg spacing of 4.2 A, a cluster of bulky amino acid residues and the head group of the BR chromophore are apparent in the electron density map projected along the membrane normal. Thus, a combination of synchrotron X-rays and large Guinier camera can be used for analyzing the conformational changes of BR in the intact state. In addition, the method might be extended to the structural analysis of film materials composed of two-dimensional arrays of nanoparticles.

Equipment Design↗

PubMed2006

Retrograde transport on the COG railway.

The conserved oligomeric Golgi (COG) complex is essential for establishing and/or maintaining the structure and function of the Golgi apparatus. The Golgi apparatus, in turn, has a central role in protein sorting and glycosylation within the eukaryotic secretory pathway. As a consequence, COG mutations can give rise to human genetic diseases known as congenital disorders of glycosylation. We review recent results from studies of yeast, worm, fly and mammalian COG that provide evidence that COG might function in retrograde vesicular trafficking within the Golgi apparatus. This hypothesis explains the impact of COG mutations by postulating that they impair the retrograde flow of resident Golgi proteins needed to maintain normal Golgi structure and function.

Adaptor Proteins, Vesicular Transport↗

PubMed2005

Conformational dynamics of complementarity-determining region H3 of an anti-dansyl Fv fragment in the presence of its hapten.

Antigen-induced structural changes in the Fv fragment of an anti-dansyl immunoglobulin G were studied by X-ray crystallography and stopped-flow fluorescence measurement. The crystal structure of the Fv fragment complexed with dansyl-lysine was determined at a resolution of 1.85 A. The dansyl-lysine molecule bound to a narrow cavity formed by the complementarity-determining regions H3 and H1, the N-terminal region of the VH domain and L2 of the VL domain. The structure of the binding site in the crystal structure explained well the results of the previous nuclear magnetic resonance measurements. The hapten binding caused remarkable conformational changes in H3 and its environmental structures, including the hydration structure from those observed in the unliganded state. The tip of H3 moved about 12 A from its position in the unliganded state. In addition, because of the contacts of H3 with the VL domain at the domain interface, the conformational changes of H3 resulted in the relative rotation of the variable domains by 5 degrees from their association observed in the unliganded state. The hydrophobic interactions at the domain interface seemed to be particularly important for the mutual rotation of the domains. The stopped-flow fluorescence measurement monitoring the interaction of the dansyl group and the binding pocket revealed that H3 was in a conformational equilibrium of three consecutive conformational states in the presence of dansyl-lysine in solution; an unliganded state preventing the access of the hapten, another unliganded state able to bind the hapten and the complex. The conformational dynamics of H3 in recognizing and binding the hapten molecule are discussed on the basis of the structural information from the present and previous studies.

Binding Sites↗

PubMed2005

Genetic analysis of the subunit organization and function of the conserved oligomeric golgi (COG) complex: studies of COG5- and COG7-deficient mammalian cells.

The conserved oligomeric Golgi (COG) complex is an eight-subunit (Cog1-8) peripheral Golgi protein involved in Golgi-associated membrane trafficking and glycoconjugate synthesis. We have analyzed the structure and function of COG using Cog1 or Cog2 null Chinese hamster ovary cell mutants, fibroblasts from a patient with Cog7-deficient congenital disorders of glycosylation, and stable Cog5-deficient HeLa cells generated by RNA interference. Although the dilation of some Golgi cisternae in Cog5-deficient cells resembled that observed in Cog1- or Cog2-deficient cells, their global glycosylation defects (less severe) and intracellular processing and function of low density lipoprotein receptors (essentially normal) differed from Cog1- and Cog2-deficient cells. Immunoblotting, gel filtration, and immunofluorescence microscopy analyses of the COG-deficient cells and cell extracts indicated that 1) Cog2-4 and Cog5-7 form stable subcomplexes, 2) Cog1 mediates Golgi association of a Cog2-4 plus Cog8 subcomplex, 3) Cog8 associates stably with both Cog5-7 and Cog1-4 subcomplexes, and thus 4) Cog8 helps assemble the Cog1-4 and Cog5-7 subcomplexes into the complete COG complex. This model of the subunit organization of COG is in excellent agreement with in vitro data presented in an accompanying paper (Ungar, D., Oka, T., Vasile, E., Krieger, M., and Hughson, F. M. (2005) J. Biol. Chem. 280, 32729-32735). Only one or two of the seven Cog1- or Cog2-dependent Golgi membrane proteins called GEARs are also sensitive to Cog5 or Cog7 deficiency, indicating that the COG subunits play distinctive roles in controlling Golgi structure and function.

Adaptor Proteins, Vesicular Transport↗

PubMed2005

Subunit architecture of the conserved oligomeric Golgi complex.

The conserved oligomeric Golgi (COG) complex is thought to function in intra-Golgi retrograde trafficking mediated by coat protein I vesicles, a pathway essential for the proper structure and function of the Golgi apparatus. Previous work suggested that COG might act as a tethering factor to mediate the initial attachment between coat protein I vesicles and Golgi membranes. Here, we present extensive in vitro co-translation and immunoprecipitation experiments leading to a new model for the overall architecture of the mammalian COG complex. The eight COG subunits (Cog1-8) are found to form two heterotrimeric subassemblies (Cog2/3/4 and Cog5/6/7) linked by a heterodimer composed of the remaining subunits (Cog1/8). This model is in excellent agreement with in vivo data presented in an accompanying paper (Oka, T., Vasile, E., Penman, M., Novina, C. D., Dykxhoorn, D. M., Ungar, D., Hughson, F. M., and Krieger, M. (2005) J. Biol. Chem. 280, 32736-32745).

Adaptor Proteins, Vesicular Transport↗

PubMed2005

A railroad switch in mitochondrial protein import.

Chacinska et al., (2005) recently clarified how translocation machineries of the mitochondrial outer and inner membranes cooperate to correctly sort preproteins destined for the mitochondrial matrix and inner membrane.

Biological Transport↗

PubMed2005

Multi-component protein complexes and Golgi membrane trafficking.

Several large cytosolic protein complexes with multiple components have been proposed to play key roles in mediating or controlling membrane trafficking. Among these complexes, TRAPP, COG and GARP/VFT have been implicated in multiple steps of Golgi membrane trafficking. The importance of these complexes for Golgi function has been established using in vitro biochemical assays and yeast and mammalian somatic cell genetics. Furthermore, mutations in the genes encoding subunits of either TRAPP or the COG complex have been shown to be responsible for human genetic disorders. We here review recent studies exploring the structures and functions of these three oligomeric complexes.

Animals↗

PubMed2004

Structural transition of bacteriorhodopsin is preceded by deprotonation of Schiff base: microsecond time-resolved x-ray diffraction study of purple membrane.

The structural changes in the photoreaction cycle of bacteriorhodopsin, a light-driven proton pump, was investigated at a resolution of 7 angstroms by a time-resolved x-ray diffraction experiment utilizing synchrotron x rays from an undulator of SPring-8. The x-ray diffraction measurement system, used in coupling with a pulsed YAG laser, enabled us to record a diffraction pattern from purple membrane film at a time-resolution of 6 micros over the time domain of 5 micros to 500 ms. In the time domain, the functionally most important M-intermediate appears. A series of time-resolved x-ray diffraction data after photo-excitation showed clear intensity changes caused by the conformational changes of helix G in the M-intermediate. The population of the reaction intermediate was prominently observed at approximately 5 ms after a photo-stimulus. In contrast, absorption measurement indicated the deprotonation of the Schiff base predominantly occurred at approximately 300 micros after a photo-stimulus. These results showed that the conformational changes characterizing structurally the M-intermediate predominantly occur at a later stage of the deprotonation of the Schiff base. Thus, the M-intermediate can be divided into two metastable stages with different physical characteristics.

Bacteriorhodopsins↗

PubMed2004

CCD-based X-ray area detector for time-resolved diffraction experiments.

A fast X-ray area detector for diffraction, scattering and imaging experiments at microsecond to millisecond time resolution has been developed. The key element of the detector is a fast (291 frames s(-1)) framing camera with three CCDs. A prism forms identical images on the CCDs and the frame rate is increased three times by reading them alternately. In order to convert X-rays into visible light that is detectable with the CCDs, an X-ray image intensifier is used. The camera can also be used with a high-resolution X-ray detector. In both cases it was found to be important to use a phosphor with a short decay time to fully make use of the high-speed framing capability of the camera. Preliminary results of a fibre diffraction experiment on a skeletal muscle and coronary angiography are presented.

Animals↗

PubMed2004

Picometer-scale dynamical observations of individual membrane proteins: the case of bacteriorhodopsin.

In vivo measurements of dynamical conformational changes in single biomolecules under functional conditions have had a tremendous impact on molecular and cell biology. However, even single-molecule fluorescent resonance energy transfer cannot easily monitor the intramolecular dynamics in cell systems due to shortcomings in monitoring precision. Here, we report dynamical observations of irreversible intramolecular conformational changes in a single-membrane protein [bacteriorhodopsin (BR)] using diffracted x-ray tracking. The light-driven proton pump BR is the best-characterized membrane protein. The position of BR's 35th amino acid, which is located farthest from retinal, exhibits a momentary positional jump of 0.73+/-0.48 A due to the expression of its function. Following that, we observed Brownian motion without the diffracted spots returning to their initial positions. The average width of this jump is about 14 times larger than that of thermal Brownian motion and agrees with estimated movements from known x-ray crystallography data. This result is an important step toward realizing in vivo observations of single-molecular conformational changes in membrane proteins.

Bacteriorhodopsins↗

PubMed2004

The COG and COPI complexes interact to control the abundance of GEARs, a subset of Golgi integral membrane proteins.

The conserved oligomeric Golgi (COG) complex is a soluble hetero-octamer associated with the cytoplasmic surface of the Golgi. Mammalian somatic cell mutants lacking the Cog1 (ldlB) or Cog2 (ldlC) subunits exhibit pleiotropic defects in Golgi-associated glycoprotein and glycolipid processing that suggest COG is involved in the localization, transport, and/or function of multiple Golgi processing proteins. We have identified a set of COG-sensitive, integral membrane Golgi proteins called GEARs (mannosidase II, GOS-28, GS15, GPP130, CASP, giantin, and golgin-84) whose abundances were reduced in the mutant cells and, in some cases, increased in COG-overexpressing cells. In the mutants, some GEARs were abnormally localized in the endoplasmic reticulum and were degraded by proteasomes. The distributions of the GEARs were altered by small interfering RNA depletion of epsilon-COP in wild-type cells under conditions in which COG-insensitive proteins were unaffected. Furthermore, synthetic phenotypes arose in mutants deficient in both epsilon-COP and either Cog1 or Cog2. COG and COPI may work in concert to ensure the proper retention or retrieval of a subset of proteins in the Golgi, and COG helps prevent the endoplasmic reticulum accumulation and degradation of some GEARs.

Acetylcysteine↗

PubMed2003

From lysosomes to the plasma membrane: localization of vacuolar-type H+ -ATPase with the a3 isoform during osteoclast differentiation.

Osteoclasts generate a massive acid flux to mobilize bone calcium. Local extracellular acidification is carried out by vacuolar type H+-ATPase (V-ATPase) localized in the plasma membrane. We have shown that a3, one of the four subunit a isoforms (a1, a2, a3, and a4), is a component of the plasma membrane V-ATPase (Toyomura, T., Oka, T., Yamaguchi, C., Wada, Y., and Futai, M. (2000) J. Biol. Chem. 275, 8760-8765). To establish the unique localization of V-ATPase, we have used a murine macrophage cell line, RAW 264.7, that can differentiate into multinuclear osteoclast-like cells on stimulation with RANKL (receptor activator of nuclear factor kappaB ligand). The V-ATPase with the a3 isoform was localized to late endosomes and lysosomes, whereas those with the a1 and a2 isoforms were localized to organelles other than lysosomes. After stimulation, the V-ATPase with the a3 isoform was immunochemically colocalized with lysosome marker lamp2 and was detected in acidic organelles. These organelles were also colocalized with microtubules, and the signals of lamp2 and a3 were dispersed by nocodazole, a microtubule depolymerizer. In RAW-derived osteoclasts cultured on mouse skull pieces, the a3 isoform was transported to the plasma membrane facing the bone and accumulated inside podosome rings. These findings indicate that V-ATPases with the a3 isoform localized in late endosomes/lysosomes are transported to the cell periphery during differentiation and finally assembled into the plasma membrane of mature osteoclasts.

3T3 Cells↗

PubMed2002

Oligosaccharide specificity of galectins: a search by frontal affinity chromatography.

Galectins are widely distributed sugar-binding proteins whose basic specificity for beta-galactosides is conserved by evolutionarily preserved carbohydrate-recognition domains (CRDs). Although they have long been believed to be involved in diverse biological phenomena critical for multicellular organisms, in only few a cases has it been proved that their in vivo functions are actually based on specific recognition of the complex carbohydrates expressed on cell surfaces. To obtain clues to understand the physiological roles of diverse members of the galectin family, detailed analysis of their sugar-binding specificity is necessary from a comparative viewpoint. For this purpose, we recently reinforced a conventional system for frontal affinity chromatography (FAC) [J. Chromatogr., B, Biomed. Sci. Appl. 771 (2002) 67-87]. By using this system, we quantitatively analyzed the interactions at 20 degrees C between 13 galectins including 16 CRDs originating from mammals, chick, nematode, sponge, and mushroom, with 41 pyridylaminated (PA) oligosaccharides. As a result, it was confirmed that galectins require three OH groups of N-acetyllactosamine, as had previously been denoted, i.e., 4-OH and 6-OH of Gal, and 3-OH of GlcNAc. As a matter of fact, no galectin could bind to glycolipid-type glycans (e.g., GM2, GA2, Gb3), complex-type N-glycans, of which both 6-OH groups are sialylated, nor Le-related antigens (e.g., Le(x), Le(a)). On the other hand, considerable diversity was observed for individual galectins in binding specificity in terms of (1) branching of N-glycans, (2) repeating of N-acetyllactosamine units, or (3) substitutions at 2-OH or 3-OH groups of nonreducing terminal Gal. Although most galectins showed moderately enhanced affinity for branched N-glycans or repeated N-acetyllactosamines, some of them had extremely enhanced affinity for either of these multivalent glycans. Some galectins also showed particular preference for alpha1-2Fuc-, alpha1-3Gal-, alpha1-3GalNAc-, or alpha2-3NeuAc-modified glycans. To summarize, galectins have evolved their sugar-binding specificity by enhancing affinity to either "branched", "repeated", or "substituted" glycans, while conserving their ability to recognize basic disaccharide units, Galbeta1-3/4GlcNAc. On these bases, they are considered to exert specialized functions in diverse biological phenomena, which may include formation of local cell-surface microdomains (raft) by sorting glycoconjugate members for each cell type.

Animals↗

PubMed2002

Characterization of a mammalian Golgi-localized protein complex, COG, that is required for normal Golgi morphology and function.

Multiprotein complexes are key determinants of Golgi apparatus structure and its capacity for intracellular transport and glycoprotein modification. Three complexes that have previously been partially characterized include (a) the Golgi transport complex (GTC), identified in an in vitro membrane transport assay, (b) the ldlCp complex, identified in analyses of CHO cell mutants with defects in Golgi-associated glycosylation reactions, and (c) the mammalian Sec34 complex, identified by homology to yeast Sec34p, implicated in vesicular transport. We show that these three complexes are identical and rename them the conserved oligomeric Golgi (COG) complex. The COG complex comprises four previously characterized proteins (Cog1/ldlBp, Cog2/ldlCp, Cog3/Sec34, and Cog5/GTC-90), three homologues of yeast Sec34/35 complex subunits (Cog4, -6, and -8), and a previously unidentified Golgi-associated protein (Cog7). EM of ldlB and ldlC mutants established that COG is required for normal Golgi morphology. "Deep etch" EM of purified COG revealed an approximately 37-nm-long structure comprised of two similarly sized globular domains connected by smaller extensions. Consideration of biochemical and genetic data for mammalian COG and its yeast homologue suggests a model for the subunit distribution within this complex, which plays critical roles in Golgi structure and function.

Adaptor Proteins, Vesicular Transport↗

PubMed2002

Light-induced unfolding of photoactive yellow protein mutant M100L.

Light-activation of the PAS domain protein photoactive yellow protein (PYP) is believed to trigger a negative phototactic response in the phototropic bacterium Halorhodospira halophila. To investigate transient conformational changes of the PYP photocycle, we utilized the PYP mutant M100L that displays an increased lifetime of the putative signaling-state photointermediate PYP(M) by 3 orders of magnitude, as previously reported for the M100A mutant [Devanathan, S., Genick, U. K., Canestrelli, I. L., Meyer, T. E., Cusanovich, M. A., Getzoff, E. D., and Tollin, G. Biochemistry (1998) 37, 11563-11568]. The FTIR difference spectrum of PYP(M) and the ground state of M100L demonstrated extensive peptide-backbone structural changes as observed in the FTIR difference spectrum of the wild-type protein and PYP(M). The conformational change investigated by CD spectroscopy in the far-UV region showed reduction of the alpha-helical content by approximately 40%, indicating a considerable amount of changes in the secondary structure. The optical activity of the p-coumaric acid chromophore completely vanished upon PYP(M) in contrast to the dark state, indicating deformation of the binding pocket structure in PYP(M). The tertiary structural changes were further monitored by small-angle X-ray scattering measurements, which demonstrated a significant increase of the radius of gyration of the molecule by approximately 5% in PYP(M). These structural changes were reversed concomitantly with the chromophore anionization upon the dark state recovery. The observed changes of the quantities provided a more vivid view of the structural changes of the mutant PYP in going from PYP(M) to PYP(dark), which can be regarded as a process of folding of the secondary and the tertiary structures of the "PAS" domain structure, coupled with the p-coumaric acid chromophore deprotonation and isomerization.

Bacterial Proteins↗