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We investigated the structures of normal (type I) bicontinuous cubic phases in hexa-, hepta-, and octaethylene glycol dodecyl ether/water mixtures by small-angle X-ray crystallography of single-crystal domains. Reconstructed electron densities showed that the hydrophilic chains with high electron density are confined to a film centered on the surface of the Gyroid (a triply periodic minimal surface), while hydrophobic chains with low electron density are distributed within the pair of interwoven labyrinths carved out by the Gyroid. Further, the local minimum within the high electron density region, due to bulk water, coincides precisely with the Gyroid. This minimum is less pronounced in mixtures with longer ethylene glycol chains, consistent with their decreased water content. Our analysis clearly shows that the polar–nonpolar interfaces are parallel to the Gyroid surface in all mixtures. The repulsive hydration or overlapping force between the pair of facing monolayers of ethylene glycol chains on either side of the Gyroid surface is the likely origin of the parallel interfaces.

Among the photoproducts of vertebrate rhodopsin, only metarhodopsin II (Meta-II) preferentially adopts the active structure in which transmembrane helices are rearranged. Light-induced helical rearrangement of rhodopsin in membrane-embedded form was directly monitored by wide-angle X-ray scattering (WAXS) using nanodiscs. The change in the WAXS curve for the formation of Meta-II was characterized by a peak at 0.2 Å–1and a valley at 0.6 Å–1, which were not observed in metarhodopsin I and opsin. However, acid-induced active opsin (Opsin*) showed a 0.2 Å–1peak, but no 0.6 Å–1valley. Analyses using the model structures based on the crystal structures of dark state and Meta-II suggest that the outward movement of helix VI occurred in Opsin*. However, the displaced helices III and V in Meta-II resulting from the disruption of cytoplasmic ionic lock were restored in Opsin*, which is likely to destabilize the G-protein-activating structure of opsin.

We investigated two distinct lyotropic liquid crystal inverse bicontinuous cubic phases of phytantriol/water mixtures by small-angle X-ray crystallography of the single-crystal regions. Reconstructed electron density maps revealed hydrophilic head and hydrophobic tail regions of the phytantriol bilayer membranes and water regions. The bilayer membranes are shown to be located on the D and gyroid triply periodic minimal surfaces. To investigate the structures of the polar–nonpolar interfaces, we optimized two models: a parallel surface model and a constant mean curvature surface model. The parallel surface model agreed well with the X-ray data, and the Rfactors, which show the degree of agreement between those structural models and the data, were less than 0.04. In stark contrast, the constant mean curvature surface model deviated significantly from the data, and the Rfactors were around 0.15. We therefore conclude that the polar–nonpolar interface of the inverse bicontinuous cubic phase of the phytantriol/water system is close to a parallel surface to a triply periodic minimal surface.

Mitochondria are highly dynamic organelles that undergo frequent fusion and fission. Optic atrophy 1 (OPA1) is an essential GTPase protein for both mitochondrial inner membrane (IM) fusion and cristae morphology. Under mitochondria-stress conditions, membrane-anchored L-OPA1 is proteolytically cleaved to form peripheral S-OPA1, leading to the selection of damaged mitochondria for mitophagy. However, molecular details of the selective mitochondrial fusion are less well understood. Here, we showed that L-OPA1 and cardiolipin (CL) cooperate in heterotypic mitochondrial IM fusion. We reconstituted an in vitro membrane fusion reaction using purified human L-OPA1 protein expressed in silkworm, and found that L-OPA1 on one side of the membrane and CL on the other side are sufficient for fusion. GTP-independent membrane tethering through L-OPA1 and CL primes the subsequent GTP-hydrolysis-dependent fusion, which can be modulated by the presence of S-OPA1. These results unveil the most minimal intracellular membrane fusion machinery. In contrast, independent of CL, a homotypic trans-OPA1 interaction mediates membrane tethering, thereby supporting the cristae structure. Thus, multiple OPA1 functions are modulated by local CL conditions for regulation of mitochondrial morphology and quality control.

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

Mitochondrial morphology dynamically changes in a balance of membrane fusion and fission in response to the environment, cell cycle, and apoptotic stimuli. Here, we report that a novel mitochondrial protein, MICS1, is involved in mitochondrial morphology in specific cristae structures and the apoptotic release of cytochrome c from the mitochondria. MICS1 is an inner membrane protein with a cleavable presequence and multiple transmembrane segments and belongs to the Bi-1 super family. MICS1 down-regulation causes mitochondrial fragmentation and cristae disorganization and stimulates the release of proapoptotic proteins. Expression of the anti-apoptotic protein Bcl-XL does not prevent morphological changes of mitochondria caused by MICS1 down-regulation, indicating that MICS1 plays a role in maintaining mitochondrial morphology separately from the function in apoptotic pathways. MICS1 overproduction induces mitochondrial aggregation and partially inhibits cytochrome c release during apoptosis, regardless of the occurrence of Bax targeting. MICS1 is cross-linked to cytochrome c without disrupting membrane integrity. Thus, MICS1 facilitates the tight association of cytochrome c with the inner membrane. Furthermore, under low-serum condition, the delay in apoptotic release of cytochrome c correlates with MICS1 up-regulation without significant changes in mitochondrial morphology, suggesting that MICS1 individually functions in mitochondrial morphology and cytochrome c release.

Mitochondrial morphology dynamically changes in a balance of membrane fusion and fission in response to the environment, cell cycle, and apoptotic stimuli. Here, we report that a novel mitochondrial protein, MICS1, is involved in mitochondrial morphology in specific cristae structures and the apoptotic release of cytochrome c from the mitochondria. MICS1 is an inner membrane protein with a cleavable presequence and multiple transmembrane segments and belongs to the Bi-1 super family. MICS1 down-regulation causes mitochondrial fragmentation and cristae disorganization and stimulates the release of proapoptotic proteins. Expression of the anti-apoptotic protein Bcl-XL does not prevent morphological changes of mitochondria caused by MICS1 down-regulation, indicating that MICS1 plays a role in maintaining mitochondrial morphology separately from the function in apoptotic pathways. MICS1 overproduction induces mitochondrial aggregation and partially inhibits cytochrome c release during apoptosis, regardless of the occurrence of Bax targeting. MICS1 is cross-linked to cytochrome c without disrupting membrane integrity. Thus, MICS1 facilitates the tight association of cytochrome c with the inner membrane. Furthermore, under low-serum condition, the delay in apoptotic release of cytochrome c correlates with MICS1 up-regulation without significant changes in mitochondrial morphology, suggesting that MICS1 individually functions in mitochondrial morphology and cytochrome c release.

The structural changes in the photoreaction cycle of bacteriorhodopsin, a light-driven proton pump, was investigated at a resolution of 7Å 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 6μs over the time domain of 5μs to 500ms. 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 ∼5ms after a photo-stimulus. In contrast, absorption measurement indicated the deprotonation of the Schiff base predominantly occurred at ∼300μs 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.

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 ϵ-COP in wild-type cells under conditions in which COG-insensitive proteins were unaffected. Furthermore, synthetic phenotypes arose in mutants deficient in both ϵ-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.

D96N bacteriorhodopsin has two photointermediates with the deprotonated Schiff base: the M and MN intermediates. We measure the time-resolved x-ray diffraction of the D96N purple membrane after flash photoexcitation (pH 7.0, 25°C). The data clearly show the M-MN transition during the D96N photocycle. Low-resolution projection maps of these states show that the F helix of the MN intermediate shifts from its original position and this shift is much larger than that of the M intermediate. This indicates that the F helix moves in the M-MN transition of the D96N bacteriorhodopsin photocycle. Moreover, the existence of the MN intermediate in the D96N photocycle under neutral pH indicates that the MN intermediate is not peculiar to the alkaline condition. It is notable that the structural transition of M-MN is independent of the protonation state of the Schiff base. Therefore, the F helix movement precedes reprotonation of the Schiff base in the bacteriorhodopsin photocycle. Our previous study showed that the M-MN transition is hydration-dependent and that the MN intermediate is more hydrated than the M intermediate. Considering this together with the present results, we conclude that the movement of the F helix causes hydration of the cytoplasmic side, which promotes the reprotonation of the Schiff base.

The vacuolar-type H+-ATPase (V-ATPase) translocates protons across membranes. Here, we have identified a mouse cDNA coding for a fourth isoform (a4) of the membrane sector subunit aof V-ATPase. This isoform was specifically expressed in kidney, but not in the heart, brain, spleen, lung, liver, muscle, or testis. Immunoprecipitation experiments, together with sequence similarities for other isoforms (a1, a2, anda3), indicate that the a4 isoform is a component of V-ATPase. Moreover, histochemical studies show thata4 is localized in the apical and basolateral plasma membranes of cortical α- and β-intercalated cells, respectively. These results suggest that the V-ATPase, with the a4 isoform, is important for renal acid/base homeostasis.

We have identified four genes (vha-5, vha-6, vha-7,and unc-32) coding for vacuolar-type proton-translocating ATPase (V-ATPase) subunit ainCaenorhabditis elegans, the first example of four distinct isoforms in eukaryotes. Their products had nine putative transmembrane regions, exhibited 43–60% identity and 62–84% similarity with the bovine subunit a1 isoform, and retained 11 amino acid residues essential for yeast V-ATPase activity (Leng, X. H., Manolson, M. F., and Forgac, M. (1998) J. Biol. Chem.273, 6717–6723). The similarities, together with the results of immunoprecipitation, suggest that these isoforms are components of V-ATPase. Transgenic and immunofluorescence analyses revealed that these genes were strongly expressed in distinct cells;vha-5was strongly expressed in an H-shaped excretory cell,vha-6was strongly expressed in intestine,vha-7was strongly expressed in hypodermis, andunc-32was strongly expressed in nerve cells. Furthermore, the vha-7and unc-32genes were also expressed in the uteri of hermaphrodites. RNA interference analysis showed that the double-stranded RNA for unc-32caused embryonic lethality similar to that seen with other subunit genes (vha-1, vha-4,and vha-11) (Oka, T., and Futai, M. (2000)J. Biol. Chem.275, 29556–29561). The progenies of worms injected with the vha-5or vha-6double-stranded RNA became died at a specific larval stage, whereas thevha-7double-stranded RNA showed no effect on development. These results suggest that V-ATPases with these isoforms generate acidic compartments essential for worm development in a cell-specific manner.

Immunofluorescence analysis indicated that VHA-11, the Csubunit of Caenorhabditis elegansV-ATPase, was localized in dot-like structures around the nuclei of early embryonic cells and was also detected in embryonic intestinal cells after comma stage. Vital staining with acridine orange showed that the intestinal cells had acidic compartments generated by V-ATPase, consistent with the intracellular localization of VHA-11. RNA interference could efficiently silence vha-11gene expression: introduction of vha-11double strand RNA led to embryonic lethality. Worms injected with the vha-11double strand RNA produced embryos that became lethal. The development of embryos was arrested at various stages. However, their numbers gradually decreased, and the worms eventually became sterile due to the failure of ovulation. Similar results were obtained for RNA interference of the V-ATPase proteolipid genes. These results suggest that V-ATPases, and thus inside-acidic organelles, are required for ovulation and embryogenesis.

Vacuolar H+-ATPase (V-ATPase) is a multi-subunit enzyme with a membrane peripheral catalytic (V1) and an intrinsic (Vo) sector. We have identified three cDNA clones coding for isoforms of mouse Vosubunit a(a1, a2, and a3). They exhibit 48–52% identity with each other and high similarity to subunit aof other species. Thea1 isoform was mainly expressed in brain and liver. Thea2 isoform was observed in heart and kidney in addition to brain and liver. Transcripts for the a3 isoform were strongly expressed in heart and liver. The a3 isoform was induced during osteoclast differentiation, and localized in the plasma membrane and cytoplasmic filamentous structures. In contrast toa3, the a1 isoform was constitutively expressed and localized in the cytoplasmic endomembrane compartments of the same cells. These findings suggest that the a3 isoform is a component of the plasma membrane V-ATPase essential for bone resorption.

According to the current structural model of bacteriorhodopsin, Ile222 is located at the cytoplasmic end of helix G. We labeled the single cysteine of the site-directed mutant Ile222 → Cys with p-chloromercuribenzoic acid and determined the position of the labeled mercury by x-ray diffraction in the unphotolyzed state, and in the MN photointermediate accumulated in the presence of guanidine hydrochloride at pH 9.5. According to the difference Fourier maps between the MN intermediate and the unphotolyzed state, the structural change in the MN intermediate was not affected by mercury labeling. The difference Fourier map between the labeled and the unlabeled I222C gave the position of the mercury label. This information was obtained for both the unphotolyzed state and the MN intermediate. We found that the position of the mercury at residue 222 is shifted by 2.1±0.8Å in the MN intermediate. This agrees with earlier results that suggested a structural change in the G helix. The movement of the mercury label is so large that it must originate from a cooperative conformational change in the helix G at its cytoplasmic end, rather than from displacement of residue 222. Because Ile222 is located at the same level on the z coordinate as Asp96, the structural change in the G helix could have the functional role of perturbing the environment and therefore the pKa of this functionally important aspartate.

Abstract: Uniparental inheritance of the chloroplast genome has been observed in a wide variety of green plants. In Chlamydomonas this phenomenon, which can be selectively inhibited by UV irradiation of mt + gametes, has been shown cytologically to be due to the preferential degradation of mt −-derived chloroplast nucleoids in young zygotes. The zygote-specific pair of zys1 genes, zys1A and zys1B, is expressed earliest among five genes isolated from a “10-min” zygote library. We report here that the ZYS1 protein, which is encoded by the invertedly duplicated zys1 gene, accumulates in zygotes and is localized in nuclei. In addition, when mt + gametes (but not mt − gametes) are UV-irradiated before mating, only very limited accumulation of ZYS1 protein can be detected in the resulting zygotes.

We cloned the clh-1 gene coding for a putative ClC chloride channel in Caenorhabditis elegans. The gene product exhibited a high degree of homology with human ClC-1 and ClC-2. The clh-1 gene was predominantly expressed in the hypodermis, including seam cells. Null mutations of clh-1 caused a significantly wider body and an abnormal alae structure. High osmolarity in the culture medium restored the normal body width of the clh-1mutants. These results suggest that the clh-1 gene contributes to maintenance of the body width through regulation of osmolarity.

In the vacuolar-type H+-ATPase (V-ATPase), highly hydrophobic subunits known as the proteolipids are components of the integral membrane V0sector. Previously, we described the identification of three different proteolipid genes inCaenorhabditis elegans(Oka, T., Yamamoto, R., and Futai, M. (1997) J. Biol. Chem.272, 24387–24392):vha-1and vha-2encoded 16-kDa subunits, andvha-4, a 23-kDa isoform. We report here that a third 16-kDa gene, vha-3, has been identified on chromosome IV. This is the first example in which four proteolipid genes have been found in a single organism. vha-2and vha-3exhibited 85% nucleotide identity within the open reading frames which encoded the identical amino acid sequence. Northern blot analysis indicated that all four genes were expressed in a similar pattern during the worm life cycle; however, studies with transgenic worms indicated that thevha-3gene was expressed differently from other proteolipid genes in a cell-specific manner. These results implied that the isoforms of the proteolipids may be related to functional differences of V-ATPases in various cell types.

A cDNA encoding a putative copper chaperone protein, CUC‐1, was cloned from Caenorhabditis elegans. CUC‐1 had the characteristic motifs of MTCXXC and KKTGK, and showed 49.3 and 39.1% sequence identity with yeast Atx1p and human HAH1, respectively. Expression of CUC‐1 cDNA complemented a null atx1mutant, the yeast copper chaperone gene, thus demonstrating that CUC‐1 is a functional copper chaperone. Studies with transgenic worms indicated that cuc‐1and cua‐1, which encodes the copper transporting ATPase, are expressed together in intestinal cells of adult and hypodermal cells in the larvae. cua‐1was also expressed in pharyngeal muscle but cuc‐1was not. These results suggest that CUC‐1 and CUA‐1 constitute a copper trafficking pathway similar to the yeast counterparts in intestinal and hypodermal cells, and CUA‐1 may have a different function in pharyngeal muscle.

A complementary DNA clone preferentially expressed in the gastrointestinal tract was obtained from a rat stomach library. The protein coded by the clone had a single carbohydrate recognition domain having conserved motifs for β-galactoside binding and showed 67% amino acid identity with human galectin-2. The recombinant protein synthesized inEscherichia colicould bind to an asialofetuin column and was eluted with β-galactopyranoside. From these observations, we named the protein rat galectin-2 coded by the cDNA. The rat galectin-2 was predominantly expressed in the epithelial cells of stomach. Thus this protein may form a mucin layer cross-linking with the β-galactoside moiety of glycoproteins.

The proteolipids of the vacuolar-type H+-ATPase (V-ATPase) are major components of the integral membrane sector. The vha-1and vha-2(vacuolar-typeH+-ATPase) genes inCaenorhabditis elegansencode putative 16-kDa proteolipids and are tandemly localized on chromosome III. The vha-2gene has three exons, whereas vha-1has no introns. The deduced amino acid sequences of the two genes exhibit about 60% identity with the homologues from yeast, mouse, and cow. The mRNAs of both vhagenes are trans-spliced to spliced leaders, suggesting that these genes constitute a polycistronic transcriptional unit. The vha-4gene consists of four exons and is very similar to the yeast VMA16gene that codes for the 23-kDa proteolipid. This is the first example of three distinct V-ATPase proteolipids being identified in higher eukaryotes. Northern blot and transgenic analyses show that the three vhagenes may be highly expressed in the H-shaped excretory cell, rectum, and a pair of cells posterior to the anus. These results suggest that the V-ATPase activity may be important for exporting toxic compounds or metabolic wastes in this organism.