Dopamine release via the vacuolar ATPase V0 sector c-subunit, confirmed in N18 neuroblastoma cells, results in behavioral recovery in hemiparkinsonian mice.

A 16-kDa proteolipid, mediatophore, in Torpedo electric organs mediates Ca(2+)-dependent acetylcholine release. Mediatophore is identical to the pore-forming stalk c-subunit of the V0 sector of vacuolar proton ATPase (ATP6V0C). The function of ATP6V0C in the mammalian central nervous system is not clear. Here, we report transfection of adeno-associated viral vectors harboring rat ATP6V0C into the mouse substantia nigra, in which high potassium stimulation increased overflow of endogenous dopamine (DA) measured in the striatum by in vivo microdialysis. Next, in the striatum of 6-hydroxydopamine-lesioned mice, a model of Parkinson's disease (PD), human tyrosine hydroxylase, aromatic l-amino-acid decarboxylase and guanosine triphosphate cyclohydrolase 1, together with or without ATP6V0C, were expressed in the caudoputamen for rescue. Motor performance on the accelerating rotarod test and amphetamine-induced ipsilateral rotation were improved in the rescued mice coexpressing ATP6V0C. [(3)H]DA, taken up into cultured N18 neuronal tumor cells transformed to express ATP6V0C, was released by potassium stimulation. These results indicated that ATP6V0C mediates DA release from nerve terminals in the striatum of DA neurons of normal mice and from gene-transferred striatal cells of parkinsonian mice. The results suggested that ATP6V0C may be useful as a rescue molecule in addition to DA-synthetic enzymes in the gene therapy of PD.

Autophagosome-lysosome fusion depends on the pH in acidic compartments in CHO cells.

Autophagy is the bulk degradation of cytoplasmic constituents in response to starvation and other environmental or intracellular cues. During this process, most of the cytoplasm is sequestered into autophagosomes, which then fuse with lysosomes where the degradation of the sequestered material proceeds. We investigated the relationship between autophagosome-lysosome fusion and the pH in acidic compartments by visualizing the fusion process using fluorescence in CHO cells. In this experiment, mitochondria were labeled with GFP by transfecting CHO cells with the presequence of ornithine transcarbamylase, and lysosomes were labeled with Texas Red Dextran; any fusion was identified by the colocalization of mitochondria (in autophagosomes) and lysosomes using fluorescence microscopy. When CHO cells were treated with rapamycin or starvation medium to induce autophagy, the colocalization of fluorescence was observed. Whereas when they were treated with 3-MA, an inhibitor of autophagy, the colocalization disappeared. We conclude that the colocalization reflects the fusion of autophagosomes and lysosomes. Moreover, when the CHO cells were treated with drugs that increase the pH of acidic compartments, the colocalization disappeared. This suggests that the autophagosome-lysosome fusion is inhibited by increasing pH in acidic compartments independently of V-ATPase activity in CHO cells.

Knockdown of mitochondrial heat shock protein 70 promotes progeria-like phenotypes in caenorhabditis elegans.

Mitochondrial heat shock protein 70 (mthsp70) functions as a mitochondrial import motor and is essential in mitochondrial biogenesis and energy generation in eukaryotic cells. HSP-6 (hsp70F) is a nematode orthologue of mthsp70. Knockdown of HSP-6 by RNA interference in young adult nematodes caused a reduction in the levels of ATP-2, HSP-60 and CLK-1, leading to abnormal mitochondrial morphology and lower ATP levels. As a result, RNA interference-treated worms had lower motility, defects in oogenesis, earlier accumulation of autofluorescent material, and a shorter life span. These are the major phenotypes observed during the aging of worms, suggesting that the reduction of HSP-6 causes early aging or progeria-like phenotypes. The amount of HSP-6 became dramatically reduced at the expected mean life span in not only wild-type but also in long and short life span mutant worms (wild-type, daf-2, and daf-16). Mitochondrial HSP-60 and ATP-2 were also reduced following the reduction of HSP-6 during aging. These results suggest that the reduction of HSP-6 causes defects in mitochondrial function at the final stage of aging, leading to mortality.

Quantitative monitoring of autophagic degradation.

We developed a quantitative method for analyzing the induction of autophagy using a CHO-K1 cell line stably expressing a green fluorescent protein (GFP) in mitochondrial matrix (mtGFP-CHO). When mtGFP-CHO cells were incubated with a medium depleted of amino acids and serum, the GFP fluorescence was decreased concomitant with degradation of the protein. Biochemical and morphological analyses strongly suggested the degradation of mtGFP was mediated by bulk and non-selective degradation of mitochondria by autophagy. Quantitative measurement of the mtGFP degradation was performed by measuring the GFP fluorescence and DNA content by a fluorometric method and calculating the relative GFP intensity of DNA content, which approximated mean GFP fluorescence per cell. Using this method, we showed for the first time that different inducers, such as amino acids and serum starvation or rapamycin treatment, promote autophagy with different kinetics. This method is easy, relatively quick, and may be easily adapted to high throughput screening for novel drugs that enhance or inhibit autophagy, and also for genes that regulate or modulate autophagy.

Convergence of cell cycle regulation and growth factor signals on GRASP65.

Together with other Golgi matrix components, GRASP65 contributes to the stacking of Golgi cisternae in interphase cells. During mitosis, GRASP65 is heavily phosphorylated, and in turn, cisternal stacking is inhibited leading to the breakdown of the Golgi apparatus. Here we show that GRASP65 is phosphorylated on serine 277 in interphase cells, and this is strongly enhanced in response to the addition of serum or epidermal growth factor. This is directly mediated by ERK suggesting that GRASP65 has some role in growth factor signal transduction. Phosphorylation of Ser-277 is also dramatically increased during mitosis, however this is mediated by Cdk1 and not by ERK. The microinjection of recombinant GRASP65 without N-terminal myristoylation or a peptide fragment containing Ser-277 into the cytosol of normal rat kidney cells inhibits passage through mitosis. This effect is abolished when Ser-277 is replaced with alanine suggesting the phosphorylation of Ser-277 plays an important role in cell cycle regulation. The convergence of cell cycle regulation and growth factor signals on GRASP65 Ser-277 suggests that GRASP65 may function as a signal integrator controlling the cell growth.

Subunit arrangement in V-ATPase from Thermus thermophilus.

The V0V1-ATPase of Thermus thermophilus catalyzes ATP synthesis coupled with proton translocation. It consists of an ATPase-active V1 part (ABDF) and a proton channel V0 part (CLEGI), but the arrangement of each subunit is still largely unknown. Here we found that acid treatment of V0V1-ATPase induced its dissociation into two subcomplexes, one with subunit composition ABDFCL and the other with EGI. Exposure of the isolated V0 to acid or 8 m urea also produced two subcomplexes, EGI and CL. Thus, the C subunit (homologue of d subunit, yeast Vma6p) associates with the L subunit ring tightly, and I (homologue of 100-kDa subunit, yeast Vph1p), E, and G subunits constitute a stable complex. Based on these observations and our recent demonstration that D, F, and L subunits rotate relative to A3B3 (Imamura, H., Nakano, M., Noji, H., Muneyuki, E., Ohkuma, S., Yoshida, M., and Yokoyama, K. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 2312-2315; Yokoyama, K., Nakano, M., Imamura, H., Yoshida, M., and Tamakoshi, M. (2003) J. Biol. Chem. 278, 24255-24258), we propose that C, D, F, and L subunits constitute the central rotor shaft and A, B, E, G, and I subunits comprise the surrounding stator apparatus in the V0V1-ATPase.

Different roles of proteolipids and 70-kDa subunits of V-ATPase in growth and death of cultured human cells.

BACKGROUND: The vacuolar-type proton-translocating adenosine triphosphatase (V-ATPase) plays important roles in cell growth and tumour progression. V-ATPase is composed of two distinct structures, a hydrophilic catalytic cytosolic sector (V(1)) and a hydrophobic transmembrane sector (V(0)). The V(1) sector is composed of 5-8 different subunits with the structure A(3)B(3)C(1)D(1)E(1)F(1)G(1)H(1). The V0 sector is composed of 5 different subunits with the structure 1161381191166. The over-expression of 16-kDa proteolipid subunit of V-ATPase in the perinuclear region of the human adventitial fibroblasts promotes phenotypic modulation that contributes to neointimal formation and medial thickening. A relationship between oncogenicity and the expression of the 16-kDa proteolipid has also been suggested in human pancreatic carcinoma tissue. RESULTS: We found that the mRNA levels of the 16-kDa proteolipid but not of the 70-kDa subunit of V-ATPase in human myofibroblasts were more abundant in serum-containing medium (MF(+) cells) than serum-free medium (MF(-) cells). In HeLa cells, the levels of mRNA and protein of the 16-kDa, 21-kDa or 70-kDa were clearly suppressed when the corresponding anti-sense oligonucleotides were administered to the culture medium. The growth rate and viability (mostly due to necrosis) of HeLa cells were reduced markedly by the 16-kDa and 21-kDa anti-sense, but little by the 70-kDa anti-sense, and not at all by any sense oligonucleotides. The localization of 16-kDa/21-kDa proteolipid subunits was different from that of the 70-kDa subunit in HeLa cells. CONCLUSION: These results suggest that the 16-kDa and 21-kDa proteolipid subunits of the V0 sector play crucial roles in growth and death of cultured human cells. Our results may provide new insights into the mechanism and therapeutic implications for vessel wall hyperplasia and tumorigenesis.

Existence of catalase-less peroxisomes in Sf21 insect cells.

Catalase activity, a peroxisomal marker enzyme, was not detectable in any of the subcellular fractions of Spodoptera frugiperda (Sf) 21 insect cells, although marker enzymes in other organelles were distributed in the fractions in a manner similar to that seen in mammalian cells. When a green fluorescent protein fused with peroxisome targeting signal 1 at the C-terminal (GFP-SKL) was expressed in Sf21 cells, punctate fluorescent dots were observed in the cytoplasm. The fraction where GFP-SKL was concentrated exhibited long-chain and very-long-chain fatty acid beta-oxidation activities in the presence of KCN and the density of this fraction was slightly higher than that of mitochondria. Immunoelectron microscopy studies with anti-SKL antibody demonstrated that Sf21 cells have immunoreactive peroxisome-like organelles which are structurally distinct from mitochondria, endoplasmic reticulum, and lysosomes. In contrast to peroxisomal matrix proteins, adrenoleukodystrophy protein, a peroxisomal membrane protein, was not located to peroxisomes. This suggests that the targeting signal for PMP in insect cells is distinct from that in mammalian cells. These results demonstrate that Sf21 insect cells have unique catalase-less peroxisomes capable of beta-oxidation of fatty acids.

Evidence for rotation of V1-ATPase.

V(o)V(1)-ATPase is responsible for acidification of eukaryotic intracellular compartments and ATP synthesis of Archaea and some eubacteria. From the similarity to F(o)F(1)-ATP synthase, V(o)V(1)-ATPase has been assumed to be a rotary motor, but to date there are no experimental data to support this. Here we visualized the rotation of single molecules of V(1)-ATPase, a catalytic subcomplex of V(o)V(1)-ATPase. V(1)-ATPase from Thermus thermophilus was immobilized onto a glass surface, and a bead was attached to the D or F subunit through the biotin-streptavidin linkage. In both cases we observed ATP-dependent rotations of beads, the direction of which was always counterclockwise viewed from the membrane side. Given that three ATP molecules are hydrolyzed per one revolution, rates of rotation agree consistently with rates of ATP hydrolysis at saturating ATP concentrations. This study provides experimental evidence that V(o)V(1)-ATPase is a rotary motor and that both D and F subunits constitute a rotor shaft.

In bafilomycin A1-resistant cells, bafilomycin A1 raised lysosomal pH and both prodigiosins and concanamycin A inhibited growth through apoptosis.

In bafilomycin A(1)-resistant cells (Vero-317 and MC-3T3-E1), bafilomycin A(1) neither inhibited cell growth, induced cell death, nor activated caspase-3. However, 100 nM bafilomycin A(1) did raise the lysosomal pH similar to 10 mM NH(4)Cl. Prodigiosins, H(+)/Cl(-) symporters that raise the lysosomal pH, inhibited cell growth through apoptosis and caused the activation of caspase-3. Concanamycin A also inhibited the growth of these cells through apoptosis. 10 mM NH(4)Cl inhibited the growth of these cells as well, but cytostatically. These results suggest that plecomacrolides inhibited cell growth apoptotically through specific site(s), in contrast to the cytostatic effect of 10 mM NH(4)Cl, besides raising the lysosomal pH.

Inhibition of weak-base amine-induced lysis of lysosomes by cytosol.

Certain amines known to be concentrated in lysosomes, termed "lysosomotropic amines," cause the formation of lysosomal vacuoles. A cell-free system was established to examine the effects of basic substances and acidic ionophores. In this system, the drugs not only increased the internal pH, but also caused a disruption of lysosomes. The osmotic swelling of lysosomes induced by protonated bases or cations for particular ionophores, which had accumulated within lysosomes driven by the proton pump, caused the osmotic lysis of lysosomes. The lysosomal disruption was inhibited upon the addition of the cytosol fraction. This phenomenon provides an in vitro system for studying the osmo-regulation and intercellular dynamics of the lysosomal system, including membrane fusion. The lysosomal stabilization factor was purified from the cytosol fraction and identified as ATP-stimulated glucocorticoid receptor translocation promoter (ASTP).

GTP gamma S-stimulated lysosomal lysis dependent on the assembly of adaptor protein on lysosome.

Cytosol treated with guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) destroys the dextran-loaded lysosomes (J. Biochem., 123, 637 (1998)). The transfer of the ADP-ribosylation factor (ARF) from the cytosol to the lysosomal membrane is necessary for this lysis to occur. The role of ARF in the biogenesis of the Golgi complex is to generate high-affinity membrane-binding sites for the heterotetrameric adaptor protein complex in Golgi membranes. We have found that ARF also recruits the adaptor protein to lysosomes. The recruitment of protein coats for vesicles is necessary for the GTPgammaS-stimulated lysis of lysosomes. The GTPgammaS-induced lysis proceeded via a process similar to that for the assembly of coated proteins to coated vesicles, which serve to transport proteins between intracellular organelles.

BE-18591 as a new H(+)/Cl(-) symport ionophore that inhibits immunoproliferation and gastritis.

In our previous papers [e.g. Sato et al., J. Biol. Chem. 273 (1998) 21455-21462], we have shown that prodigiosins can uncouple various H(+)-ATPases through their H(+)/Cl(-) symport activity. BE-18591 is an enamine of 4-methoxy-2,2'-bipyrrole-5-carboxyaldehyde (tambjamine group antibiotics) which resembles the prodigiosins. We found that BE-18591 was a new group of antibiotics that uncouples various H(+)-ATPases: it inhibited proton pump activities with IC(50)s of about 1-2 nM (about 20 pmol/mg protein) for submitochondrial particles as well as gastric vesicles and of 230 nM (about 230 pmol/mg protein) for lysosomes, but it had little effect on their ATP hydrolyses (up to 10 microM), a property of H(+)/Cl(-) symport activity. At low concentrations (<1 microM), BE-18591 inhibited immunoproliferation, the IC(50) of lipopolysaccharide-stimulated mouse splenocytes was 38 nM, that of Concanavalin A-stimulated cells was 230 nM. Gastritis of rabbits was also inhibited. At higher concentrations (>1 microM), BE-18591 induced neurite outgrowth (15% induction in 48 h at 4 microM), inhibited bone resorption (approximately 35% in 48 h at 10 microM) and caused cell death (approximately 30% in 48 h at 4 microM) but with little apoptosis.

Extended longevity of Caenorhabditis elegans by knocking in extra copies of hsp70F, a homolog of mot-2 (mortalin)/mthsp70/Grp75.

The Caenorhabditis elegans homolog of mortalin/mthsp70/Grp75 (called mot-2 hereafter) was isolated by screening of a nematode cDNA library with mouse mot-2 cDNA. The isolated clone matched to hsp70F of C. elegans. Analysis with two of the antibodies raised against hsp70F revealed that unlike mammalian mot-2, it is heat inducible. Transient induction of hsp70F by heat shock led to a slight (<13%) extension in the C. elegans life span. The transgenic worms that constitutively over-expressed hsp70F predominantly in muscle showed life span extension (approximately 43% for mean and approximately 45% for maximum life span) as compared to the wild-type and green fluorescent protein-transgenic worms. Life span extension of human cells was obtained by over-expression of mot-2 [Kaul et al. (2000) FEBS Lett. 474, 159-164]. Our results show, for the first time, that this member of the hsp70 family governs the longevity of worms and thus there are common pathways that determine mammalian and worm longevity.