Localization of the Wilson’s disease protein product to mitochondria

Wilson’s disease (WND) is an inherited disorder of copper homeostasis characterized by abnormal accumulation of copper in several tissues, particularly in the liver, brain, and kidney. The disease-associated gene encodes a copper-transporting P-type ATPase, the WND protein, the subcellular location of which could be regulated by copper. We demonstrate that the WND protein is present in cells in two forms, the 160-kDa and the 140-kDa products. The 160-kDa product was earlier shown to be targeted to trans-Golgi network. The 140-kDa product identified herein is located in mitochondria as evidenced by the immunofluorescent staining of HepG2 cells with specific mitochondria markers and polyclonal antibody directed against the C terminus of the WND molecule. The mitochondrial location for the 140-kDa WND product was confirmed by membrane fractionation and by analysis of purified human mitochondria. The antibody raised against a repetitive sequence in the N-terminal portion of the WND molecule detects an additional 16-kDa protein, suggesting that the 140-kDa product was formed after proteolytic cleavage of the full-length WND protein at the N terminus. Thus, the WND protein is a P-type ATPase with an unusual subcellular localization. The mitochondria targeting of the WND protein suggests its important role for copper-dependent processes taking place in this organelle.

Raft association of SNAP receptors acting in apical trafficking in Madin–Darby canine kidney cells

We have investigated the relationships between the apical sorting mechanism using lipid rafts and the soluble N-ethyl maleimide-sensitive factor attachment protein receptor (SNARE) machinery, which is involved in membrane docking and fusion. We first confirmed that anti-alpha-SNAP antibodies inhibit the apical pathway in Madin– Darby canine kidney (MDCK) cells; in addition, we report that a recombinant SNAP protein stimulates the apical transport whereas a SNAP mutant inhibits this transport step. Based on t-SNARE overexpression experiments and the effect of botulinum neurotoxin E, syntaxin 3 and SNAP-23 have been implicated in apical membrane trafficking. Here, we show in permeabilized MDCK cells that antisyntaxin 3 and anti-SNAP-23 antibodies lower surface delivery of an apical reporter protein. Moreover, using a similar approach, we show that tetanus toxin-insensitive, vesicle-associated membrane protein (TI-VAMP; also called VAMP7), a recently described apical v-SNARE, is involved. Furthermore, we show the presence of syntaxin 3 and TI-VAMP in isolated apical carriers. Polarized apical sorting has been postulated to be mediated by the clustering of apical proteins into dynamic sphingolipid-cholesterol rafts. We provide evidence that syntaxin 3 and TI-VAMP are raft-associated. These data support a raft-based mechanism for the sorting of not only apically destined cargo but also of SNAREs having functions in apical membrane-docking and fusion events.

Secondary absence of mitochondria in Giardia lamblia and Trichomonas vaginalis revealed by valyl-tRNA synthetase phylogeny

Nuclear-coded valyl-tRNA synthetase (ValRS) of eukaryotes is regarded of mitochondrial origin. Complete ValRS sequences obtained by us from two amitochondriate protists, the diplomonad, Giardia lamblia and the parabasalid, Trichomonas vaginalis were of the eukaryotic type, strongly suggesting an identical history of ValRS in all eukaryotes studied so far. The findings indicate that diplomonads are secondarily amitochondriate and give further evidence for such conclusion reached recently concerning parabasalids. Together with similar findings on other amitochondriate groups (microsporidia and entamoebids), this work provides critical support for the emerging notion that no representatives of the premitochondrial stage of eukaryotic phylogenesis exist among the species living today.

A cytomegalovirus-encoded mitochondria-localized inhibitor of apoptosis structurally unrelated to Bcl-2

Human cytomegalovirus (CMV), a herpesvirus that causes congenital disease and opportunistic infections in immunocompromised individuals, encodes functions that facilitate efficient viral propagation by altering host cell behavior. Here we show that CMV blocks apoptosis mediated by death receptors and encodes a mitochondria-localized inhibitor of apoptosis, denoted vMIA, capable of suppressing apoptosis induced by diverse stimuli. vMIA, a product of the viral UL37 gene, inhibits Fas-mediated apoptosis at a point downstream of caspase-8 activation and Bid cleavage but upstream of cytochrome c release, while residing in mitochondria and associating with adenine nucleotide translocator. These functional properties resemble those ascribed to Bcl-2; however, the absence of sequence similarity to Bcl-2 or any other known cell death suppressors suggests that vMIA defines a previously undescribed class of anti-apoptotic proteins.

Molecular cloning of a peroxisomal Ca2+-dependent member of the mitochondrial carrier superfamily

A cDNA from a novel Ca2+-dependent member of the mitochondrial solute carrier superfamily was isolated from a rabbit small intestinal cDNA library. The full-length cDNA clone was 3,298 nt long and coded for a protein of 475 amino acids, with four elongation factor-hand motifs located in the N-terminal half of the molecule. The 25-kDa N-terminal polypeptide was expressed in Escherichia coli, and it was demonstrated that it bound Ca2+, undergoing a reversible and specific conformational change as a result. The conformation of the polypeptide was sensitive to Ca2+ which was bound with high affinity (Kd ≈ 0.37 μM), the apparent Hill coefficient for Ca2+-induced changes being about 2.0. The deduced amino acid sequence of the C-terminal half of the molecule revealed 78% homology to Grave disease carrier protein and 67% homology to human ADP/ATP translocase; this sequence homology identified the protein as a new member of the mitochondrial transporter superfamily. Northern blot analysis revealed the presence of a single transcript of about 3,500 bases, and low expression of the transporter could be detected in the kidney but none in the liver. The main site of expression was the colon with smaller amounts found in the small intestine proximal to the ileum. Immunoelectron microscopy localized the transporter in the peroxisome, although a minor fraction was found in the mitochondria. The Ca2+ binding N-terminal half of the transporter faces the cytosol.

Na,K-ATPase transport from endoplasmic reticulum to Golgi requires the Golgi spectrin–ankyrin G119 skeleton in Madin Darby canine kidney cells

Spectrin (βIΣ∗) and ankyrin (AnkG119) associate with Golgi membranes and the dynactin complex, but their role in vesicle trafficking remains uncertain. We find that the actin-binding domain and membrane-association domain 1 (MAD1) of βI spectrin together form a constitutive Golgi targeting signal in transfected MDCK cells. Expression of this signal in transfected cells disrupts the endogenous Golgi spectrin skeleton and blocks transport of α- and β-Na,K-ATPase and vesicular stomatitis virus-G protein from the endoplasmic reticulum (ER) but does not disrupt the formation of Golgi stacks, the distribution of β-COP, or the transport and surface display of E-cadherin. The Golgi spectrin skeleton is thus required for the transport of a subset of membrane proteins from the ER to the Golgi. We postulate that together with polyfunctional adapter proteins such as AnkG119, Golgi spectrin forms a docking complex that acts prior to the cis-Golgi, presumably with vesicular–tubular clusters (VTCs or ERGIC), to sequester specific membrane proteins into vesicles transiting between the ER and Golgi, and subsequently (probably involving other isoforms of spectrin and ankyrin) to mediate cargo transport within the Golgi and to other membrane compartments. We hypothesize that this vesicular spectrin–ankyrin adapter-protein trafficking (or tethering) system (SAATS) mediates the capture and transport of many membrane proteins and acts in conjunction with vesicle-targeting molecules to effect the efficient transport of cargo proteins.

The cluA− mutant of Dictyostelium identifies a novel class of proteins required for dispersion of mitochondria

The cluA gene of Dictyostelium discoideum encodes a novel 150-kDa protein. Disruption of cluA results in clustering of mitochondria near the cell center. This is a striking difference from normal cells, whose mitochondria are dispersed uniformly throughout the cytoplasm. The mutant cell populations also exhibit an increased frequency of multinucleated cells, suggesting an impairment in cytokinesis. Both phenotypes are reversed by transformation of cluA− cells with a plasmid carrying a constitutively expressed cluA gene. The predicted sequence of the cluA gene product is homologous to sequences encoded by open reading frames in the genomes of Saccharomyces cerevisiae and Caenorhabditis elegans, but not to any known protein. The only exception is a short region with some homology to the 42-residue imperfect repeats present in the kinesin light chain, which probably function in protein–protein interaction. These studies identify a new class of proteins that appear to be required for the proper distribution of mitochondria.

Transfection of rat kidney with human 15-lipoxygenase suppresses inflammation and preserves function in experimental glomerulonephritis

The human 15-lipoxygenase (15-LO) gene was transfected into rat kidneys in vivo via intra-renal arterial injection. Three days later, acute (passive) or accelerated forms of antiglomerular basement membrane antibody-mediated glomerulonephritis were induced in transfected and nontransfected or sham-transfected controls. Studies of glomerular functions (filtration and protein excretion) and ex vivo glomerular leukotriene B4 biosynthesis at 3 hr, and up to 4 days, after induction of nephritis revealed preservation or normalization of these parameters in transfected kidneys that expressed human 15-LO mRNA and mature protein, but not in contralateral control kidneys or sham-transfected animals. The results provide in vivo-derived data supporting a direct anti-inflammatory role for 15-LO during immune-mediated tissue injury.

Explosive invasion of plant mitochondria by a group I intron

Group I introns are mobile, self-splicing genetic elements found principally in organellar genomes and nuclear rRNA genes. The only group I intron known from mitochondrial genomes of vascular plants is located in the cox1 gene of Peperomia, where it is thought to have been recently acquired by lateral transfer from a fungal donor. Southern-blot surveys of 335 diverse genera of land plants now show that this intron is in fact widespread among angiosperm cox1 genes, but with an exceptionally patchy phylogenetic distribution. Four lines of evidence—the intron’s highly disjunct distribution, many incongruencies between intron and organismal phylogenies, and two sources of evidence from exonic coconversion tracts—lead us to conclude that the 48 angiosperm genera found to contain this cox1 intron acquired it by 32 separate horizontal transfer events. Extrapolating to the over 13,500 genera of angiosperms, we estimate that this intron has invaded cox1 genes by cross-species horizontal transfer over 1,000 times during angiosperm evolution. This massive wave of lateral transfers is of entirely recent occurrence, perhaps triggered by some key shift in the intron’s invasiveness within angiosperms.

Withdrawal of IL-7 induces Bax translocation from cytosol to mitochondria through a rise in intracellular pH

IL-7 functions as a trophic factor during T lymphocyte development by a mechanism that is partly based on the induction of Bcl-2, which protects cells from apoptosis. Here we report a mechanism by which cytokine withdrawal activates the prodeath protein Bax. On loss of IL-7 in a dependent cell line, Bax protein translocated from the cytosol to the mitochondria, where it integrated into the mitochondrial membrane. This translocation was attributable to a conformational change in the Bax protein itself. We show that a rise in intracellular pH preceded mitochondrial translocation and triggered the change in Bax conformation. Intracellular pH in the IL-7-dependent cells rose steadily to peak over pH 7.8 by 6 hr after cytokine withdrawal, paralleling the time point of Bax translocation (a similar alkalinization and Bax translocation was also observed after IL-3 withdrawal from a dependent cell line). The conformation of Bax was directly altered by pH of 7.8 or higher and was demonstrated by increased protease sensitivity, exposure of N terminus epitopes, and exposure of a hydrophobic domain in the C terminus. Eliminating charged amino acids at the C or N termini of Bax induced a conformational change similar to that induced by raising pH, implicating these residues in the pH effect. Therefore, we have shown that by either cytokine withdrawal, experimental manipulation of pH, or site-directed mutagenesis, Bax protein changes conformation, exposing membrane-seeking domains, thereby inducing mitochondrial translocation and initiating the cascade of events leading to apoptotic death.

Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria

Cytochrome c release and the mitochondrial permeability transition (PT), including loss of the transmembrane potential (Δψ), play an important role in apoptosis. Using isolated mitochondria, we found that recombinant Bax and Bak, proapoptotic members of the Bcl-2 family, induced mitochondrial Δψ loss, swelling, and cytochrome c release. All of these changes were dependent on Ca2+ and were prevented by cyclosporin A (CsA) and bongkrekic acid, both of which close the PT pores (megachannels), indicating that Bax- and Bak-induced mitochondrial changes were mediated through the opening of these pores. Bax-induced mitochondrial changes were inhibited by recombinant Bcl-xL and transgene-derived Bcl-2, antiapoptotic members of the Bcl-2 family, as well as by oligomycin, suggesting a possible regulatory effect of F0F1-ATPase on Bax-induced mitochondrial changes. Proapoptotic Bax- and Bak-BH3 (Bcl-2 homology) peptides, but not a mutant BH3 peptide nor a mutant Bak lacking BH3, induced the mitochondrial changes, indicating an essential role of the BH3 region. A coimmunoprecipitation study revealed that Bax and Bak interacted with the voltage-dependent anion channel, which is a component of PT pores. Taken together, these findings suggest that proapoptotic Bcl-2 family proteins, including Bax and Bak, induce the mitochondrial PT and cytochrome c release by interacting with the PT pores.

Epigenetic changes at the insulin-like growth factor II/H19 locus in developing kidney is an early event in Wilms tumorigenesis

Relaxation of imprinting at the insulin-like growth factor II (IFG-II)/H19 locus is a major mechanism involved in the onset of sporadic Wilms tumor and several other embryonal tumors. The high prevalence of histologically abnormal foci in kidney adjacent to Wilms tumors suggests that tumor-predisposing genetic/epigenetic lesion might also be found at high frequency in Wilms tumor-bearing kidneys. Focusing on Wilms tumors with relaxation of IFG-II imprinting, we determined the frequency of epigenetic change at the IFG-II/H19 locus in adjacent kidney. In all kidneys adjacent to these Wilms tumors, we detected substantial mosaicism for a population of cells with relaxation of IFG-II imprinting and biallelic H19 methylation, regardless of whether the patient had a tumor-predisposing syndrome or not. The high proportion of epigenetically modified cells among “normal” tissue indicates that the epigenetic error occurred very early in development, before the onset of Wilms tumor. Not only does this suggest that the major Wilms tumor-predisposing event occurs within the first few days of development, but it also suggests that sporadic Wilms tumor may represent one end of a spectrum of overgrowth disorders characterized by mosaic epigenetic change at the IFG-II/H19 locus.

RNA editing in Arabidopsis mitochondria effects 441 C to U changes in ORFs

On the basis of the sequence of the mitochondrial genome in the flowering plant Arabidopsis thaliana, RNA editing events were systematically investigated in the respective RNA population. A total of 456 C to U, but no U to C, conversions were identified exclusively in mRNAs, 441 in ORFs, 8 in introns, and 7 in leader and trailer sequences. No RNA editing was seen in any of the rRNAs or in several tRNAs investigated for potential mismatch corrections. RNA editing affects individual coding regions with frequencies varying between 0 and 18.9% of the codons. The predominance of RNA editing events in the first two codon positions is not related to translational decoding, because it is not correlated with codon usage. As a general effect, RNA editing increases the hydrophobicity of the coded mitochondrial proteins. Concerning the selection of RNA editing sites, little significant nucleotide preference is observed in their vicinity in comparison to unedited C residues. This sequence bias is, per se, not sufficient to specify individual C nucleotides in the total RNA population in Arabidopsis mitochondria.

Association of Rab25 and Rab11a with the Apical Recycling System of Polarized Madin–Darby Canine Kidney Cells

Recent evidence suggests that apical and basolateral endocytic pathways in epithelia converge in an apically located, pericentriolar endosomal compartment termed the apical recycling endosome. In this compartment, apically and basolaterally internalized membrane constituents are thought to be sorted for recycling back to their site of origin or for transcytosis to the opposite plasma membrane domain. We report here that in the epithelial cell line Madin–Darby Canine Kidney (MDCK), antibodies to Rab11a label an apical pericentriolar endosomal compartment that is dependent on intact microtubules for its integrity. Furthermore, this compartment is accessible to a membrane-bound marker (dimeric immunoglobulin A [IgA]) internalized from either the apical or basolateral pole, functionally defining it as the apical recycling endosome. We have also examined the role of a closely related epithelial-specific Rab, Rab25, in the regulation of membrane recycling and transcytosis in MDCK cells. When cDNA encoding Rab25 was transfected into MDCK cells, the protein colocalized with Rab11a in subapical vesicles. Rab25 transfection also altered the distribution of Rab11a, causing the coalescence of immunoreactivity into multiple denser vesicular structures not associated with the centrosome. Nevertheless, nocodazole still dispersed these vesicles, and dimeric IgA internalized from either the apical or basolateral membrane was detected in endosomes labeled with antibodies to both Rab11a and Rab25. Overexpression of Rab25 decreased the rate of IgA transcytosis and of apical, but not basolateral, recycling of internalized ligand. Conversely, expression of the dominant-negative Rab25T26N did not alter either apical recycling or transcytosis. These results indicate that both Rab11a and Rab25 associate with the apical recycling system of epithelial cells and suggest that Rab25 may selectively regulate the apical recycling and/or transcytotic pathways.

Urokinase-Type Plasminogen Activator Receptor Is Internalized by Different Mechanisms in Polarized and Nonpolarized Madin–Darby Canine Kidney Epithelial Cells

Accumulated data indicate that endocytosis of the glycosylphosphatidyl-inositol-anchored protein urokinase plasminogen activator receptor (uPAR) depends on binding of the ligand uPA:plasminogen activator inhibitor-1 (PAI-1) and subsequent interaction with internalization receptors of the low-density lipoprotein receptor family, which are internalized through clathrin-coated pits. This interaction is inhibited by receptor-associated protein (RAP). We show that uPAR with bound uPA:PAI-1 is capable of entering cells in a clathrin-independent process. First, HeLaK44A cells expressing mutant dynamin efficiently internalized uPA:PAI-1 under conditions in which transferrin endocytosis was blocked. Second, in polarized Madin–Darby canine kidney (MDCK) cells, which expressed human uPAR apically, the low basal rate of uPAR ligand endocytosis, which could not be inhibited by RAP, was increased by forskolin or phorbol ester (phorbol 12-myristate 13-acetate), which selectively up-regulate clathrin-independent endocytosis from the apical domain of epithelial cells. Third, in subconfluent nonpolarized MDCK cells, endocytosis of uPA:PAI-1 was only decreased marginally by RAP. At the ultrastructural level uPAR was largely excluded from clathrin-coated pits in these cells and localized in invaginated caveolae only in the presence of cross-linking antibodies. Interestingly, a larger fraction of uPAR in nonpolarized relative to polarized MDCK cells was insoluble in Triton X-100 at 0°C, and by surface labeling with biotin we also show that internalized uPAR was mainly detergent insoluble, suggesting a correlation between association with detergent-resistant membrane microdomains and higher degree of clathrin-independent endocytosis. Furthermore, by cryoimmunogold labeling we show that 5–10% of internalized uPAR in nonpolarized, but not polarized, MDCK cells is targeted to lysosomes by a mechanism that is regulated by ligand occupancy.