Wilson disease is a disorder of copper metabolism characterized by hepatic cirrhosis and neuronal degeneration due to inherited mutations in a gene encoding a putative copper-transporting P-type ATPase. Polyclonal antisera generated against the amino terminus of the Wilson protein detected a specific 165-kDa protein in HepG2 and CaCo cell lysates. Further analysis revealed that this protein is synthesized as a single-chain polypeptide and localized to the trans-Golgi network under steady state conditions. An increase in the copper concentration resulted in the rapid movement of this protein to a cytoplasmic vesicular compartment. This copper-specific cellular redistribution of the Wilson protein is a reversible process that occurs independent of a new protein synthesis. Expression of the wild-type but not mutant Wilson protein in the ccc2⌬ strain of Saccharomyces cerevisiae restored copper incorporation into the multicopper oxidase Fet3p, providing direct evidence of copper transport by the Wilson protein. Taken together these data reveal a remarkable evolutionary conservation in the cellular mechanisms of copper metabolism and provide a unique model for the regulation of copper transport into the secretory pathway of eucaryotic cells.Copper is an essential trace element that plays a fundamental role in biochemistry, permitting facile electron transfer reactions in diverse metabolic pathways. Despite this essential role, copper is highly reactive and potentially toxic, thus specialized pathways have evolved for the trafficking of this metal within cells (1). This is dramatically illustrated by the genetic disorders Wilson and Menkes disease as well as by recent studies revealing copper-dependent neuronal degeneration in amyotrophic lateral sclerosis and Alzheimer's disease (2, 3). Although little information is currently available about these pathways, the recent cloning of the genes involved in several of these disorders provides the opportunity to elucidate such events at the molecular level.Wilson disease is an inherited disorder resulting in hepatic cirrhosis and neuronal degeneration due to a marked impairment in biliary copper excretion. The Wilson disease gene has been cloned and shown to encode a protein with homology to the cation-transporting P-type ATPase family (4 -6). This family includes a number of membrane proteins that utilize ATPdependent phosphorylation of an invariant aspartate residue to derive energy for cation transport across membranes (7). Gene disruption studies of homologous transporters in procaryotes and yeast have shown that these genes encode proteins required to maintain cellular copper homeostasis (8, 9). Despite these sequence data there is currently no information available about the structure or function of the Wilson disease protein. This current study was undertaken to directly address these issues. EXPERIMENTAL PROCEDURESCell Culture and Antibodies-Cell lines were obtained and cultured as described previously (10). Copper concentration in the media was altered by adding CuS...

Menkes disease is a fatal neurodegenerative disorder of childhood due to the absence or dysfunction of a putative copper-transporting P-type ATPase encoded on the X chromosome. To elucidate the biosynthesis and subcellular localization of this protein, polyclonal antisera were generated against a bacterial fusion protein encoding the 4th to

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Several autophagy proteins contain an LC3-interacting region (LIR) responsible for their interaction with Atg8 homolog proteins. Here, we show that ALFY binds selectively to LC3C and the GABARAPs through a LIR in its WD40 domain. Binding of ALFY to GABARAP is indispensable for its recruitment to LC3B-positive structures and, thus, for the clearance of certain p62 structures by autophagy. In addition, the crystal structure of the GABARAP-ALFY-LIR peptide complex identifies three conserved residues in the GABARAPs that are responsible for binding to ALFY. Interestingly, introduction of these residues in LC3B is sufficient to enable its interaction with ALFY, indicating that residues outside the LIR-binding hydrophobic pockets confer specificity to the interactions with Atg8 homolog proteins.

We study entanglement entropy on the fuzzy sphere. We calculate it in a scalar field theory on the fuzzy sphere, which is given by a matrix model. We use a method that is based on the replica method and applicable to interacting fields as well as free fields. For free fields, we obtain the results consistent with the previous study, which serves as a test of the validity of the method. For interacting fields, we perform Monte Carlo simulations at strong coupling and see a novel behavior of entanglement entropy.

Betalains are one of the major plant pigment groups found in some higher plants and higher fungi. They are not produced naturally in any plant species outside of the order Caryophyllales, nor are they produced by anthocyanin-accumulating Caryophyllales. Here, we attempted to reconstruct the betalain biosynthetic pathway as a self-contained system in an anthocyanin-producing plant species. The combined expressions of a tyrosinase gene from shiitake mushroom and a DOPA 4,5-dioxygenase gene from the four-o'clock plant resulted in successful betalain production in cultured cells of tobacco BY2 and Arabidopsis T87. Transgenic tobacco BY2 cells were bright yellow because of the accumulation of betaxanthins. LC-TOF-MS analyses showed that proline-betaxanthin (Pro-Bx) accumulated as the major betaxanthin in these transgenic BY2 cells. Transgenic Arabidopsis T87 cells also produced betaxanthins, but produced lower levels than transgenic BY2 cells. These results illustrate the success of a novel genetic engineering strategy for betalain biosynthesis.

First-generation adenovirus vectors (FG AdVs) are widely used in basic studies and gene therapy. However, virus-associated (VA) RNAs that act as small-interference RNAs are indeed transcribed from the vector genome. These VA RNAs can trigger the innate immune response. Moreover, VA RNAs are processed to functional viral miRNAs and disturb the expressions of numerous cellular genes. Therefore, VA-deleted AdVs lacking VA RNA genes would be advantageous for basic studies, both in vitro and in vivo. Here, we describe an efficient method of producing VA-deleted AdVs. First, a VA RNA-substituted “pre-vector” lacking the original VA RNA genes but alternatively possessing an intact VA RNA region flanked by a pair of FRTs was constructed. VA-deleted AdVs were efficiently obtained by infecting 293hde12 cells, which highly express FLP, with the pre-vector. The resulting transduction titers of VA-deleted AdVs were sufficient for practical use. Therefore, VA-deleted AdVs may be substitute for current FG AdV.