Biochemical characterization and intracellular localization of the Menkes disease protein
- 26 November 1996
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 93 (24) , 14030-14035
- https://doi.org/10.1073/pnas.93.24.14030
Abstract
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 6th copper-binding domains in the amino terminus of the human Menkes protein. RNA blot analysis revealed abundant Menkes gene expression in several cell lines, and immunoblotting studies utilizing this antiserum readily detected a 178-kDa protein in lysates from these cells. Pulse–chase studies indicate that this protein is synthesized as a single-chain polypeptide which is modified by N-linked glycosylation to a mature endoglycosidase H-resistant form. Sucrose gradient fractionation of HeLa cell lysates followed by immunoblotting of individual fractions with antibodies to proteins of known intracellular location identified the Menkes ATPase in fractions similar to those containing the cation-independent mannose-6-phosphate receptor. Consistent with this observation, confocal immunofluorescence studies of these same cells localized this protein to the trans-Golgi network and a vesicular compartment with no expression in the nucleus or on the plasma membrane. Taken together, these data provide a unique model of copper transport into the secretory pathway of mammalian cells which is compatible with clinical observations in affected patients and with recent data on homologous proteins identified in prokaryotes and yeast.Keywords
This publication has 42 references indexed in Scilit:
- A murine model of Menkes disease reveals a physiological function of metallothioneinNature Genetics, 1996
- Molecular Structure of the Menkes Disease Gene (ATP7A)Genomics, 1995
- Dithiothreitol Treatment of Madin-Darby Canine Kidney Cells Reversibly Blocks Export from the Endoplasmic Reticulum but Does Not Affect Vectorial Targeting of Secretory ProteinsPublished by Elsevier ,1995
- Characterization of the exon structure of the Menkes disease gene using vectorette PCRGenomics, 1995
- Isolation and Characterization of a Human Liver cDNA as a Candidate Gene for Wilson DiseaseBiochemical and Biophysical Research Communications, 1993
- Human Menkes X‐chromosome disease and the staphylococcal cadmium‐resistance ATPase: a remarkable similarity in protein sequencesMolecular Microbiology, 1993
- Relations between the intracellular pathways of the receptors for transferrin, asialoglycoprotein, and mannose 6-phosphate in human hepatoma cells.The Journal of cell biology, 1989
- Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferaseGene, 1988
- The mannose 6-phosphate receptor and the biogenesis of lysosomesCell, 1988
- Isolation of biologically active ribonucleic acid from sources enriched in ribonucleaseBiochemistry, 1979