The VPS33B-binding protein VPS16B is required in megakaryocyte and platelet α-granule biogenesis
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- Denisa Urban
- Department of Biochemistry, University of Toronto, Toronto, ON;
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- Ling Li
- Program in Cell Biology, The Hospital for Sick Children, Toronto, ON;
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- Hilary Christensen
- Program in Cell Biology, The Hospital for Sick Children, Toronto, ON;
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- Fred G. Pluthero
- Program in Cell Biology, The Hospital for Sick Children, Toronto, ON;
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- Shao Zun Chen
- Department of Biochemistry, University of Toronto, Toronto, ON;
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- Michael Puhacz
- Department of Biochemistry, University of Toronto, Toronto, ON;
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- Parvesh M. Garg
- Department of Pediatrics, Neonatology Section, The Brody School of Medicine at East Carolina University, Greenville, NC;
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- Kiran K. Lanka
- Department of Pediatrics, Neonatology Section, The Brody School of Medicine at East Carolina University, Greenville, NC;
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- James J. Cummings
- Department of Pediatrics, Neonatology Section, The Brody School of Medicine at East Carolina University, Greenville, NC;
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- Helmut Kramer
- Departments of Neuroscience and Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX;
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- James D. Wasmuth
- Departments of Biochemistry & Molecular Genetics, University of Toronto, Program in Molecular Structure & Function, The Hospital for Sick Children, Toronto, ON; and
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- John Parkinson
- Departments of Biochemistry & Molecular Genetics, University of Toronto, Program in Molecular Structure & Function, The Hospital for Sick Children, Toronto, ON; and
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- Walter H. A. Kahr
- Department of Biochemistry, University of Toronto, Toronto, ON;
説明
<jats:title>Abstract</jats:title> <jats:p>Patients with platelet α or dense δ-granule defects have bleeding problems. Although several proteins are known to be required for δ-granule development, less is known about α-granule biogenesis. Our previous work showed that the BEACH protein NBEAL2 and the Sec1/Munc18 protein VPS33B are required for α-granule biogenesis. Using a yeast two-hybrid screen, mass spectrometry, coimmunoprecipitation, and bioinformatics studies, we identified VPS16B as a VPS33B-binding protein. Immunoblotting confirmed VPS16B expression in various human tissues and cells including megakaryocytes and platelets, and also in megakaryocytic Dami cells. Characterization of platelets from a patient with arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome containing mutations in C14orf133 encoding VPS16B revealed pale-appearing platelets in blood films and electron microscopy revealed a complete absence of α-granules, whereas δ-granules were observed. Soluble and membrane-bound α-granule proteins were reduced or undetectable, suggesting that both releasable and membrane-bound α-granule constituents were absent. Immunofluorescence microscopy of Dami cells stably expressing GFP-VPS16B revealed that similar to VPS33B, GFP-VPS16B colocalized with markers of the trans-Golgi network, late endosomes and α-granules. We conclude that VPS16B, similar to its binding partner VPS33B, is essential for megakaryocyte and platelet α-granule biogenesis.</jats:p>
収録刊行物
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- Blood
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Blood 120 (25), 5032-5040, 2012-12-13
American Society of Hematology