title: The VPS33B-binding protein VPS16B is required in megakaryocyte and platelet α-granule biogenesis.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Denisa
authors.initials: D
authors.lastname: Urban
authors.forename: Ling
authors.initials: L
authors.lastname: Li
authors.forename: Hilary
authors.initials: H
authors.lastname: Christensen
authors.forename: Fred G
authors.initials: FG
authors.lastname: Pluthero
authors.forename: Shao Zun
authors.initials: SZ
authors.lastname: Chen
authors.forename: Michael
authors.initials: M
authors.lastname: Puhacz
authors.forename: Parvesh M
authors.initials: PM
authors.lastname: Garg
authors.forename: Kiran K
authors.initials: KK
authors.lastname: Lanka
authors.forename: James J
authors.initials: JJ
authors.lastname: Cummings
authors.forename: Helmut
authors.initials: H
authors.lastname: Kramer
authors.forename: James D
authors.initials: JD
authors.lastname: Wasmuth
authors.forename: John
authors.initials: J
authors.lastname: Parkinson
authors.forename: Walter H A
authors.initials: WH
authors.lastname: Kahr
mesh_terms: D001176:Arthrogryposis / Q000378:metabolism / Q000473:pathology; D001792:Blood Platelets / Q000378:metabolism / Q000473:pathology*; D002352:Carrier Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002779:Cholestasis / Q000378:metabolism / Q000473:pathology; D002883:Chromosomes, Human, Pair 14 / Q000235:genetics / Q000378:metabolism; D018389:Codon, Nonsense; D005260:Female; D006056:Golgi Apparatus / Q000648:ultrastructure; D057809:HEK293 Cells; D006801:Humans; D007231:Infant, Newborn; D008533:Megakaryocytes / Q000378:metabolism / Q000473:pathology*; D016366:Open Reading Frames; D010802:Phylogeny; D011485:Protein Binding; D051437:Renal Insufficiency / Q000378:metabolism / Q000473:pathology; D022142:Secretory Vesicles / Q000378:metabolism* / Q000473:pathology; D033921:Vesicular Transport Proteins / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D018389:Codon, Nonsense; C539711:VIPAS39 protein, human; C411584:VPS33B protein, human; D033921:Vesicular Transport Proteins