The VPS33B-binding Protein VPS16B Is Required in Megakaryocyte and Platelet α-granule Biogenesis

Blood. 2012 Dec 13;120(25):5032-40. doi: 10.1182/blood-2012-05-431205. Epub 2012 Sep 21.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arthrogryposis / metabolism
  • Arthrogryposis / pathology
  • Blood Platelets / metabolism
  • Blood Platelets / pathology*
  • Carrier Proteins / analysis
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • Cell Line
  • Cholestasis / metabolism
  • Cholestasis / pathology
  • Chromosomes, Human, Pair 14 / genetics
  • Chromosomes, Human, Pair 14 / metabolism
  • Codon, Nonsense
  • Female
  • Golgi Apparatus / ultrastructure
  • HEK293 Cells
  • Humans
  • Infant, Newborn
  • Megakaryocytes / metabolism
  • Megakaryocytes / pathology*
  • Open Reading Frames
  • Phylogeny
  • Protein Binding
  • Renal Insufficiency / metabolism
  • Renal Insufficiency / pathology
  • Secretory Vesicles / metabolism*
  • Secretory Vesicles / pathology
  • Vesicular Transport Proteins / metabolism*

Substances

  • Carrier Proteins
  • Codon, Nonsense
  • VIPAS39 protein, human
  • VPS33B protein, human
  • Vesicular Transport Proteins

Supplementary concepts

  • Arthrogryposis renal dysfunction cholestasis syndrome