The VPS33B-binding protein VPS16B is required in megakaryocyte and platelet ?-granule biogenesis.

ABSTRACT: Patients with platelet ? or dense ?-granule defects have bleeding problems. Although several proteins are known to be required for ?-granule development, less is known about ?-granule biogenesis. Our previous work showed that the BEACH protein NBEAL2 and the Sec1/Munc18 protein VPS33B are required for ?-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 ?-granules, whereas ?-granules were observed. Soluble and membrane-bound ?-granule proteins were reduced or undetectable, suggesting that both releasable and membrane-bound ?-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 ?-granules. We conclude that VPS16B, similar to its binding partner VPS33B, is essential for megakaryocyte and platelet ?-granule biogenesis.

SUBMITTER: Urban D 

PROVIDER: S-EPMC3538988 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature