Platelet a-granules regulate hemostasis and myriad other physiological processes but their biogenesis is unclear. Mutations in only three proteins are known to cause a-granule defects and bleeding disorders in humans. Two such proteins, VPS16B and VPS33B, form a complex mediating transport of newly synthesized a-granule proteins through megakaryocyte endosomal compartments. It is unclear how the VPS16B/VPS33B complex accomplishes this function. Here we report VPS16B/VPS33B associates physically with Stx12, a SNARE protein that mediates vesicle fusion at endosomes. Importantly, Stx12 deficient megakaryocytes display reduced a-granule numbers and overall levels of a-granule proteins, thus revealing Stx12 as new component of the a-granule biogenesis machinery. VPS16B/VPS33B also binds CCDC22, a component of the CCC complex working at endosome exit sites. CCDC22 competes with Stx12 for binding to VPS16B/VPS33B suggesting a possible hand-off mechanism. Moreover, the major CCC form expressed in megakaryocytes contains COMMD3, one of ten COMMD proteins. Deficiency of COMMD3/CCDC22 causes reduced a-granule numbers and overall levels of a-granule proteins, establishing the COMMD3/CCC complex as a new factor in a-granule biogenesis. Furthermore, P-Selectin traffics through the cell surface in a COMMD3-dependent manner and depletion of COMMD3 results in lysosomal degradation of P-Selectin and PF4. Stx12 and COMMD3/CCC deficiency cause less severe phenotypes than VPS16B/VPS33B deficiency, suggesting Stx12 and COMMD3/CCC assist but are less important than VPS16B/VPS33B in a-granule biogenesis. Mechanistically, our results suggest VPS16B/VPS33B coordinates the endosomal entry and exit of a-granule proteins by linking the fusogenic machinery with a ubiquitous endosomal retrieval complex that is repurposed in megakaryocytes to make a-granules.
368: A specialized complex between SNARE protein syntaxin 17 And CFTR potentiates autophagosomal clearance under stress
