Specificity of membrane fusion in vesicular trafficking is dependent on proper subcellular distribution of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). Although SNARE complexes are fairly promiscuous in vitro, substantial specificity is achieved in cells owing to the spatial segregation and shielding of SNARE motifs prior to association with cognate Q-SNAREs. In this study, we identified phosphatidylinositol 4-kinase IIα (PI4K2A) as a binding partner of vesicle-associated membrane protein 3 (VAMP3), a small R-SNARE involved in recycling and retrograde transport, and found that the two proteins co-reside on tubulo-vesicular endosomes. PI4K2A knockdown inhibited VAMP3 trafficking to perinuclear membranes and impaired the rate of VAMP3-mediated recycling of the transferrin receptor. Moreover, depletion of PI4K2A significantly decreased association of VAMP3 with its cognate Q-SNARE Vti1a. Although binding of VAMP3 to PI4K2A did not require kinase activity, acute depletion of phosphatidylinositol 4-phosphate (PtdIns4P) on endosomes significantly delayed VAMP3 trafficking. Modulation of SNARE function by phospholipids had previously been proposed based on in vitro studies, and our study provides mechanistic evidence in support of these claims by identifying PI4K2A and PtdIns4P as regulators of an R-SNARE in intact cells.

Department of Molecular Genetics University of Toronto 1 King's College Circle Toronto ON M5S 1A8 Canada Program in Cell Biology The Hospital for Sick Children PGCRL 686 Bay Street Toronto ON M5G 0A4 Canada

Institute of Organic Chemistry and Biochemistry AS CR v v i Flemingovo nam 2 166 10 Prague 6 Czech Republic

Samuel Lunenfeld Research Institute 600 University Avenue Toronto ON M5G 1X5 Canada Department of Molecular Genetics University of Toronto 1 King's College Circle Toronto ON M5S 1A8 Canada

Section on Molecular Signal Transduction Program for Developmental Neuroscience NICHD NIH Bethesda MD 20892 USA

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A large scale high-throughput screen identifies chemical inhibitors of phosphatidylinositol 4-kinase type II alpha

The crystal structure of the phosphatidylinositol 4-kinase IIα