Jović Marko, Kean Michelle J, Dubankova Anna, Boura Evzen, Gingras Anne-Claude, Brill Julie A, Balla Tamas
Section on Molecular Signal Transduction, Program for Developmental Neuroscience, NICHD, NIH, Bethesda, MD 20892, USA
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.
J Cell Sci. 2014 Sep 1;127(Pt 17):3745-56. doi: 10.1242/jcs.148809. Epub 2014 Jul 7.
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.
囊泡运输中膜融合的特异性取决于可溶性N - 乙基马来酰亚胺敏感因子附着蛋白受体（SNAREs）在亚细胞中的正确分布。尽管SNARE复合物在体外相当混杂，但由于SNARE基序在与同源Q - SNARE结合之前的空间隔离和屏蔽，在细胞中实现了显著的特异性。在本研究中，我们鉴定出磷脂酰肌醇4 - 激酶IIα（PI4K2A）是囊泡相关膜蛋白3（VAMP3）的结合伴侣，VAMP3是一种参与再循环和逆行运输的小R - SNARE，并发现这两种蛋白共同存在于管状囊泡内体上。PI4K2A敲低抑制了VAMP3向核周膜的运输，并损害了VAMP3介导的转铁蛋白受体再循环速率。此外，PI4K2A的缺失显著降低了VAMP3与其同源Q - SNARE Vti1a的结合。虽然VAMP3与PI4K2A的结合不需要激酶活性，但内体上磷脂酰肌醇4 - 磷酸（PtdIns4P）的急性缺失显著延迟了VAMP3的运输。此前基于体外研究提出了磷脂对SNARE功能的调节作用，我们的研究通过鉴定PI4K2A和PtdIns4P作为完整细胞中R - SNARE的调节因子，为这些说法提供了机制证据。
