PI4KIIa and VAMP3 as Coordinate Regulators of Endocytic Transport

Wednesday, October 10, 2012 — Poster Session II
Noon – 2:00 p.m	Natcher Conference Center, Building 45	NICHD	CELLBIO-4

Authors

• M. Jovic
• M. Kean
• A. C. Gingras
• J. Brill
• T. Balla

Abstract

Phosphatidylinositol 4-kinases (PI4Ks) are essential regulators of secretory and degradative transport. The importance of PI4K localization to trans-Golgi and endosomes is underscored by its role in EGF receptor degradation, Wnt signaling, and its suspected role in the late-onset neurodegeneration In previous proteomic and biochemical studies we have identified an interaction between PI4KIIa and the vesicular fusion protein VAMP-3. Here, we demonstrate the physiological relevance of this interaction in live cells using a fluorescence recovery after photobleaching (FRAP) approach. Expression of Tetanus toxin (TeNT), which specifically cleaves VAMP-3, resulted in accumulation of PI4KIIa on endosomes rather then the Golgi. Coexpression of TeNT with TeNT-resistant VAMP-3 mutant recovered Golgi localization of PI4KIIa. Retrograde trafficking of PI4KIIa to the pericentriolar Golgi area following photobleaching was significantly decreased in the presence of TeNT. Conversely, PI4KIIa was also found to be critical for VAMP-3 distribution. Knockdown of the kinase or depletion of phosphatidylinositol 4-phosphate, significantly reduced the rate of VAMP-3 trafficking to the perinuclear region. Furthermore, PI4KIIa depletion reduced the trafficking rate of transferrin receptor, a cargo that requires VAMP-3-mediated fusion. Collectively, these data suggest that VAMP-3 and PI4KIIa are coordinate regulators of endocytic transport, revealing new aspects of phosphoinositide regulation of SNARE-mediated fusion.

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