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MBC in Press, published online ahead of print June 8, 2005
Mol. Biol. Cell 10.1091/mbc.E05-01-0020

A more recent version of this article appeared on August 1, 2005
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Submitted on January 10, 2005
Revised on May 11, 2005
Accepted on May 31, 2005

Phosphatidylinositol-4-Kinase Type II Alpha Is a Component of AP-3-derived Vesicles

Gloria Salazar,*{dagger} Branch Craige,*{dagger}{ddagger} Bruce H. Wainer,{sect} Jun Guo,|| Pietro De Camilli,|| and Victor Faundez*¶

Departments of *Cell Biology and {sect}Pathology and Laboratory Medicine, {ddagger}Graduate Division of Biological and Biomedical Sciences, and Center for Neurodegenerative Disease, Emory University, Atlanta, GA 30322; ||Howard Hughes Medical Institute and Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510

Monitoring Editor: Keith Mostov

A membrane fraction enriched in vesicles containing the AP-3 cargo ZnT3 was generated from PC12 cells, and was used to identify new components of these organelles by mass spectrometry. Proteins prominently represented in the fraction included AP-3 subunits, synaptic vesicle proteins, and lysosomal proteins known to be sorted in an AP-3-dependent way or to interact genetically with AP-3. A protein enriched in this fraction was phosphatidylinositol-4-kinase type II{alpha} (PI4KII{alpha}). Biochemical, pharmacological, and morphological analyses supported the presence of PI4KII{alpha} in AP-3-positive organelles. Furthermore, the subcellular localization of PI4KII{alpha} was altered in cells from AP-3 deficient mocha mutant mice. The PI4KII{alpha} normally present both in perinuclear and peripheral organelles was substantially decreased in the peripheral membranes of AP-3 deficient mocha fibroblasts. In addition, as is the case for other proteins sorted in an AP-3 dependent way, PI4KII{alpha} content was strongly reduced in nerve terminals of mocha hippocampal mossy fibers. The functional relationship between AP-3 and PI4KII{alpha} was further explored by PI4KII{alpha} knock-down experiments. Reduction of the cellular content of PI4KII{alpha} strongly decreased the punctate distribution of AP-3 observed in PC12 cells. These results indicate that PI4KII{alpha} is present on AP-3 organelles where it regulates AP-3 function.

{dagger}These authors contributed equally to this work.

Address correspondence to: Victor Faundez (faundez{at}cellbio.emory.edu)




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