Phosphatidylinositol-4-kinase type II α is a component of adaptor protein-3-derived vesicles

A membrane fraction enriched in vesicles containing the adaptor protein (AP) -3 cargo zinc transporter 3 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 (P14KII alpha). Biochemical, pharmacological, and morphological analyses supported the presence of P14KII alpha in AP-3-positive organelles. Furthermore, the subcellular localization of P14KII alpha was altered in cells from AP-3-deficient mocha mutant mice. The P14KII 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, P14KII alpha content was strongly reduced in nerve terminals of mocha hippocampal mossy fibers. The functional relationship between AP-3 and P14KII alpha was further explored by P14KII alpha knockdown experiments. Reduction of the cellular content of P14KII alpha strongly decreased the punctate distribution of AP-3 observed in PC12 cells. These results indicate that P14KIIa is present on AP-3 organelles where it regulates AP-3 function.