Phosphatidylinositol-4-Kinase Type II α Is a Component of Adaptor Protein-3-derived Vesicles

Abstract

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␣ (PI4KII␣). Biochemical, pharmacological, and morphological analyses supported the presence of PI4KII␣ in AP-3-positive organelles. Furthermore, the subcellular localization of PI4KII␣ was altered in cells from AP-3-deficient mocha mutant mice. The PI4KII␣ 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␣ content was strongly reduced in nerve terminals of mocha hippocampal mossy fibers. The functional relationship between AP-3 and PI4KII␣ was further explored by PI4KII␣ knockdown experiments. Reduction of the cellular content of PI4KII␣ strongly decreased the punctate distribution of AP-3 observed in PC12 cells. These results indicate that PI4KII␣ is present on AP-3 organelles where it regulates AP-3 function. INTRODUCTIONMembrane-enclosed organelles possess distinctive protein compositions that are dynamically maintained by inbound and outbound vesicle carriers. These vesicles selectively concentrate appropriate membrane proteins while leaving behind resident proteins found in the donor organelle, a process called sorting (Bonifacino and Glick, 2004). Central to membrane protein sorting and vesiculation are a family of cytosolic coat complexes that mediate vesicle budding and function as cargo-specific adaptors. These include monomeric proteins and the heterotetrameric adaptor proteins (AP)-1, -2, -3, and -4 (Bonifacino and Glick, 2004;Robinson, 2004). These coats participate in the generation of vesicles that carry a unique array of membrane protein "cargoes." The characterization of these carriers has played a major role in the functional dissection of coat-dependent sorting and vesiculation mechanisms. For example, vesicles "in a basket" isolated from brain led to the biochemical identification of the first sorting machinery, clathrin and the AP-1 and AP-2 adaptors (Kanaseki and Kadota, 1969;Pearse, 1975;Pfeffer and Kelly, 1981;Pearse and Crowther, 1987). Subsequent studies of cargo molecules in brain clathrin-coated vesicles were crucial in revealing mechanisms of synaptic vesicle recycling (Pfeffer and Kelly, 1985;Maycox et al., 1992;Blondeau et al., 2004). The advent of complete genome sequencing and the concomitant development of informatics tools has led to the rapid identification of proteins by mass spectrometry (Taylor et al., 2003). Application of these methodologies to clathrin-coated vesicles has allowed the identifi...

Mint3/X11γ Is an ADP-Ribosylation Factor-dependent Adaptor that Regulates the Traffic of the Alzheimer's Precursor Protein from theTrans-Golgi Network

“…We estimate from such data that APP and Mint3 are increased in specific activity Ͼ10-fold over cell homogenate. Although this number may seem low in comparison with a standard protein purification, it is consistent with published procedures for the purification of synaptic (15-18-fold) (Huttner et al, 1983;Floor et al, 1988) or AP-3 (ϳ16-fold; Craige et al, 2005;Salazar et al, 2005) coated vesicles. The fold purification of vesicles was estimated from that of Mint3, which in turn was determined by comparison of immunoreactivity in V6 to serial dilutions of a bacterially expressed recombinant Mint3 standard.…”

“…Magnetic beads (Dynal Biotech, Oslo, Norway), coated with either anti-mouse (M480) or anti-rabbit (M280) immunoglobulin (IgG, were incubated with either mouse monoclonal antibodies to Mint3 or rabbit polyclonal antibody to APP, respectively in PBS/5% bovine serum albumin (BSA) for 2 h at room temperature, according to the Salazar et al (2005). Controls included substitution of either mouse monoclonal or rabbit polyclonal c-myc antibodies.…”

“…Equal amounts of protein from these fractions were then analyzed by immunoblotting to determine where the APP and organelles of the secretory and endocytic membrane traffic fractionated. S2 has been shown previously to contain small vesicles, including synaptic vesicles and coated vesicles, in addition to soluble proteins Salazar et al, 2005), and so was expected to contain a subpopulation of cargos and adaptors that can be used to identify specific vesicles.…”

“…Equal amounts of protein from these fractions were then analyzed by immunoblotting to determine where the APP and organelles of the secretory and endocytic membrane traffic fractionated. S2 has been shown previously to contain small vesicles, including synaptic vesicles and coated vesicles, in addition to soluble proteins Salazar et al, 2005), and so was expected to contain a subpopulation of cargos and adaptors that can be used to identify specific vesicles.We found substantial amounts of APP and previously established binding partners, Mints (1-3) and Fe65, in S2 ( Figure 2A). The S2 contained ϳ45-50% of total cell protein under these conditions; yet, it is largely devoid of markers of secretory organelles.…”

Global Expression Profiling Applied to the Analysis of Arabidopsis Stamen Development

“…At2g40850 encodes a type II PtdIns 4-kinase, which has been previously named PI4Kg1 (Mueller-Roeber and Pical, 2002). While the function of type II PtdIns 4-kinases in plants is unknown, it has been suggested that in animals, they are involved in the regulation of intracellular trafficking (Wang et al, 2003;Salazar et al, 2005). Thus, the mutant phenotype observed for the At2g40850 insertion line might be caused by a malfunction of this essential cellular process.…”