Entry and exit of proteins into flagella is gauged by CEP290 in the transition zone.

The adaptor complex 3 (AP-3) targets membrane proteins from endosomes to lysosomes, lysosome-related organelles and synaptic vesicles. Phosphatidylinositol-4-kinase type II alpha (PI4KIIalpha) is one of several proteins possessing catalytic domains that regulate AP-3-dependent sorting. Here we present evidence that PI4KIIalpha uniquely behaves both as a membrane protein cargo as well as an enzymatic regulator of adaptor function. In fact, AP-3 and PI4KIIalpha form a complex that requires a dileucine-sorting motif present in PI4KIIalpha. Mutagenesis of either the PI4KIIalpha-sorting motif or its kinase-active site indicates that both are necessary to interact with AP-3 and properly localize PI4KIIalpha to LAMP-1-positive endosomes. Similarly, both the kinase activity and the sorting signal present in PI4KIIalpha are necessary to rescue endosomal PI4KIIalpha siRNA-induced mutant phenotypes. We propose a mechanism whereby adaptors use canonical sorting motifs to selectively recruit a regulatory enzymatic activity to restricted membrane domains.

The central apparatus is an essential component of “9+2” cilia. Zhao et al. identify more than 40 new potential components of the central apparatus of Chlamydomonas. Many are conserved and will facilitate genetic screening of patients with a form of primary ciliary dyskinesia that is difficult to diagnose.

Mutational analyses have revealed many genes that are required for proper biogenesis of lysosomes and lysosome-related organelles. The proteins encoded by these genes assemble into five distinct complexes (AP-3, BLOC-1-3, and HOPS) that either sort membrane proteins or interact with SNAREs. Several of these seemingly distinct complexes cause similar phenotypic defects when they are rendered defective by mutation, but the underlying cellular mechanism is not understood. Here, we show that the BLOC-1 complex resides on microvesicles that also contain AP-3 subunits and membrane proteins that are known AP-3 cargoes. Mouse mutants that cause BLOC-1 or AP-3 deficiencies affected the targeting of LAMP1, phosphatidylinositol-4-kinase type II alpha, and VAMP7-TI. VAMP7-TI is an R-SNARE involved in vesicle fusion with late endosomes/lysosomes, and its cellular levels were selectively decreased in cells that were either AP-3-or BLOC-1-deficient. Furthermore, BLOC-1 deficiency selectively altered the subcellular distribution of VAMP7-TI cognate SNAREs. These results indicate that the BLOC-1 and AP-3 protein complexes affect the targeting of SNARE and non-SNARE AP-3 cargoes and suggest a function of the BLOC-1 complex in membrane protein sorting. INTRODUCTIONMembrane enclosed organelles possess distinctive protein compositions maintained by vesicle formation and vesicle fusion mechanisms. Vesicles are formed by coat and coat accessory molecules that selectively regulate the concentration of specific membrane proteins into departing vesicles. Once formed, vesicle contents are delivered to target membranes by vesicle fusion. This process depends on the pairing of fusogenic membrane proteins generically known as R-or vesicle (v)-SNAREs and Q-or target (t)-SNAREs (Springer et al., 1999;Bonifacino and Glick, 2004;Hong, 2005). Vesicle formation by coats and fusion by SNAREs is likely to be coordinately regulated, as suggested by the specific subcellular localizations that coats and SNAREs possess (Robinson, 2004;Hong, 2005). Predictably, coats either interact with and/or sort SNAREs. However, SNARE sorting mechanisms have been described only for a limited number of the SNAREs known to eukaryotic cells (Gurkan et al., 2005). For example, COPII interacts/sorts the R-(v)-SNAREs Bos1p and Bet1p into vesicles (Matsuoka et al., 1998;Springer and Schekman, 1998); the adaptors GGA1-2 sort the yeast Q-(t)-SNARE Pep12p (Black and Pelham, 2000); epsinR sorts/interacts with the Q-(t)-SNARE Vti1b (Hirst et al., 2004); the adaptor complex AP-3 sorts or interacts with the R-(v)-SNAREs VAMP7-TI (Martinez-Arca et al., 2003) and engineered variants of VAMP2 (Salem et al., 1998); and the adaptor complex AP-1 sorts/interacts with VAMP4 (Peden et al., 2001). These coat-SNARE interactions define prebudding interactions of the vesicle sorting and fusion machineries. However, it is poorly understood in vertebrates whether 1) these interactions persist at later stages once a vesicle is formed and cargo concentration is completed and 2) if molecules or com...

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...