Bioinformatics revealed several potential interactors in the brain, specifically captured by the HA-ArPIKfyve-Myc-Sac3 complex. lines, including human neuronal SH-SY5Y or primary mouse cortical neurons, revealed that the ArPIKfyve-Sac3 complex specifically altered the aggregation properties of Sph1-GFP. This effect required an active Sac3 phosphatase and proceeded through mechanisms that involved increased Sph1-GFP partitioning into the cytosol and removal of Sph1-GFP aggregates by basal autophagy but not by the proteasomal system. If uncoupled from ArPIKfyve elevation, overexpressed Sac3 readily aggregated, markedly enhancing the aggregation potential of Sph1-GFP. These data identify a novel role of the ArPIKfyve-Sac3 complex in the mechanisms controlling aggregate formation of Sph1 and suggest that Sac3 protein deficiency or overproduction may facilitate aggregation of aggregation-prone proteins, thereby precipitating the onset of multiple neuronal disorders. null mice, which die soon after birth, AZ1 exhibit profound defects in the nervous system but not in other organs, liver, kidney, lung, heart, etc. (7). By contrast, AZ1 hypomorphic mice, which also die soon after birth, have prominent defects not only in the brain but also in all organs tested, including heart, lung, kidney etc., whereas a complete systemic disruption results in preimplantation mouse embryo death (17, 20). Findings for alterations in steady-state levels of PtdIns(3,5)P2 in opposite directions in mouse models of CMT neuropathies triggered by disruptions of PtdIns(3,5)P2 phosphatases Sac3 and MTMR2, respectively, also fail to provide a coherent model for the role of PtdIns(3,5)P2 in the neuropathological mechanism (21, 22). We surmised that the soluble ArPIKfyve-Sac3 complex may have yet unknown PIKfyve-independent interactions with neuron-specific proteins that could underlie ArPIKfyve-Sac3 functionality in the nervous system. To this end, here we undertook a proteomic screen of mouse brain lysates using the ArPIKfyve-Sac3 complex as bait and unraveled -synuclein-interacting protein Synphilin-1 (Sph1) (gene symbol green for GFP, red for Alexa Fluor 568, and blue for Alexa Fluor 350 signals) and the Hoffman modulation contrast system. Images were captured with a SPOT RT slider MGC129647 charge-coupled device camera (Diagnostic Instruments, Sterling Heights, MI) and processed using SPOT 3.2 and Adobe Photoshop CS6. Where indicated, coverslips were observed by motorized inverted confocal microscope (model 1X81, Olympus, Melville, AZ1 NY) with an Uplan Apo objective. Images were captured using a cooled charge-couple device 12-bit camera (Hamamatsu). Immunoprecipitation, Immunoblotting, and Protein Fractionation Cell lysates were collected in RIPA+ buffer (50 mm Tris/HCl buffer, pH 8.0, 150 mm NaCl, 1% Nonidet P-40, and 0.5% sodium deoxycholate), supplemented with 1 protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 5 g/ml leupeptin, 5 g/ml AZ1 aprotinin, 1 g/ml pepstatin, and 1 mm benzamidine). Lysates from mouse brains (male C57Bl6, 4 months old) were prepared by homogenization in RIPA2+ buffer, containing 1 x phosphatase inhibitors (1, 19). Immunoprecipitation with the indicated antibodies was performed with precleared lysates (20,000 Triton X-100-insoluble protein fractions of Sph1-GFP or Myc-Sac3 were evaluated in ArPIKfyve-HEK cells with or without ArPIKfyve induction following previously described protocols (30). Briefly, cells on parallel confluent 35-mm dishes were extracted on ice for 2 min with 0.5% Triton X-100 in PBS containing 1 protease inhibitors or directly solubilized for 10 min at 4 C in RIPA+ buffer containing 0.1% SDS to obtain Triton-soluble and total protein fractions, respectively. The Triton-insoluble protein fraction that remained on the first dish was then solubilized in RIPA+ buffer containing 0.1% SDS. Equal volumes of the respective fractions were analyzed by immunoblotting with the indicated antibodies. Sample Preparation and NanoLC-MS/MS Analysis The Myc-Sac3-HA-ArPIKfyve complex, immunopurified from transfected COS7 cells with a monoclonal anti-Myc antibody and immobilized on protein A-Sepharose CL-4B beads, was incubated with mouse brain lysates for 16 h at 4 C. Anti-Myc immune complexes immobilized on protein A-Sepharose CL-4B beads derived from lysates of nontransfected or Myc-Sac3-transfected COS7 cells were incubated with brain lysates in parallel and served as controls. After extensive washing in RIPA+ buffer, the beads were analyzed by SDS-PAGE. Gels.