The Protein Complex of Neurodegeneration-related Phosphoinositide Phosphatase Sac3 and ArPIKfyve Binds the Lewy Body-associated Synphilin-1, Preventing Its Aggregation

J Biol Chem. 2015 Nov 20;290(47):28515-28529. doi: 10.1074/jbc.M115.669929. Epub 2015 Sep 24.

Abstract

The 5-phosphoinositide phosphatase Sac3, in which loss-of-function mutations are linked to neurodegenerative disorders, forms a stable cytosolic complex with the scaffolding protein ArPIKfyve. The ArPIKfyve-Sac3 heterodimer interacts with the phosphoinositide 5-kinase PIKfyve in a ubiquitous ternary complex that couples PtdIns(3,5)P2 synthesis with turnover at endosomal membranes, thereby regulating the housekeeping endocytic transport in eukaryotes. Neuron-specific associations of the ArPIKfyve-Sac3 heterodimer, which may shed light on the neuropathological mechanisms triggered by Sac3 dysfunction, are unknown. Here we conducted mass spectrometry analysis for brain-derived interactors of ArPIKfyve-Sac3 and unraveled the α-synuclein-interacting protein Synphilin-1 (Sph1) as a new component of the ArPIKfyve-Sac3 complex. Sph1, a predominantly neuronal protein that facilitates aggregation of α-synuclein, is a major component of Lewy body inclusions in neurodegenerative α-synucleinopathies. Modulations in ArPIKfyve/Sac3 protein levels by RNA silencing or overexpression in several mammalian cell 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.

Keywords: ArPIKfyve-Sac3 complex; Parkinson disease; Synphilin-1; aggregation; aggresome; neurodegenerative disease; neuron; phosphatidylinositol phosphatase; phospholipid; proteasome.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • COS Cells
  • Carrier Proteins / metabolism*
  • Chlorocebus aethiops
  • Flavoproteins / metabolism*
  • Green Fluorescent Proteins / metabolism
  • HEK293 Cells
  • Humans
  • Intracellular Signaling Peptides and Proteins
  • Lewy Bodies / metabolism*
  • Membrane Proteins / metabolism*
  • Nerve Tissue Proteins / metabolism*
  • Neurodegenerative Diseases / enzymology
  • Neurodegenerative Diseases / metabolism*
  • Phosphoric Monoester Hydrolases / metabolism*
  • Protein Binding

Substances

  • Carrier Proteins
  • Flavoproteins
  • Intracellular Signaling Peptides and Proteins
  • Membrane Proteins
  • Nerve Tissue Proteins
  • SNCAIP protein, human
  • VAC14 protein, human
  • Green Fluorescent Proteins
  • FIG4 protein, human
  • Phosphoric Monoester Hydrolases