Lys-63-linked ubiquitination by E3 ubiquitin ligase Nedd4-1 facilitates endosomal sequestration of internalized α-synuclein.
α-Synuclein (aS) is a major constituent of Lewy bodies, which are not only a pathological marker for Parkinson disease but also a trigger for neurodegeneration. Cumulative evidence suggests that aS spreads from cell to cell and thereby propagates neurodegeneration to neighboring cells. Recently, Nedd4-1 (neural precursor cell expressed developmentally down-regulated protein 4-1), an E3 ubiquitin ligase, was shown to catalyze the Lys-63-linked polyubiquitination of intracellular aS and thereby facilitate aS degradation by the endolysosomal pathway. Because Nedd4-1 exerts its activity in close proximity to the inner leaflet of the plasma membrane, we speculate that after the internalization of aS the membrane resident aS is preferentially ubiquitinated by Nedd4-1. To clarify the role of Nedd4-1 in aS internalization and endolysosomal sequestration, we generated aS mutants, including ΔPR1(1-119 and 129-140), ΔC(1-119), and ΔPR2(1-119 and 134-140), that lack the proline-rich sequence, a putative Nedd4-1 recognition site. We show that wild type aS, but not ΔPR1, ΔPR2, or ΔC aS, is modified by Nedd4-1 in vitro, acquiring a Lys-63-linked ubiquitin chain. Compared with the mutants lacking the proline-rich sequence, wild type-aS is preferentially internalized and translocated to endosomes. The overexpression of Nedd4-1 increased aS in endosomes, whereas RNAi-mediated silencing of Nedd4-1 decreased endosomal aS. Although aS freely passes through plasma membranes within minutes, a pulse-chase experiment revealed that the overexpression of Nedd4-1 markedly decreased the re-secretion of internalized aS. Together, these findings demonstrate that Nedd4-1-linked Lys-63 ubiquitination specifies the fate of extrinsic and de novo synthesized aS by facilitating their targeting to endosomes.
From the Division of Neurology, Department of Neuroscience and Sensory Organs, Tohoku University Graduate School of Medicine, Sendai 980-8574, Japan.
Naoto
N
Sugeno
From the Division of Neurology, Department of Neuroscience and Sensory Organs, Tohoku University Graduate School of Medicine, Sendai 980-8574, Japan, thasegawa@med.tohoku.ac.jp.
Takafumi
T
Hasegawa
the Division of Cancer Biology and Therapeutics, Miyagi Cancer Center Research Institute, Natori 981-1293, Japan.
Nobuyuki
N
Tanaka
the Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Sendai 980-8578, Japan.
Mitsunori
M
Fukuda
the Department of Neuropathology, Institute of Brain Science, Hirosaki University School of Medicine, Hirosaki 036-8562, Japan.
Koichi
K
Wakabayashi
From the Division of Neurology, Department of Neuroscience and Sensory Organs, Tohoku University Graduate School of Medicine, Sendai 980-8574, Japan, the Division of Cancer Biology and Therapeutics, Miyagi Cancer Center Research Institute, Natori 981-1293, Japan.
Ryuji
R
Oshima
From the Division of Neurology, Department of Neuroscience and Sensory Organs, Tohoku University Graduate School of Medicine, Sendai 980-8574, Japan.
Masashi
M
Konno
From the Division of Neurology, Department of Neuroscience and Sensory Organs, Tohoku University Graduate School of Medicine, Sendai 980-8574, Japan.
Emiko
E
Miura
From the Division of Neurology, Department of Neuroscience and Sensory Organs, Tohoku University Graduate School of Medicine, Sendai 980-8574, Japan.
Akio
A
Kikuchi
From the Division of Neurology, Department of Neuroscience and Sensory Organs, Tohoku University Graduate School of Medicine, Sendai 980-8574, Japan.
Toru
T
Baba
the Department of Pediatrics, Kumamoto University School of Medicine, Kumamoto 860-0811, Japan.
Tadashi
T
Anan
the Department of Medical Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 860-0811, Japan, and.
Mitsuyoshi
M
Nakao
the Laboratory of Functional Neurogenetics, Department for Neurodegenerative Diseases, Hertie Institute for Clinical Brain Research, University of Tübingen, German Centre for Neurodegenerative Diseases, Tübingen 72076, Germany.
Sven
S
Geisler
From the Division of Neurology, Department of Neuroscience and Sensory Organs, Tohoku University Graduate School of Medicine, Sendai 980-8574, Japan.
Masashi
M
Aoki
From the Division of Neurology, Department of Neuroscience and Sensory Organs, Tohoku University Graduate School of Medicine, Sendai 980-8574, Japan.
Atsushi
A
Takeda
D020816:Amino Acid Motifs; D045744:Cell Line, Tumor; D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics / Q000378:metabolism*; D011992:Endosomes / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D000075702:Nedd4 Ubiquitin Protein Ligases; D010300:Parkinson Disease / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D051844:alpha-Synuclein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D056827:Endosomal Sorting Complexes Required for Transport; D051844:alpha-Synuclein; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; D044767:Ubiquitin-Protein Ligases; D008239:Lysine
E3 Ubiquitin Ligase; Membrane Trafficking; Neurobiology; Parkinson Disease; Propagation; Ubiquitination; α-Synuclein (aS)