Identification of PSD-95 Depalmitoylating Enzymes.
Postsynaptic density (PSD)-95, the most abundant postsynaptic scaffolding protein, plays a pivotal role in synapse development and function. Continuous palmitoylation cycles on PSD-95 are essential for its synaptic clustering and regulation of AMPA receptor function. However, molecular mechanisms for palmitate cycling on PSD-95 remain incompletely understood, as PSD-95 depalmitoylating enzymes remain unknown. Here, we isolated 38 mouse or rat serine hydrolases and found that a subset specifically depalmitoylated PSD-95 in heterologous cells. These enzymes showed distinct substrate specificity. α/β-Hydrolase domain-containing protein 17 members (ABHD17A, 17B, and 17C), showing the strongest depalmitoylating activity to PSD-95, showed different localization from other candidates in rat hippocampal neurons, and were distributed to recycling endosomes, the dendritic plasma membrane, and the synaptic fraction. Expression of ABHD17 in neurons selectively reduced PSD-95 palmitoylation and synaptic clustering of PSD-95 and AMPA receptors. Furthermore, taking advantage of the acyl-PEGyl exchange gel shift (APEGS) method, we quantitatively monitored the palmitoylation stoichiometry and the depalmitoylation kinetics of representative synaptic proteins, PSD-95, GluA1, GluN2A, mGluR5, Gαq, and HRas. Unexpectedly, palmitate on all of them did not turn over in neurons. Uniquely, most of the PSD-95 population underwent rapid palmitoylation cycles, and palmitate cycling on PSD-95 decelerated accompanied by its increased stoichiometry as synapses developed, probably contributing to postsynaptic receptor consolidation. Finally, inhibition of ABHD17 expression dramatically delayed the kinetics of PSD-95 depalmitoylation. This study suggests that local palmitoylation machinery composed of synaptic DHHC palmitoylating enzymes and ABHD17 finely controls the amount of synaptic PSD-95 and synaptic function.



Protein palmitoylation, the most common lipid modification, dynamically regulates neuronal protein localization and function. Its unique reversibility is conferred by DHHC-type palmitoyl acyl transferases (palmitoylating enzymes) and still controversial palmitoyl-protein thioesterases (depalmitoylating enzymes). Here, we identified the membrane-anchored serine hydrolases, ABHD17A, 17B, and 17C, as the physiological PSD-95 depalmitoylating enzymes that regulate PSD-95 palmitoylation cycles in neurons. This study describes the first direct evidence for the neuronal depalmitoylating enzyme and provides a new aspect of the dynamic regulatory mechanisms of synaptic development and synaptic plasticity. In addition, our established APEGS assay, which provides unbiased and quantitative information about the palmitoylation state and dynamics, revealed the distinct regulatory mechanisms for synaptic palmitoylation.
Division of Membrane Physiology, Department of Molecular and Cellular Physiology and Department of Physiological Sciences, School of Life Science, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Aichi 444-8787, Japan.
Norihiko
N
Yokoi
Division of Membrane Physiology, Department of Molecular and Cellular Physiology and Department of Physiological Sciences, School of Life Science, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Aichi 444-8787, Japan mfukata@nips.ac.jp yfukata@nips.ac.jp.
Yuko
Y
Fukata
Division of Membrane Physiology, Department of Molecular and Cellular Physiology and Department of Physiological Sciences, School of Life Science, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Aichi 444-8787, Japan.
Atsushi
A
Sekiya
Division of Membrane Physiology, Department of Molecular and Cellular Physiology and Department of Physiological Sciences, School of Life Science, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Aichi 444-8787, Japan.
Tatsuro
T
Murakami
Section of Viral Vector Development, Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences (NIPS), National Institutes of Natural Sciences (NINS), and Department of Physiological Sciences, School of Life Science, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Aichi 444-8787, Japan.
Kenta
K
Kobayashi
Division of Membrane Physiology, Department of Molecular and Cellular Physiology and Department of Physiological Sciences, School of Life Science, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Aichi 444-8787, Japan mfukata@nips.ac.jp yfukata@nips.ac.jp.
Masaki
M
Fukata
D000818:Animals; D002461:Cell Line, Transformed; D002531:Cerebellum / Q000378:metabolism; D002522:Chlorocebus aethiops; D000076122:Disks Large Homolog 4 Protein; D005260:Female; D051528:Guanylate Kinases / Q000235:genetics / Q000378:metabolism*; D006624:Hippocampus / Q000166:cytology; D006867:Hydrolases / Q000378:metabolism; D054878:Lipoylation / Q000502:physiology*; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008994:Monoacylglycerol Lipases / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000201:enzymology*; D010168:Palmitates / Q000378:metabolism; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012694:Serine / Q000031:analogs & derivatives* / Q000302:isolation & purification / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D013379:Substrate Specificity; D014316:Tritium / Q000378:metabolism
D000076122:Disks Large Homolog 4 Protein; C501447:Dlg4 protein, mouse; D008565:Membrane Proteins; D010168:Palmitates; D034741:RNA, Small Interfering; D014316:Tritium; C028002:serine hydroxamate; D012694:Serine; D051528:Guanylate Kinases; D006867:Hydrolases; C454240:Abhd2 protein, mouse; D008994:Monoacylglycerol Lipases
ABHD protein; ABHD17; AMPA receptor; PSD-95; palmitoylation; synapse