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Role of local palmitoylation machinery in the postsynaptic nanodomain organization

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Fukata M., Yokoi N., Fukata Y.

Acta Neurobiologiae Experimentalis

2017

tom 77

nr Suppl.1

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 palmitoyl-protein thioesterases (depalmitoylating enzymes). PSD-95 represents a major palmitoylated postsynaptic scaffolding protein that assembles various synaptic components such as AMPA- and NMDA-type glutamate receptors at the postsynaptic specialized membrane domain (postsynaptic density, PSD). Recently, we found that PSD-95 is partitioned into subsynaptic nanodomains and that PSD-95 in nanodomains undergoes continuous de/repalmitoylation cycles, thereby defining the geometry of PSDs. We found that a subset of metabolic serine hydrolases, ABHD17A, 17B and 17C, specifically depalmitoylate PSD-95 in heterologous cells. Expression of the plasma membrane-localized ABHD17 in hippocampal neurons directly binds to PSD-95 through its catalytic region and dramatically disperses PSD-95 clusters. 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. Uniquely, most of the PSD‑95 population undergoes rapid palmitoylation cycles. We also found that inhibition of ABHD17 expression dramatically delays the kinetics of PSD-95 depalmitoylation. We propose that local palmitoylation machinery composed of synaptic DHHC palmitoylating enzymes and ABHD17 finely controls the amount of synaptic PSD-95 and thereby organizes the postsynaptic nanodomains. FINANCIAL SUPPORT: This work was supported by the Ministry of Education, Culture, Sports, Science and Technology (Grant numbers 15H04279, 15H01299 to Y.F; 26291045, 15H01570, 16H01371, 16K14560 to M.F.); Takeda Science Foundation to Y.F and M.F.; and The Naito Foundations to M.F.

Assessment of palmitoylation status and cycles on PSD 95 in neurons and in vivo

100%

Fukata Y., Yokoi N., Kobayashi K., Fukata M.

Acta Neurobiologiae Experimentalis

2017

tom 77

nr Suppl.1

INTRODUCTION: Precise synaptic function requires spatio-temporally regulated protein localization. Protein palmitoylation, a reversible lipid modification, represents one such mechanism. Although numerous synaptic palmitoylated proteins have been identified, the physiological importance of their palmitoylation remains incompletely understood due to the lack of quantitative information. AIM(S): To determine the actual palmitoylation stoichiometry and state (for example, mono-, di-, tri-) of representative synaptic proteins in the rat brain, and to examine how dynamically the palmitoyl-turnover on proteins is regulated. METHOD(S): We used recently developed acyl-PEGyl exchange gel‑shift (APEGS) assay to profile palmitoylation stoichiometry of synaptic proteins and their dynamic changes, especially for PSD-95, in rat cultured hippocampal neurons and in rat brain. RESULTS: We found that individual palmitoylated proteins have the distinct palmitoylation site occupancy and the kinetics in rat cultured hippocampal neurons. Unexpectedly, palmitate on synaptic proteins did not all turn over. Of particular importance however is uniquely robust and dynamic palmitoylation for a postsynaptic scaffold PSD-95. In young neurons the stoichiometry of PSD‑95 palmitoylation was about 60% with the rapid palmitate cycling, whereas palmitate cycling on PSD-95 significantly decelerated accompanied by the increased stoichiometry in neurons and in vivo. Furthermore, we found that the sensitivity against recently discovered PSD-95 depalmitoylating enzyme, ABHD17, well correlated with the speed of palmitoyl cycling and cluster formation of PSD-95. CONCLUSIONS: This study suggests that the palmitoylation stoichiometry and kinetics of PSD-95 could be tightly controlled in response to the physiological contexts during synapse development by the specific palmitoylating/depalmitoylating enzymes. FINANCIAL SUPPORT: The Ministry of Education, Culture, Sports, Science and Technology – Japan (15H04279, 15H01299)

Ograniczanie wyników

2 Acta Neurobiologiae Experimentalis

2 Fukata M.

2 Fukata Y.

2 Yokoi N.

1 Kobayashi K.

2 2017

Wyniki wyszukiwania

Role of local palmitoylation machinery in the postsynaptic nanodomain organization

Assessment of palmitoylation status and cycles on PSD 95 in neurons and in vivo