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Mobile DHHC palmitoylating enzyme mediates activity-sensitive synaptic targeting of PSD-95.

Noritake J, Fukata Y, Iwanaga T, Hosomi N, Tsutsumi R, Matsuda N, Tani H, Iwanari H, Mochizuki Y, Kodama T, Matsuura Y, Bredt DS, Hamakubo T, Fukata M - J. Cell Biol. (2009)

Bottom Line: We found that blocking synaptic activity rapidly induces PSD-95 palmitoylation and mediates synaptic clustering of PSD-95 and associated AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-type glutamate receptors.Upon activity blockade, DHHC2 translocates to the postsynaptic density to transduce this effect.These data demonstrate that individual DHHC members are differentially regulated and that dynamic recruitment of protein palmitoylation machinery enables compartmentalized regulation of protein trafficking in response to extracellular signals.

View Article: PubMed Central - PubMed

Affiliation: Division of Membrane Physiology, Department of Cell Physiology, National Institute for Physiological Sciences, Okazaki, Aichi, Japan.

ABSTRACT
Protein palmitoylation is the most common posttranslational lipid modification; its reversibility mediates protein shuttling between intracellular compartments. A large family of DHHC (Asp-His-His-Cys) proteins has emerged as protein palmitoyl acyltransferases (PATs). However, mechanisms that regulate these PATs in a physiological context remain unknown. In this study, we efficiently monitored the dynamic palmitate cycling on synaptic scaffold PSD-95. We found that blocking synaptic activity rapidly induces PSD-95 palmitoylation and mediates synaptic clustering of PSD-95 and associated AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-type glutamate receptors. A dendritically localized DHHC2 but not the Golgi-resident DHHC3 mediates this activity-sensitive palmitoylation. Upon activity blockade, DHHC2 translocates to the postsynaptic density to transduce this effect. These data demonstrate that individual DHHC members are differentially regulated and that dynamic recruitment of protein palmitoylation machinery enables compartmentalized regulation of protein trafficking in response to extracellular signals.

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DHHC2 is essential for activity-sensitive PSD-95 palmitoylation. (A and B) DHHC2 but not DHHC3 mediates palmitoylation of PSD-95 upon activity blockade. Knockdown of DHHC2 but not DHHC3 inhibited Kyn-induced PSD-95–GFP recruitment at the synaptic membrane. miDHHC2-resistant DHHC2 (WT) but not PAT-inactive DHHC2 (CS) rescued Kyn-induced PSD-95 accumulation. (A) TIRFM intensity of representative five punctae from a neuron was plotted with time. (B) n = 3 each; ***, P < 0.001. Error bars indicate SD. Bar, 5 µm.
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fig5: DHHC2 is essential for activity-sensitive PSD-95 palmitoylation. (A and B) DHHC2 but not DHHC3 mediates palmitoylation of PSD-95 upon activity blockade. Knockdown of DHHC2 but not DHHC3 inhibited Kyn-induced PSD-95–GFP recruitment at the synaptic membrane. miDHHC2-resistant DHHC2 (WT) but not PAT-inactive DHHC2 (CS) rescued Kyn-induced PSD-95 accumulation. (A) TIRFM intensity of representative five punctae from a neuron was plotted with time. (B) n = 3 each; ***, P < 0.001. Error bars indicate SD. Bar, 5 µm.

Mentions: DHHC2 or -3 knockdown by microRNA (miRNA; miDHHCs) greatly reduced the number of PSD-95 punctae (Fig. 4, A and B). Importantly, knockdown of DHHC2 but not DHHC3 prevented Kyn- or TTX-induced increase of endogenous PSD-95 accumulation at synaptic sites (Fig. 4, A, C and D) and Kyn-induced augmentation of PSD-95–GFP accumulation (Fig. 5 and Video 3). The inhibitory effect of miDHHC2 was rescued by miDHHC2-resistant WT DHHC2 (WT) but not by PAT-inactive DHHC2 (CS) (Fig. 5 and Fig. S4 C). These results indicate that DHHC3 localizes to the Golgi apparatus and mediates constitutive palmitoylation of various substrates, including PSD-95, Gαq, and GABAA receptor-γ subunit (Fukata et al., 2004; Keller et al., 2004; Tsutsumi et al., 2009). In contrast, dendritic DHHC2 mediates activity-sensitive PSD-95 palmitoylation.


Mobile DHHC palmitoylating enzyme mediates activity-sensitive synaptic targeting of PSD-95.

Noritake J, Fukata Y, Iwanaga T, Hosomi N, Tsutsumi R, Matsuda N, Tani H, Iwanari H, Mochizuki Y, Kodama T, Matsuura Y, Bredt DS, Hamakubo T, Fukata M - J. Cell Biol. (2009)

DHHC2 is essential for activity-sensitive PSD-95 palmitoylation. (A and B) DHHC2 but not DHHC3 mediates palmitoylation of PSD-95 upon activity blockade. Knockdown of DHHC2 but not DHHC3 inhibited Kyn-induced PSD-95–GFP recruitment at the synaptic membrane. miDHHC2-resistant DHHC2 (WT) but not PAT-inactive DHHC2 (CS) rescued Kyn-induced PSD-95 accumulation. (A) TIRFM intensity of representative five punctae from a neuron was plotted with time. (B) n = 3 each; ***, P < 0.001. Error bars indicate SD. Bar, 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2712995&req=5

fig5: DHHC2 is essential for activity-sensitive PSD-95 palmitoylation. (A and B) DHHC2 but not DHHC3 mediates palmitoylation of PSD-95 upon activity blockade. Knockdown of DHHC2 but not DHHC3 inhibited Kyn-induced PSD-95–GFP recruitment at the synaptic membrane. miDHHC2-resistant DHHC2 (WT) but not PAT-inactive DHHC2 (CS) rescued Kyn-induced PSD-95 accumulation. (A) TIRFM intensity of representative five punctae from a neuron was plotted with time. (B) n = 3 each; ***, P < 0.001. Error bars indicate SD. Bar, 5 µm.
Mentions: DHHC2 or -3 knockdown by microRNA (miRNA; miDHHCs) greatly reduced the number of PSD-95 punctae (Fig. 4, A and B). Importantly, knockdown of DHHC2 but not DHHC3 prevented Kyn- or TTX-induced increase of endogenous PSD-95 accumulation at synaptic sites (Fig. 4, A, C and D) and Kyn-induced augmentation of PSD-95–GFP accumulation (Fig. 5 and Video 3). The inhibitory effect of miDHHC2 was rescued by miDHHC2-resistant WT DHHC2 (WT) but not by PAT-inactive DHHC2 (CS) (Fig. 5 and Fig. S4 C). These results indicate that DHHC3 localizes to the Golgi apparatus and mediates constitutive palmitoylation of various substrates, including PSD-95, Gαq, and GABAA receptor-γ subunit (Fukata et al., 2004; Keller et al., 2004; Tsutsumi et al., 2009). In contrast, dendritic DHHC2 mediates activity-sensitive PSD-95 palmitoylation.

Bottom Line: We found that blocking synaptic activity rapidly induces PSD-95 palmitoylation and mediates synaptic clustering of PSD-95 and associated AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-type glutamate receptors.Upon activity blockade, DHHC2 translocates to the postsynaptic density to transduce this effect.These data demonstrate that individual DHHC members are differentially regulated and that dynamic recruitment of protein palmitoylation machinery enables compartmentalized regulation of protein trafficking in response to extracellular signals.

View Article: PubMed Central - PubMed

Affiliation: Division of Membrane Physiology, Department of Cell Physiology, National Institute for Physiological Sciences, Okazaki, Aichi, Japan.

ABSTRACT
Protein palmitoylation is the most common posttranslational lipid modification; its reversibility mediates protein shuttling between intracellular compartments. A large family of DHHC (Asp-His-His-Cys) proteins has emerged as protein palmitoyl acyltransferases (PATs). However, mechanisms that regulate these PATs in a physiological context remain unknown. In this study, we efficiently monitored the dynamic palmitate cycling on synaptic scaffold PSD-95. We found that blocking synaptic activity rapidly induces PSD-95 palmitoylation and mediates synaptic clustering of PSD-95 and associated AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-type glutamate receptors. A dendritically localized DHHC2 but not the Golgi-resident DHHC3 mediates this activity-sensitive palmitoylation. Upon activity blockade, DHHC2 translocates to the postsynaptic density to transduce this effect. These data demonstrate that individual DHHC members are differentially regulated and that dynamic recruitment of protein palmitoylation machinery enables compartmentalized regulation of protein trafficking in response to extracellular signals.

Show MeSH
Related in: MedlinePlus