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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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Activity-sensitive synaptic translocation of DHHC2. (A) No change in DHHC autopalmitoylation (detected by the ABE method) was seen upon activity blockade (TTX or Kyn) of hippocampal neurons, suggesting that DHHC activity remains constant. (B and C) TIRFM imaging revealed that treatment with Kyn or TTX translocated DHHC2-GFP near the plasma membrane. n = 3; *, P < 0.05; **, P < 0.01 compared with control. Kymographs (pseudocolor) represent the changes in the intensity of DHHC2-GFP over time. (D) The translocation of DHHC2-GFP induced by Kyn treatment was reversible upon washing out Kyn. (E and F) Colocalization of endogenous DHHC2 with PSD-95 steadily increased over prolonged TTX or Kyn treatment. (F) n = 5–7 each; **, P < 0.01; ***, P < 0.001. (C and F) Error bars indicate SD. Bars: (B) 2 µm; (D) 5 µm; (E [left]) 3 µm; (E [right]) 1 µm.
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fig6: Activity-sensitive synaptic translocation of DHHC2. (A) No change in DHHC autopalmitoylation (detected by the ABE method) was seen upon activity blockade (TTX or Kyn) of hippocampal neurons, suggesting that DHHC activity remains constant. (B and C) TIRFM imaging revealed that treatment with Kyn or TTX translocated DHHC2-GFP near the plasma membrane. n = 3; *, P < 0.05; **, P < 0.01 compared with control. Kymographs (pseudocolor) represent the changes in the intensity of DHHC2-GFP over time. (D) The translocation of DHHC2-GFP induced by Kyn treatment was reversible upon washing out Kyn. (E and F) Colocalization of endogenous DHHC2 with PSD-95 steadily increased over prolonged TTX or Kyn treatment. (F) n = 5–7 each; **, P < 0.01; ***, P < 0.001. (C and F) Error bars indicate SD. Bars: (B) 2 µm; (D) 5 µm; (E [left]) 3 µm; (E [right]) 1 µm.

Mentions: We next investigated whether DHHC2 PAT activity, monitored by autopalmitoylation (Fukata et al., 2004), was regulated by synaptic activity. Whereas PSD-95 palmitoylation increased upon TTX or Kyn treatment, autopalmitoylation of DHHC2 and -3 did not change (Fig. 6 A), suggesting that DHHC2 activity may remain constant. We then investigated whether DHHC2 localization is regulated by synaptic activity. TIRFM imaging revealed that more DHHC2 was recruited near the membrane upon Kyn or TTX treatment (Fig. 6, B and C; and Video 4), where PSD-95 localized (Fig. S4 A). This translocation was activity sensitive as it was reversible upon washing out of Kyn (Fig. 6 D). Furthermore, we found that Kyn or TTX steadily induced colocalization of endogenous DHHC2 with PSD-95 over 48 h (Fig. 6, E and F), whereas DHHC3 remained at the Golgi apparatus (Fig. S4 B).


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)

Activity-sensitive synaptic translocation of DHHC2. (A) No change in DHHC autopalmitoylation (detected by the ABE method) was seen upon activity blockade (TTX or Kyn) of hippocampal neurons, suggesting that DHHC activity remains constant. (B and C) TIRFM imaging revealed that treatment with Kyn or TTX translocated DHHC2-GFP near the plasma membrane. n = 3; *, P < 0.05; **, P < 0.01 compared with control. Kymographs (pseudocolor) represent the changes in the intensity of DHHC2-GFP over time. (D) The translocation of DHHC2-GFP induced by Kyn treatment was reversible upon washing out Kyn. (E and F) Colocalization of endogenous DHHC2 with PSD-95 steadily increased over prolonged TTX or Kyn treatment. (F) n = 5–7 each; **, P < 0.01; ***, P < 0.001. (C and F) Error bars indicate SD. Bars: (B) 2 µm; (D) 5 µm; (E [left]) 3 µm; (E [right]) 1 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2712995&req=5

fig6: Activity-sensitive synaptic translocation of DHHC2. (A) No change in DHHC autopalmitoylation (detected by the ABE method) was seen upon activity blockade (TTX or Kyn) of hippocampal neurons, suggesting that DHHC activity remains constant. (B and C) TIRFM imaging revealed that treatment with Kyn or TTX translocated DHHC2-GFP near the plasma membrane. n = 3; *, P < 0.05; **, P < 0.01 compared with control. Kymographs (pseudocolor) represent the changes in the intensity of DHHC2-GFP over time. (D) The translocation of DHHC2-GFP induced by Kyn treatment was reversible upon washing out Kyn. (E and F) Colocalization of endogenous DHHC2 with PSD-95 steadily increased over prolonged TTX or Kyn treatment. (F) n = 5–7 each; **, P < 0.01; ***, P < 0.001. (C and F) Error bars indicate SD. Bars: (B) 2 µm; (D) 5 µm; (E [left]) 3 µm; (E [right]) 1 µm.
Mentions: We next investigated whether DHHC2 PAT activity, monitored by autopalmitoylation (Fukata et al., 2004), was regulated by synaptic activity. Whereas PSD-95 palmitoylation increased upon TTX or Kyn treatment, autopalmitoylation of DHHC2 and -3 did not change (Fig. 6 A), suggesting that DHHC2 activity may remain constant. We then investigated whether DHHC2 localization is regulated by synaptic activity. TIRFM imaging revealed that more DHHC2 was recruited near the membrane upon Kyn or TTX treatment (Fig. 6, B and C; and Video 4), where PSD-95 localized (Fig. S4 A). This translocation was activity sensitive as it was reversible upon washing out of Kyn (Fig. 6 D). Furthermore, we found that Kyn or TTX steadily induced colocalization of endogenous DHHC2 with PSD-95 over 48 h (Fig. 6, E and F), whereas DHHC3 remained at the Golgi apparatus (Fig. S4 B).

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