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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 and -3 are differently involved in PSD-95 trafficking. (A and B) In the DHHC2 or -3 knocked down neurons (labeled with mCherry), the number of native PSD-95 puncta (green) was significantly decreased. n = 5 neurons; ***, P < 0.001. (C and D) Knockdown of DHHC2 but not DHHC3 prevented TTX- or Kyn-induced augmentation of endogenous PSD-95 accumulation. Dashed lines (100%) indicate the normalized control level. ***, P < 0.001. (C) Analyzed by confocal laser-scanning microscopy (CLSM). n = 10–15 neurons. (D) Analyzed by TIRFM. n = 5 neurons. miLacZ is a control miRNA targeting LacZ (β-galactosidase). (B–D) Error bars indicate SD. Bar, 5 µm.
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fig4: DHHC2 and -3 are differently involved in PSD-95 trafficking. (A and B) In the DHHC2 or -3 knocked down neurons (labeled with mCherry), the number of native PSD-95 puncta (green) was significantly decreased. n = 5 neurons; ***, P < 0.001. (C and D) Knockdown of DHHC2 but not DHHC3 prevented TTX- or Kyn-induced augmentation of endogenous PSD-95 accumulation. Dashed lines (100%) indicate the normalized control level. ***, P < 0.001. (C) Analyzed by confocal laser-scanning microscopy (CLSM). n = 10–15 neurons. (D) Analyzed by TIRFM. n = 5 neurons. miLacZ is a control miRNA targeting LacZ (β-galactosidase). (B–D) Error bars indicate SD. Bar, 5 µm.

Mentions: To follow changes in synaptic PSD-95 accumulation over time, we first performed time-lapse imaging of cultured hippocampal neurons transfected with PSD-95–GFP by TIRFM, which excites only molecules within 100 nm of the cover glass. TIRFM preferentially visualizes wild-type (WT) PSD-95–GFP as discrete punctae on dendrites, which are not seen with cytosolic palmitoylation–deficient (CS) mutant PSD-95 or GFP (Fig. 1, A and B). We confirmed comparable expression levels of PSD-95 (WT) and PSD-95 (CS) in transfected culture (Fig. 1 B). These data confirm that palmitoylation mediates membrane trafficking and synaptic clustering of PSD-95 (Topinka and Bredt, 1998). Because PSD-95 visualized by TIRFM apposes presynaptic synaptophysin and VGLUT1 and overlaps postsynaptic NR1 NMDA receptor (Fig. 1 C), TIRFM tracks synaptic PSD-95. When ionotropic glutamate receptor activity was blocked by kynurenic acid (Kyn), the intensity of PSD-95–GFP by TIRFM steadily increased over 2 h, whereas the intensity of PSD-95 (CS) did not change (Fig. 1, D and E; and Video 1). This Kyn-induced PSD-95 increase was blocked by coapplication of 2-bromopalmitate (2-BP), which is a palmitoyl acyl transfer inhibitor. PSD-95 signals did not detectably change within 2 h of 2-BP treatment alone (Fig. 1 E). These results indicate that newly occurring palmitoylation mediates this synaptic accumulation of PSD-95. Tetrodotoxin (TTX), a blocker of action potentials, also increased PSD-95 accumulation. The dynamic change of PSD-95 intensity was specific to palmitoylation as the localizations of GFP-Rac1-CLLL (Cys-Leu-Leu-Leu), a geranylgeranylated CaaL motif, and synaptophysin-GFP, a presynaptic protein, did not change upon Kyn treatment (Fig. 1 E). Synaptic PSD-95 accumulation upon activity blockade was also confirmed by antibody staining of native PSD-95 (see Fig. 4, C and D). The effect of Kyn or TTX on PSD-95 accumulation does not reflect newly synthesized PSD-95, as cycloheximide (CHX), an inhibitor of protein synthesis, did not affect the Kyn- or TTX-induced PSD-95 increase (Fig. S1, A and B; and Video 2). Thus, PSD-95 palmitoylation increases at the postsynaptic membrane upon activity blockade. These results are complementary to receptor activation–induced depalmitoylation of PSD-95 (El-Husseini et al., 2002).


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 and -3 are differently involved in PSD-95 trafficking. (A and B) In the DHHC2 or -3 knocked down neurons (labeled with mCherry), the number of native PSD-95 puncta (green) was significantly decreased. n = 5 neurons; ***, P < 0.001. (C and D) Knockdown of DHHC2 but not DHHC3 prevented TTX- or Kyn-induced augmentation of endogenous PSD-95 accumulation. Dashed lines (100%) indicate the normalized control level. ***, P < 0.001. (C) Analyzed by confocal laser-scanning microscopy (CLSM). n = 10–15 neurons. (D) Analyzed by TIRFM. n = 5 neurons. miLacZ is a control miRNA targeting LacZ (β-galactosidase). (B–D) 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

fig4: DHHC2 and -3 are differently involved in PSD-95 trafficking. (A and B) In the DHHC2 or -3 knocked down neurons (labeled with mCherry), the number of native PSD-95 puncta (green) was significantly decreased. n = 5 neurons; ***, P < 0.001. (C and D) Knockdown of DHHC2 but not DHHC3 prevented TTX- or Kyn-induced augmentation of endogenous PSD-95 accumulation. Dashed lines (100%) indicate the normalized control level. ***, P < 0.001. (C) Analyzed by confocal laser-scanning microscopy (CLSM). n = 10–15 neurons. (D) Analyzed by TIRFM. n = 5 neurons. miLacZ is a control miRNA targeting LacZ (β-galactosidase). (B–D) Error bars indicate SD. Bar, 5 µm.
Mentions: To follow changes in synaptic PSD-95 accumulation over time, we first performed time-lapse imaging of cultured hippocampal neurons transfected with PSD-95–GFP by TIRFM, which excites only molecules within 100 nm of the cover glass. TIRFM preferentially visualizes wild-type (WT) PSD-95–GFP as discrete punctae on dendrites, which are not seen with cytosolic palmitoylation–deficient (CS) mutant PSD-95 or GFP (Fig. 1, A and B). We confirmed comparable expression levels of PSD-95 (WT) and PSD-95 (CS) in transfected culture (Fig. 1 B). These data confirm that palmitoylation mediates membrane trafficking and synaptic clustering of PSD-95 (Topinka and Bredt, 1998). Because PSD-95 visualized by TIRFM apposes presynaptic synaptophysin and VGLUT1 and overlaps postsynaptic NR1 NMDA receptor (Fig. 1 C), TIRFM tracks synaptic PSD-95. When ionotropic glutamate receptor activity was blocked by kynurenic acid (Kyn), the intensity of PSD-95–GFP by TIRFM steadily increased over 2 h, whereas the intensity of PSD-95 (CS) did not change (Fig. 1, D and E; and Video 1). This Kyn-induced PSD-95 increase was blocked by coapplication of 2-bromopalmitate (2-BP), which is a palmitoyl acyl transfer inhibitor. PSD-95 signals did not detectably change within 2 h of 2-BP treatment alone (Fig. 1 E). These results indicate that newly occurring palmitoylation mediates this synaptic accumulation of PSD-95. Tetrodotoxin (TTX), a blocker of action potentials, also increased PSD-95 accumulation. The dynamic change of PSD-95 intensity was specific to palmitoylation as the localizations of GFP-Rac1-CLLL (Cys-Leu-Leu-Leu), a geranylgeranylated CaaL motif, and synaptophysin-GFP, a presynaptic protein, did not change upon Kyn treatment (Fig. 1 E). Synaptic PSD-95 accumulation upon activity blockade was also confirmed by antibody staining of native PSD-95 (see Fig. 4, C and D). The effect of Kyn or TTX on PSD-95 accumulation does not reflect newly synthesized PSD-95, as cycloheximide (CHX), an inhibitor of protein synthesis, did not affect the Kyn- or TTX-induced PSD-95 increase (Fig. S1, A and B; and Video 2). Thus, PSD-95 palmitoylation increases at the postsynaptic membrane upon activity blockade. These results are complementary to receptor activation–induced depalmitoylation of PSD-95 (El-Husseini et al., 2002).

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