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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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Differential subcellular distribution of PSD-95 palmitoylating enzymes. (A) Specificity of antibodies to DHHC2 and -3. The bands detected by anti-DHHC2 (closed arrowheads) and anti-DHHC3 (open arrowhead) antibodies disappeared when protein expression was knocked down by siRNAs. IB, immunoblot; scr, scramble. (B) DHHC2 was enriched in the postsynaptic density (PSD) fractions (Triton X-100–insoluble postsynaptic; closed arrowheads), whereas DHHC3 was detected in only the P3 fraction (open arrowhead). H, homogenate; S, supernatant; P, precipitate; Syn, synaptosome; Sol, Triton X-100 soluble; Ins, Triton X-100–insoluble postsynaptic density fractions. (C) DHHC2 localized in dendrites and the cell body as small vesicular structures, whereas DHHC3 specifically localized at the Golgi apparatus in 18-DIV hippocampal neurons. (D) Effective knockdown of endogenous DHHC2 and -3. Cultured hippocampal neurons were transfected with mCherry-miR RNAi (miDHHC2 and -3) expression vectors at 10 DIV. 18-DIV neurons were stained by DHHC2 or -3 antibody. Note that somatodendritic DHHC2 vesicles and Golgi DHHC3 (arrows) were not stained in mCherry-expressing knocked down neurons (red). Bars: (C [left] and D) 20 µm; (C [right]) 5 µm.
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fig3: Differential subcellular distribution of PSD-95 palmitoylating enzymes. (A) Specificity of antibodies to DHHC2 and -3. The bands detected by anti-DHHC2 (closed arrowheads) and anti-DHHC3 (open arrowhead) antibodies disappeared when protein expression was knocked down by siRNAs. IB, immunoblot; scr, scramble. (B) DHHC2 was enriched in the postsynaptic density (PSD) fractions (Triton X-100–insoluble postsynaptic; closed arrowheads), whereas DHHC3 was detected in only the P3 fraction (open arrowhead). H, homogenate; S, supernatant; P, precipitate; Syn, synaptosome; Sol, Triton X-100 soluble; Ins, Triton X-100–insoluble postsynaptic density fractions. (C) DHHC2 localized in dendrites and the cell body as small vesicular structures, whereas DHHC3 specifically localized at the Golgi apparatus in 18-DIV hippocampal neurons. (D) Effective knockdown of endogenous DHHC2 and -3. Cultured hippocampal neurons were transfected with mCherry-miR RNAi (miDHHC2 and -3) expression vectors at 10 DIV. 18-DIV neurons were stained by DHHC2 or -3 antibody. Note that somatodendritic DHHC2 vesicles and Golgi DHHC3 (arrows) were not stained in mCherry-expressing knocked down neurons (red). Bars: (C [left] and D) 20 µm; (C [right]) 5 µm.

Mentions: We next examined the cellular locus for PSD-95 palmitoylation. We focused on DHHC2 and -3, as hippocampal neurons express these PATs but much less DHHC7 and -15 (Fig. S3 A). Immunoblotting with specific antibodies (Fig. 3 A) showed that DHHC2 occurred in the postsynaptic density fraction, whereas DHHC3 was present only in the P3 fraction, which contains Golgi proteins (Fig. 3 B). Consistent with this finding, DHHC3 specifically localizes to the somatic Golgi apparatus (Keller et al., 2004; Tsutsumi et al., 2009), whereas DHHC2 distributes in the dendrites and cell body as small vesicular-like structures (Fig. 3 C). These signals are specific, as the staining completely disappeared in the validated knockdown vector–transfected neuron (Fig. 3 D).


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)

Differential subcellular distribution of PSD-95 palmitoylating enzymes. (A) Specificity of antibodies to DHHC2 and -3. The bands detected by anti-DHHC2 (closed arrowheads) and anti-DHHC3 (open arrowhead) antibodies disappeared when protein expression was knocked down by siRNAs. IB, immunoblot; scr, scramble. (B) DHHC2 was enriched in the postsynaptic density (PSD) fractions (Triton X-100–insoluble postsynaptic; closed arrowheads), whereas DHHC3 was detected in only the P3 fraction (open arrowhead). H, homogenate; S, supernatant; P, precipitate; Syn, synaptosome; Sol, Triton X-100 soluble; Ins, Triton X-100–insoluble postsynaptic density fractions. (C) DHHC2 localized in dendrites and the cell body as small vesicular structures, whereas DHHC3 specifically localized at the Golgi apparatus in 18-DIV hippocampal neurons. (D) Effective knockdown of endogenous DHHC2 and -3. Cultured hippocampal neurons were transfected with mCherry-miR RNAi (miDHHC2 and -3) expression vectors at 10 DIV. 18-DIV neurons were stained by DHHC2 or -3 antibody. Note that somatodendritic DHHC2 vesicles and Golgi DHHC3 (arrows) were not stained in mCherry-expressing knocked down neurons (red). Bars: (C [left] and D) 20 µm; (C [right]) 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig3: Differential subcellular distribution of PSD-95 palmitoylating enzymes. (A) Specificity of antibodies to DHHC2 and -3. The bands detected by anti-DHHC2 (closed arrowheads) and anti-DHHC3 (open arrowhead) antibodies disappeared when protein expression was knocked down by siRNAs. IB, immunoblot; scr, scramble. (B) DHHC2 was enriched in the postsynaptic density (PSD) fractions (Triton X-100–insoluble postsynaptic; closed arrowheads), whereas DHHC3 was detected in only the P3 fraction (open arrowhead). H, homogenate; S, supernatant; P, precipitate; Syn, synaptosome; Sol, Triton X-100 soluble; Ins, Triton X-100–insoluble postsynaptic density fractions. (C) DHHC2 localized in dendrites and the cell body as small vesicular structures, whereas DHHC3 specifically localized at the Golgi apparatus in 18-DIV hippocampal neurons. (D) Effective knockdown of endogenous DHHC2 and -3. Cultured hippocampal neurons were transfected with mCherry-miR RNAi (miDHHC2 and -3) expression vectors at 10 DIV. 18-DIV neurons were stained by DHHC2 or -3 antibody. Note that somatodendritic DHHC2 vesicles and Golgi DHHC3 (arrows) were not stained in mCherry-expressing knocked down neurons (red). Bars: (C [left] and D) 20 µm; (C [right]) 5 µm.
Mentions: We next examined the cellular locus for PSD-95 palmitoylation. We focused on DHHC2 and -3, as hippocampal neurons express these PATs but much less DHHC7 and -15 (Fig. S3 A). Immunoblotting with specific antibodies (Fig. 3 A) showed that DHHC2 occurred in the postsynaptic density fraction, whereas DHHC3 was present only in the P3 fraction, which contains Golgi proteins (Fig. 3 B). Consistent with this finding, DHHC3 specifically localizes to the somatic Golgi apparatus (Keller et al., 2004; Tsutsumi et al., 2009), whereas DHHC2 distributes in the dendrites and cell body as small vesicular-like structures (Fig. 3 C). These signals are specific, as the staining completely disappeared in the validated knockdown vector–transfected neuron (Fig. 3 D).

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