DHHC5 Interacts with PDZ Domain 3 of Post-synaptic Density-95 (PSD-95) Protein and Plays a Role in Learning and Memory^*

Materials and General Methods

Protein G-agarose beads were obtained from Pierce. FuGENE 6 was obtained from Roche Applied Science. All restriction enzymes were obtained from New England Biolabs (Ipswich, MA). All other reagents and chemicals were from Sigma. Protein concentrations were determined by the DC protein assay (Bio-Rad).

Antibodies

Chicken anti-mouse DHHC5 IgY antibodies were produced (Genetel Laboratories) using a peptide consisting of amino acids 245–261 of mouse DHHC5 (CSSPAPRYLGRPKKEKT) and affinity purified using a peptide column. This antibody was used for immunoblotting following in vitro co-immunoprecipitation assays at a dilution of 10:000. A rabbit anti-mouse DHHC5 polyclonal antibody was obtained from Sigma (catalogue no. HPA014670, distributed in Europe by Atlas Antibodies, Stockholm, Sweden). Immunoblotting with this antibody was performed using a dilution factor of 1:1000. The mouse anti-rat PSD-95 monoclonal antibody (6G6–1C9), the rabbit anti-rat VAMP1 polyclonal antibody, and the rabbit anti-rat calnexin polyclonal antibody were from Abcam (Cambridge, MA). A rabbit anti-rat PSD-95 polyclonal antibody was obtained from Synaptic Systems (Goettingen, Germany). The rabbit anti-human GPRIN1 polyclonal antibody was obtained from Proteintech Group (Chicago, IL). The Alexa Fluor 568 goat anti-mouse IgG secondary antibody was obtained from Invitrogen. Anti-HA² ascites was obtained from Covance (Emeryville, CA). The anti-Myc mouse monoclonal antibody (9E10), the anti-Myc rabbit polyclonal antibody, the normal mouse IgG control, the anti-rat GAP43 monoclonal antibody, and the anti-bovine COXIV monoclonal antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Plasmids

A full-length mouse DHHC5 cDNA clone was obtained from Open Biosystems (catalogue number MMM1013-65887); the sequence corresponds to the mouse DHHC5 cDNA in GenBank^TM BC_020051. For yeast two-hybrid screening, sequences corresponding to the amino acids 220–715 (the carboxyl-terminal cytosolic domain) were cloned into the pLexN vector (Invitrogen) using EcoRI and BamHI sites. Full-length mouse PSD-95, PICK1, and GRIP1 cDNAs were obtained from Open Biosystems (catalogue numbers EMM1002-11769, MMM1013-9200227, and MMM1013-98478796, respectively, corresponding to GenBank^TM BC_014807, GenBank^TM BC_048788, and BC_067398). The full-length PSD-95 cDNA and fragments corresponding to amino acids 245–517, 245–447, and 397–721 were cloned into pVP16-3 (Clontech) using NotI and XbaI for use in two-hybrid assays. The mouse PICK1 cDNA (and a fragment corresponding to amino acids 1–200) and GRIP 1 cDNA (and fragment corresponding to amino acids 401–800) were cloned into pVP16-3 at EcoRI and BglII sites. The mouse DHHC5 carboxyl-terminal fragment (amino acids 220–715) was cloned into the pCMV-HA vector (Clontech) using EcoRI and BglII sites to generate the plasmid pHA-CtermDHHC5. Plasmid pHA-CtermDHHC5ΔEISV that deletes the extreme carboxyl terminus of DHHC5 was generated from the parent plasmid by site-directed mutagenesis (QuikChange II XL, Stratagene). The full-length mouse PSD95 was cloned into pcDNA-Myc-His vector (Invitrogen) using NheI and XhoI sites and into pEFGP-C1 (Clontech) using XhoI and SacII sites. The coding regions of all the above plasmids were sequenced to ensure integrity of the constructs. Further details of plasmid construction including primer sequences are available upon request.

Generation of DHHC5-deficient Mice

A mouse embryonic stem cell line (RRD553, strain 129/Ola) with an insertional mutation in DHHC5 was obtained from BayGenomics (24) through the International Gene Trap Consortium (25, 26). The gene-trapping vector, pGT11xf, was designed to introduce an in-frame fusion between the 5′ exons of the trapped gene and a reporter, βgeo (a fusion of β-galactosidase and neomycin phosphotransferase II). To determine the location of the genomic insertion site in the RRD553 stem cell line, genomic DNA was extracted from the embryonic stem cells using the DNeasy blood and tissue kit (Qiagen). PCR was then performed using primers fp1 (within exon 3 of DHHC5, 5′-CCAGGACTAAGCCTGAATGTGTCACC-3′) and rp1 (within the βgeo gene of the gene-trapping vector, 5′-TGCCCAGTCATAGCCGAATA-3′), and the PCR product was sequenced to determine the insertion site. The embryonic stem cells were injected into C57BL/6 blastocysts to create chimeric mice, which were bred with C57BL/6 mice to generate heterozygous DHHC5-deficient mice. The heterozygous mice were then interbred to generate all genotypes of DHHC5-deficient mice. The mice were weaned at 21 days of age and housed in an approved barrier facility with a 12-h light/dark cycle. All animal experiments were performed with the approval of the Institutional Animal Care and Research Advisory Committee at the University of Texas Southwestern Medical Center at Dallas.

Genotyping by Southern Blot

Genomic DNA was extracted from mouse tail tips using the Puregene Core Kit A (Qiagen). Approximately 10–20 μg of genomic DNA was digested with EcoRV and PvuII and analyzed by Southern blot. A probe (163 bp) was generated by polymerase chain amplification from mouse genomic DNA using primers 5′-CAGGTGTCCAGGACTAAGCC-3′ and 5′-CAACAGGGAGCTTACATGAGA-3′ derived from sequences within DHHC5 exon 3 (NCBI Entrez Gene ID 228136). The wild-type and mutant alleles are detected as 6.0- and 4.7-kb bands, respectively.

Quantitative Reverse Transcription-PCR

Total RNA was extracted from whole mouse brains using an RNeasy Lipid Tissue Midi kit (Qiagen) following the manufacturer's protocol. First-strand synthesis was performed using RNA (1 μg) and random primers using the iScript cDNA synthesis kit (Bio-Rad). Quantitative PCR was performed using iTaq Supermax with ROX (Bio-Rad). The TaqMan primers, corresponding to sequences in exons 3 and 4 of DHHC5, were obtained from Applied Biosystems (assay ID Mm00523158_m1). Glyceraldehyde-3-phosphate dehydrogenase sequences were used for normalization (assay ID Mm03302249_g1). The thermal cycling conditions were 3 min at 95 °C followed by 50 cycles of denaturation for 15 s at 95 °C and annealing for 45 s at 60 °C. Normalized data were used to compare relative levels of DHHC5 in +/+, +/gt, and gt/gt samples using ΔΔCt analysis (27).

Whole Membrane Lysates

Mice were euthanized by carbon dioxide narcosis, and tissues were quickly extracted on ice. Each tissue sample was homogenized in four volumes of 20 mm HEPES, pH 7.4, 150 mm NaCl, 1 mm EDTA with 1× complete protease inhibitors (Roche Applied Science) using a Brinkman Polytron homogenizer. Unbroken tissue and cells were removed by centrifugation at 1000 × g for 15 min at 4 °C. The supernatant was further centrifuged at 100,000 × g for 1 h. The whole membrane lysate was extracted from the pellet using radioimmune precipitation assay buffer (50 mm Tris, pH 7.2, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing 1× complete protease inhibitors at 4 °C for 1 h. The protein sample was then centrifuged at 16,000 × g for 10 min at 4 °C. The cleared supernatant was transferred to a clean Eppendorf tube. The proteins were separated by SDS-PAGE and analyzed by immunoblotting.

Synaptic Plasma Membrane Fractionation

Synaptic plasma membrane fractionation was performed according to a previously published method with some modifications (28). Procedures were carried out at 4 °C, and all buffers contained complete protease inhibitors (Roche Applied Science). Briefly, whole brain tissues were pooled from six adult mice and homogenized in 15 volumes of Tris acetate (TA) buffer (50 mm Tris acetate, pH 7.4, 2 mm CaCl₂, 10% (w/v) sucrose) using a glass-Teflon homogenizer. Homogenates were centrifuged at 1000 × g for 15 min to obtain a crude nuclear pellet (P1) and supernatant (S1). The S1 fraction was then centrifuged at 16,000 × g for 30 min, and the resulting mitochondrial pellet (P2) was resuspended in 20 mm HEPES, pH 7.4, and 2 mm CaCl₂ and centrifuged at 16,000 × g for 20 min. The resulting pellet (P3) was resuspended in 5 volumes of hypotonic TA buffer (5 mm Tris acetate, pH 8.1, 2 mm CaCl₂) and incubated on ice for 45 min. Solid sucrose was added to this fraction to 35% (w/v) and then layered on the bottom of a discontinuous sucrose density gradient. TA buffer containing 20 and 10% (w/v) sucrose constituted the middle and upper layers, respectively. The gradient was centrifuged at 60,000 × g for 2 h in a swinging bucket rotor, and the synaptosomal fraction was collected from the 35–20% sucrose interface. The synaptosomal fraction was diluted with 3 volumes of TA buffer and centrifuged at 30,000 × g for 30 min. The resulting synaptosomal pellet was resuspended in TA buffer containing 1% Triton X-100 and centrifuged at maximum speed in a microcentrifuge for 30 min. The supernatant containing the presynaptic membrane fraction was recovered, and the pellet containing the postsynaptic membrane fraction was resuspended in radioimmune precipitation assay buffer (50 mm Tris, pH 7.2, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS). Equal amounts of protein (20 μg) were loaded onto SDS-PAGE gels, transferred to Immobilon membranes, and subjected to immunoblotting.

Yeast Two-hybrid Assays

A rat brain cDNA library (postnatal day 8) cloned into the prey vector pVP16-3 was a generous gift of Dr. Thomas Sudhof (29). L40 yeast strains harboring the bait vector (pLexN) expressing the carboxyl-terminal fragment of mouse DHHC5 protein (amino acids 220–715 cloned into EcoRI and BamHI sites) and prey vector (pVP16-3) containing the rat brain cDNA library were grown on CSM-WLH (−Trp/−Leu/−His) plates (Clontech). In this protocol, only those clones that contain DHHC5-interacting partners will induce the transcription of the HIS3 gene and survive selection in the absence of histidine. A total of 100 clones were randomly selected for sequencing. The yeast DNA was extracted and subjected to PCR using primers within the vector sequence; V1 (5′-GTTTACCGATGCCCTTGG-3′) and V2 (5′-CGTTGTAAAACGACGGCC-3′). The PCR products were sequenced using V1 primer. Two rounds of selection and sequencing were performed.

To confirm the interactions between DHHC5 and PDZ domain-containing proteins, similarly, L40 yeast strains harboring the bait vector (pLexN) expressing amino acids 220–715 of the mouse DHHC5 protein and prey vectors (pVP16-3) expressing fragments of the PDZ domain-containing proteins were grown on CSM-WLH (−Trp/−Leu/−His) plates. Interactions in yeast were confirmed using a semi-quantitative β-galactosidase assay (29).

Co-immunoprecipitation

For co-immunoprecipitation of DHHC5 and PSD-95 in transfected cells, HEK 293 cells were transfected either with PSD-95-Myc or HA-CtermDHHC5 or HA-CtermDHHC5ΔEISV as indicated. After 48 h, cells were washed twice with cold phosphate-buffered saline and lysed with four volumes of lysis buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 1% Triton X-100, 1 mm EDTA) containing a complete protease inhibitor mixture (Roche Applied Science). For the co-immunoprecipitation assay, 500 μg of cell lysate was diluted to 500 μl using Buffer A (50 mm Tris, pH 7.5, 150 mm NaCl, 1% Triton X-100, 1 mm EDTA, 0.25% gelatin, 0,02% sodium azide) and pre-cleared with 25 μl of Protein G-agarose beads (Pierce) at 4 °C for 1 h. The pre-cleared solution was incubated with either anti-Myc mouse monoclonal antibody (9E10) (3 μg, Santa Cruz) or anti-HA ascites (6 μg, Covance) at 4 °C for 1 h followed by incubation with 25 μl of Protein G-agarose beads (Pierce) at 4 °C overnight. After incubation, the beads were washed three times with Buffer A and then eluted with 30 μl of SDS-PAGE sample buffer (62.5 mm Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, and 0.001% bromphenol blue) containing 5% β-mercaptoethanol. Eluted samples were analyzed by immunoblotting.

To analyze co-immunoprecipitation of DHHC5 and PSD-95 in vivo, mouse brain tissues were homogenized in four volumes of buffer containing 20 mm HEPES, 150 mm NaCl, 1 mm EDTA, pH 7.4, with 1× complete protease inhibitors (Roche Applied Science) using a motor-driven homogenizer. The unbroken tissue and cells were removed by centrifugation at 1000 × g for 15 min at 4 °C. The supernatant was further centrifuged at 100,000 - g for 1 h. The whole membrane lysate was extracted from the pellet using Buffer B (50 mm Tris, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100, 1% sodium deoxycholate) containing 1× complete protease inhibitors at 4 °C for 1 h. The protein sample was then centrifuged at 16,000 × g for 10 min at 4 °C. The cleared supernatant was transferred to a clean Eppendorf tube. For co-immunoprecipitation assay, 600 μg of brain membrane lysate was pre-cleared with 25 μl of Protein G-agarose beads (Pierce) at 4 °C for 1 h. The pre-cleared solution was incubated with anti-PSD 95 monoclonal antibody (1:40 dilution, 6G6–1C9, Abcam) at 4 °C for 1 h followed by an incubation with 25 μl of Protein G-agarose beads (Pierce) at 4 °C overnight. Normal mouse IgG (Santa Cruz sc-2025) at the same concentration was used as a negative control. After incubation, the beads were washed 3 times with Buffer B and then eluted with 30 μl of SDS-PAGE sample buffer (62.5 mm Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, and 0.001% bromphenol blue) containing 5% β-mercaptoethanol. Eluted samples were subjected to immunoblotting using the anti-DHHC5 (Sigma HPA014670) or anti-PSD-95 (Synaptic Systems) rabbit polyclonal antibodies.

Behavioral Testing

All mice were male and female littermates ∼10–12 weeks of age at the time of testing. Mice were group housed on a 12-h light/dark cycle with food and water available ad libitum. Behavioral testing occurred during the light cycle. Tests for locomotor activity, open field behavior, motor coordination (Rotarod), and fear conditioning were performed; all mice received all tests. Locomotor activity was assessed by placing mice individually into a clean, plastic mouse cage (18 × 28 cm) with a small amount of bedding that was located inside a dark Plexiglas box. Movement was monitored by 5 photobeams in one dimension (Photobeam Activity System, San Diego Instruments, San Diego, CA) for 2 h, with the number of beam breaks recorded every 5 min. For open field activity testing, mice were individually placed in the periphery of a novel open field environment (44 × 44 cm, walls 30 cm high) and allowed to explore for 5 min. The animals were monitored from above by a video camera connected to a computer running video tracking software (Ethovision 3.0, Noldus, Leesburg, VA) to determine the time, distance moved, and number of entries into two areas: the periphery (5 cm from the walls) and the center (14 × 14 cm). The open field arenas were wiped with disinfectant and allowed to dry between mice. For motor coordination testing, mice were placed on a rotarod (model 755, IITC Life Science Inc., Woodland Hills, CA). The rod was then accelerated from 5 to 45 rpm over 5 min. The time that each mouse fell from the rod was recorded. If a mouse held onto the rod and rotated completely around two times, this was recorded as a fall from the rod at that time. Each mouse was tested four times (15–30 min inter-trial interval) each day for two consecutive days. Fear conditioning was measured in boxes equipped with a metal grid floor connected to a scrambled shock generator (Med Associates Inc., St. Albans, VT). On the first day (training) mice were individually placed in the testing chamber and after 2 min received 2 tone-shock pairings (30-s white noise, 95-db tone co-terminated with a 2-s, 0.5-mA foot-shock, 1-min intertrial interval) and were removed from the chamber 1 min after the last shock. Twenty-four hours after training, memory of the context was measured by placing the mice into the same chambers, and freezing was manually measured every 5 s for 5 min. Forty-eight hours after training, memory for the white noise cue was measured by placing the mice in a box with altered floors and walls, lighting, and a vanilla smell. Freezing was manually measured every 5 s for 3 min, then the noise cue was turned on for an additional 3 min, and freezing was measured every 5 s. The experimenter rating the freezing was blind to the genotype of the animals. Approximately 1 week after fear conditioning, the animals were assessed for foot-shock sensitivity. The mice were placed into the same chambers and given a foot-shock every 15 s. For this test, the first foot-shock was 0.05 mA and increased by 0.05-mA intervals. The first shock that evoked a flinch response and jump, and vocalization was recorded.

Immunohistochemistry

To investigate the extent of the DHHC5 immunoreactivity within the brains of wild-type and DHHC5 gt/gt mice, a one-in-six series of free-floating 40-μm frozen coronal sections from each animal was stained via a modified version of a previously published immunoperoxidase protocol (30). Sections underwent antigen retrieval via heating in citrate buffer, pH 6.0, for 30 min at 80 °C and were allowed to cool to room temperature. To quench endogenous peroxidase activity, sections were incubated for 30 min in 1% H₂O₂ and subsequently blocked for 40 min in 15% normal goat serum in Tris-buffered saline, 0.3% Triton X-100 (TBS-T). Sections were incubated overnight at 4 °C in polyclonal rabbit anti-DHHC5 (1:250) diluted in TBS-T containing 10% normal goat serum. Subsequently, sections were rinsed in TBS and incubated for 2 h with secondary antibody (biotinylated goat anti-rabbit IgG, Vector Laboratories, Peterborough, UK, 1:1000) diluted in TBS-T containing 10% normal goat serum. After rinsing in TBS, sections were incubated for 2 h in avidin-biotin-peroxidase complex (Vectastain Elite ABC kit, Vector Laboratories) diluted in TBS. After rinsing in TBS, DHHC5 immunoreactivity was visualized by incubation in 0.05% diaminobenzidine tetrahydrochloride (Sigma) and 0.001% H₂0₂ for 25 min, a sufficient time to ensure saturation for this reaction. Sections were rinsed in TBS, mounted onto slides, air-dried, cleared in xylene, and coverslipped in DPX (Sigma). Appropriate negative controls where the primary antibody was omitted were included in each experiment.

Generation of DHHC5 Gene-trapped Mice

To study the function of the DHHC5 gene in vivo, DHHC5-deficient mice were derived from a mouse embryonic cell line (RRD553) generated by random insertional mutagenesis of 129/Ola embryonic stem cells at BayGenomics and distributed by the International Gene Trap Consortium (25, 26). Genomic DNA obtained from the cell line (and subsequent tail DNA) was found to contain the β-geo cassette insertion in intron 3 on the mouse DHHC5 gene, between nucleotides 5′-cctgtattatcacttatttt-3′ and 5′-tcttttttttttttaattag-3′ (25579683 and 25579684 in GenBank^TM accession no. NT_039207.7) (Fig. 1A). The RRD553 cell line was injected into C57BL/6J blastocysts to create chimeric male mice, which were bred with C57BL/6J females to produce an F₁ line. A long polypyrimidine tract located immediately 5′ to the insertion site precluded routine PCR as a genotyping method. Therefore, as shown in Fig. 1B, Southern blotting was used for the subsequent genotyping, and wild-type, heterozygous, and homozygous mice were clearly distinguished. DHHC5 heterozygote males and females from the F₁ generation were bred to obtain an F₂ generation with all genotypes.

Genotypes produced by early heterozygous crosses deviated significantly from Hardy-Weinberg equilibrium (+/+, 97 (34%); +/gt, 154 (53%); gt/gt, 37 (13%), n = 288, p < 0.0001), consistent with ∼50% loss of gt/gt homozygotes before birth. The modest decrease in expected heterozygotes did not reach statistical significance (p = 0.08). An increased number of resorption sites was seen in heterozygous matings (data not shown). However, live-born homozygotes appear healthy and reproduce sufficiently to maintain the line in the homozygous state.

The β-geo cassette consists of a splice acceptor site fused in frame to a stop codon, which leads to truncation of the normal protein product. However, inefficient use of the splice acceptor site may in some instances cause a small amount of normal mRNA to be produced (31). Therefore, we determined the DHHC5 mRNA level in all three genotypes using quantitative reverse transcriptase-PCR. Indeed, a residual level of DHHC5 mRNA in the homozygous mice (about 16% compared with wild-type) was detected (Fig. 1C). The amount of DHHC5 protein produced in numerous experiments was determined to be no greater than 7% in brain tissue, as estimated by immunoblotting (Fig. 1D). For this reason, the insertion should be considered a hypomorphic allele and is designated “gt” (for gene-trapped allele).

DHHC5 Is Enriched in Brain and Localized to Synaptic Membranes

Extracts prepared from various wild-type and DHHC5 gt/gt tissues were analyzed by immunoblotting using a rabbit polyclonal anti-DHHC5 antibody (Fig. 2A). In wild-type mice, the protein was found to be highly enriched in brain, detectable in liver and heart, and undetectable in a number of other tissues. No detectable DHHC5 was found in gt/gt homozygotes in any tissue examined except the brain. No striking differences were seen upon gross dissection of different brain regions (Fig. 2B).

To further localize DHHC5 within brain tissue, we used a synaptic fractionation protocol to prepare pre- and post-synaptic membrane fractions to examine the distribution of DHHC5. As shown in Fig. 2C, a clean separation between pre- and post-synaptic densities was achieved; for example, VAMP1, a presynaptic membrane marker, was only detected in the presynaptic membrane fraction (lane 5), whereas PSD-95, a post-synaptic membrane marker, was only found in the post-synaptic membrane fraction (lane 6). DHHC5 was clearly detected in these synaptic membrane fractions, predominantly in the post-synaptic density.

Effect of DHHC5 Deficiency on Hippocampal-dependent Learning and Memory

To further delineate the role of DHHC5 in brain function, we subjected the DHHC5 gt/gt and littermate control mice to several standard behavioral tests (Fig. 3). Contextual fear-conditioning is a hippocampus-dependent learning task in which animals associate a specific location and its surroundings with an electric shock. The standard protocol involves a learning (acquisition) phase and a testing phase. Mice tested at 10–12 weeks showed no significant differences in the acquisition phase of testing (data not shown). However, as shown in Fig. 3A, 24 h after training, there was a significant reduction in the amount of freezing for DHHC5 gt/gt mice in the context where they previously received the foot-shocks. There was no difference in the amount of freezing in a context not previously paired to foot-shock, and the freezing to the tone (cued condition) was normal. Foot-shock sensitivity was unimpaired (Fig. 4B), as were a number of other behavioral tests, such as open field activity, locomotion, and motor coordination and learning (Fig. 3, C–E). These results suggest that DHHC5 plays a role in hippocampal learning.

Candidate DHHC5 Interacting Proteins

To further delineate the role of DHHC5 in brain, the 496-amino acid carboxyl terminus of DHHC5 was cloned into a bait vector for yeast two-hybrid screening against a library derived from whole rat brain cDNA. This large domain is predicted to reside in the cytosol based on comparison with homologous proteins (32, 33). In two independent trials, 100 positive clones were recovered from a total of 12 × 10⁶ clones, and 64 of these were sequenced for identification.

As shown in supplemental Table 1, the clones fell into two major classes, PDZ-domain containing proteins and cytoskeletal proteins. The most frequently identified clone was PSD-95 (12/64 hits). Other PDZ domain family members recovered were protein that interacts with C kinase 1 (PICK1) (2 hits), glutamate receptor interacting protein-1 (GRIP1) (1 hit), and GRIP2 (1 hit). These findings suggested that the interactions may be mediated through a PDZ binding motif of DHHC5. Indeed, the last four amino acids of DHHC5, EISV, form a canonical class II PDZ binding motif (XΦXΦCOOH) (34).

Interaction of PSD-95 and DHHC5 in Transfected Cells and in Mouse Brain

As PSD-95 was the most frequently identified DHHC5 interacting partner in the yeast two-hybrid assay, we further tested its interaction with DHHC5 in a mammalian system using a co-immunoprecipitation assay. As we were unable to solubilize full-length DHHC5 when it was overexpressed in heterologous cells, we used the large carboxyl-terminal fragment (amino acids 220–715) with an amino-terminal epitope tag that was similar to that used in the yeast two hybrid screen. A custom made chicken anti-mouse DHHC5 IgY was used in these assays to detect the DHHC5 carboxyl terminus. As shown in Fig. 4A, lane 1, both expressed proteins could be detected in whole cell extracts (wce) when 293 cells were co-transfected with PDS95-myc and the HA-tagged DHHC5 carboxyl terminus (designated HA-CtermDHHC5). The DHHC5 carboxyl terminus appeared as a doublet, which was resistant to alkaline phosphatase or endoglycosidase H or peptide N-glycosidase F digestion (data not shown). Immunoprecipitation using anti-Myc antibodies to pull down PSD-95 were also able to co-immunoprecipitate DHHC5 (lane 2), confirming that the interaction occurs in transfected cells. This interaction was not seen when an empty vector control was used (lanes 3 and 6) or when the final four amino acids of DHHC5 (the PDZ binding domain) were deleted (lane 5). Fig. 4A, lanes 7–12, demonstrate the same interaction when the antibodies are reversed, i.e. when the HA epitope tag is used to pull down the DHHC5 carboxyl terminus and PSD-95 is detected in the immunoprecipitate. These data suggest that PSD-95 and the carboxyl terminus of DHHC5 have the capacity to interact in a mammalian cell and that the interaction is mediated through the DHHC5 carboxyl-terminal PDZ binding domain. Significant colocalization of DHHC5 and PSD-95 could also be demonstrated in transfected CHOK1 cells (supplemental Fig. 1).

To determine whether an endogenous interaction between DHHC5 and PSD-95 could be detected, mouse brain membranes were extracted using a stringent protocol that employs membrane solubilization in 1% sodium deoxycholate. As shown in Fig. 4B, when PSD-95 was immunoprecipitated from brain lysates, DHHC5 was also found in the immunoprecipitates (Fig. 4B, lanes 1–3). The DHHC5 signal was absent from similarly prepared brain lysates obtained from gene-targeted DHHC5 mice (Fig. 4B, lanes 4–6). We were unable to test whether PSD-95 co-immunoprecipitates with endogenous DHHC5 as neither antibody that recognizes DHHC5 on immunoblots would pull down DHHC5 from brain lysates.

To determine potential sites of interaction between DHHC5 and PSD-95, we performed individual yeast two-hybrid assays using selected regions of PSD-95 and the DHHC5 C carboxyl-terminal bait. As shown in Fig. 4C, all portions of PSD-95 were dispensable for binding to DHHC5 with the exception of the third PSD-95 PDZ domain, which was sufficient for binding. PDZ domains most commonly bind to carboxyl-terminal tails of proteins, with the binding partner tail inserting into a groove within the PDZ domain (35). Deletion of the carboxyl-terminal four amino acids of DHHC5 (EISV) completely abolished its interaction with PSD-95 in this assay as well (data not shown), again suggesting that this interaction with PSD-95 is mediated through DHHC5 carboxyl-terminal PDZ-binding motif, a result we had also obtained in transfected mammalian cells (Fig. 4A).

As DHHC5 is a member of a family of protein palmitoyltransferases based on sequence homology, we wondered whether the S-acylation of PSD-95 or other palmitoylated proteins would be altered in the brains of DHHC5 gt/gt mice. We, therefore, analyzed whole brain lysates using the acyl-biotin exchange method (36), which uses hydroxylamine at neutral pH to remove fatty acids from modified cysteines, exchanging them for HDPD-biotin, which can be captured to a solid-phase support and detected by immunoblotting. No change in the level of S-acylation of PSD-95 or several synaptic vesicles proteins (GAP43, GPRIN1, and VAMP-1) was seen, at least when averaged over the entire brain (supplemental Fig. 2). Of note, DHHC5 was itself shown to be S-acylated in these experiments.

DHHC5 Immunolocalization

Immunohistochemical staining of brain tissue revealed pronounced changes in the intensity and distribution of staining for DHHC5 in the hippocampus and cortex of DHHC5 gt/gt mice in comparison with wild-type mice (Fig. 5). The staining was virtually absent from the neuropil of these mutant mice, whereas expression was either decreased or retained in regions of the hippocampus, consistent with regional expression from the hypomorphic allele. Decreased DHHC5 immunoreactivity was most pronounced in neurons of the dentate gyrus and CA1 subfields of the hippocampal formation (Fig. 5, B, D, G, and H) but was also evident in the cortex (Fig. 5, J and L). These results are consistent with the notion that DHHC5 acts within neuronal populations of the hippocampus to affect learning.