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Originally published In Press as doi:10.1074/mcp.M400045-MCP200 on May 28, 2004.
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Molecular & Cellular Proteomics 3:857-871, 2004.
© 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Identification and Verification of Novel Rodent Postsynaptic Density Proteins*,S

Bryen A. Jordan{ddagger}, Brian D. Fernholz§, Muriel Boussac, Chongfeng Xu, Gabriela Grigorean, Edward B. Ziff{ddagger},§ and Thomas A. Neubert,||

From the {ddagger} Department of Biochemistry, § Department of Physiology and Neuroscience, and Department of Pharmacology and Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY 10016


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The postsynaptic density (PSD) is a cellular structure specialized in receiving and transducing synaptic information. Here we describe the identification of 452 proteins isolated from biochemically purified PSD fractions of rat and mouse brains using nanoflow HPLC coupled to electrospray tandem mass spectrometry (LC-MS/MS). Fluorescence microscopy and Western blotting were used to verify that many of the novel proteins identified exhibit subcellular distributions consistent with those of PSD-localized proteins. In addition to identifying most previously described PSD components, we also detected proteins involved in signaling to the nucleus as well as regulators of ADP-ribosylation factor signaling, ubiquitination, RNA trafficking, and protein translation. These results suggest new mechanisms by which the PSD helps regulate synaptic strength and transmission.

Neurons are highly polarized cells, specializing in the reception of numerous, independent signal inputs and rapid integration of these inputs into an electrochemical response. The major sites of signal input are synapses, which are highly ordered cell junctions formed between two neurons and are typically unidirectional in fast excitatory chemical neurotransmission in the mammalian CNS. The response to neurotransmitter (NT)1 release at the synapse is provided by a protein matrix of NT receptors and supporting proteins collectively known as the postsynaptic density (PSD) (for review, see Refs. 1–3). The PSD has several proposed functions including: signal amplification, cytoskeletal anchorage, biochemical signaling regulation, and NT receptor clustering (1, 4–6). Changes in size and composition of the PSD correlate with changes in synaptic strength (7, 8), including alterations that are stably maintained such as long-term potentiation (LTP), a physiologically relevant increase in synaptic efficacy and a model for learning and memory (9, 10). Therefore, an understanding of the protein composition of the PSD is a prerequisite for modeling the molecular interactions regulating synaptic strength.

The structure of PSDs purified from rodent brains using gradient centrifugation and Triton X-100 extraction has been shown by electron microscopy (EM) to be virtually identical to the "in vivo" PSD structure (4, 11). Gel electrophoresis, enzymatic activity assays, and EM experiments have demonstrated that this procedure yields a highly pure, membrane-free PSD fraction (11, 12). Recent proteomic studies have investigated the composition of the PSD by SDS-PAGE or two-dimensional gel electrophoresis (2DE) coupled with MS (13–16). Li et al. (16) also performed shotgun proteomics using cysteine-containing peptides selected using ICAT techniques. However, each of these investigations identified less than one-third of previously described and biochemically confirmed PSD components, pointing to limitations in the techniques used. A recent paper by Yoshimura et al. (17) reports the identification by mass spectrometry of 492 proteins in the PSD, which suggests that the PSD is more complex than previously thought. However, the study was of PSDs from a subset of whole brain, and no attempt was made to confirm the localization of these proteins by independent means.

In our study, we have taken advantage of increased sensitivity in protein identification afforded by the use of SDS-PAGE to fractionate proteins followed by in-gel tryptic digestion and nanoflow LC-MS/MS (for review, see Ref. 18). LC-MS/MS provides separation in a second dimension without the loss of hydrophobic or basic proteins as with 2DE (19). We report the identification of 452 proteins in PSDs isolated from whole brain using stringent statistical criteria for validation of MS-based matches. These proteins include over 90% of published, biochemically confirmed PSD components, in addition to 307 proteins not previously shown to be in the PSD and including 75 previously uncharacterized proteins. We have expressed 16 of the novel proteins as recombinant fluorescent proteins in neurons and confirmed their localization in dendritic spines. Furthermore, subcellular fractions from our PSD purification probed with antisera against 18 additional novel proteins demonstrate that all of these proteins are present and many are enriched in the PSD fraction. Western blots of known pre- and postsynaptic proteins confirm the purity of the biochemically prepared PSD fraction used in this study. Taken together, these experiments validate the protein identifications obtained by MS. This analysis of the fundamental constituents of the PSD provides new insights into its multiple functions including protein translation, trafficking, and turnover and increases our understanding of the molecular components of learning and memory.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
PSD Purification—
Our protocol is based on that of Cohen and Carlin (11, 12). Rats or mice were euthanized by CO 2 in compliance with New York University Medical Center’s Institutional Animal Care and Use Committee, and whole brains were rapidly removed and placed in ice-cold solution A (0.32 M sucrose, 1 mM NaHCO 3, 1 mM MgCl 2, 0.5 mM CaCl 2, 0.1 mM PMSF (Sigma, St. Louis, MO) and 1x Complete Protease Inhibitors (Roche Applied Science, Indianapolis, IN). The brains were subjected to dounce homogenization in 40 ml of solution A per 10 g of wet brain tissue. The homogenates were diluted to 10% weight/volume with solution A and centrifuged at 1,400 x g for 10 min. The supernatant solution was saved, and the pellet was rehomogenized in 10% solution A per 10 g of initial weight and subjected to a centrifugation at 710 x g for 10 min. Supernates were pooled and subjected to a second centrifugation at 710 x g for 10 min. The supernates were then spun at 30,000 x g for 15 min to obtain a crude P2 fraction. The pellet was resuspended in solution B (0.32 M sucrose, 1 mM NaHCO 3) using 24 ml of solution B per 10 g of starting material. This homogenate was layered on top of a 10-ml 0.85 M sucrose, 10-ml 1 M sucrose, and 10-ml 1.2 M sucrose step sucrose gradient and centrifuged at 82,500 x g for 2 h. Purified synaptosomes were collected at the 1 M and 1.2 M sucrose interface by syringe aspiration. If the "short" procedure was used, we proceeded directly to the Triton extraction section. If the "long" method was used, the synaptosomes were resuspended with 4 volumes of solution B and collected by centrifugation at 48,200 x g for 20 min. We hypotonically lysed synaptosomes by resuspending the pellet in 10 ml of 6 mM Tris pH 8.1 per gram of starting material and rocking at 4 °C for 45 min. The lysed synaptosomes were then collected by centrifugation at 48,200 x g for 20 min followed by the Triton extraction method below.

Triton Extraction—
Synaptosomes were diluted to 60 ml of Solution B per 10 g of initial starting material and an equal volume of 1% Triton X-100, 0.32 M sucrose, 12 mM Tris pH 8.1, was added and rocked at 4 °C for 15 min. These lysed synaptosomes were centrifuged at 32,800 x g for 25 min, and the pellet was resuspended in 2.5 ml of solution B per 10 g of starting material. The suspension was layered on a second sucrose step gradient of 3 ml of 1 M sucrose, 3 ml of 1.5 M sucrose, and 4 ml of 2 M sucrose and centrifuged at 200,000 x g for 2 h. The PSD fraction was collected at the 1.5 M and 2 M sucrose interface and diluted to 6 ml with solution B. An equal volume of 1% Triton X-100, 150 mM KCl was added to obtain the "two-Triton" PSD fraction. These purified PSDs were then collected by centrifugation at 200,000 x g for 20 min. PSDs were solubilized using either an IEF rehydration buffer (9 M urea, 2 M thiourea, 4% CHAPS, 0.5% Triton X-100 in the rat "short" version) or a 2% SDS-Tris buffer in the "long" and mouse "short" protocols. The purity of the fractions was assessed by Western blots for CaMKII, NMDA receptor 1, synaptophysin, synaptic vesicle 2, and PSD-95. We isolated PSDs from rats using the "long" and "short" procedure, and from mice, we obtained PSDs using the "short" procedure. PSDs obtained from rats using the "short" procedure were solubilized using the IEF buffer described above containing urea.

Identification of Proteins from SDS-PAGE Gels by MS—
Proteins were separated by SDS-PAGE on 10% Criterion gels (Bio-Rad, Hercules, CA). After electrophoresis the proteins were visualized by Coomassie blue staining. Twenty-five (rat PSD "long" and "short" protocols) to 50 (mouse PSD "short" protocol) evenly spaced gel bands were excised from each lane without consideration of staining intensity, destained, and the proteins digested in-gel with trypsin under a tissue culture hood to minimize contamination (20). The resulting peptides were extracted and dried under vacuum, then resuspended in 5–10 µl of 0.1% TFA. The peptide mixtures were analyzed using nanoflow LC/ESI-MS-MS. The peptides were loaded onto a 0.3 x 1-mm C18 nano-precolumn (LC Packings, Sunnyvale, CA), then washed 5 min with 2% ACN in 0.1% formic acid at a flow rate of 20 µl/min. After washing, flow was reversed through the precolumn and the peptides eluted with a gradient of 2–81% ACN in 0.1% formic acid. The gradient was delivered over 80 or 120 min by a CapLC (Waters, MA) HPLC system at a flow rate of 200 nl/min, obtained by a 15:1 precolumn flow split, through a 75-µm x 15-cm fused silica capillary C18 HPLC column (LC Packings PepMap) to a fused silica distal end-coated tip nano-electrospray needle (New Objective, Woburn, MA). The Q-TOF 1 (Micromass, Manchester, United Kingdom) data acquisition involved MS survey scans and automatic data-dependent MS/MS acquisitions, which were invoked after selected ions met preset parameters of minimum signal intensity of 8 counts per second, ion charge state 2+, 3+, or 4+, and appropriate retention time. Survey scans of 1 s were followed by CID of the four most intense ions for up to 11 s each, or until 5,000 total MS/MS ion counts per precursor peptide were achieved. The raw MS data were subsequently processed using manufacturer-supplied ProteinLynx software, which generated DTA files based on each MS/MS spectrum. Control experiments demonstrate the sensitivity of this system is better than 100 fmol of each protein in the gel, and 10 fmol of each peptide injected onto the HPLC columns.

Cloning—
cDNA was obtained from Kazusa Research Institute (21) ("KIAA" prefix) or cloned from a rat brain cDNA library constructed with Superscript 3 (Invitrogen, Carlsbad, CA) and using PCR with Deep Vent (New England Biolabs, Beverly, MA) or Accuprime (Invitrogen) and gene specific primers. PCR products were digested and ligated into pEGFP vectors: N1 or N3 and C1 or C3 (Clontech, Palo Alto, CA) and pcDNA3.1/myc-His(-)A (Invitrogen).

Cell Culture, Transfection, and Immunochemistry—
Dissociated neuronal cultures were prepared as previously described (22) with the following modification: an equal amount of neocortex was included with the hippocampus. Three- to 4-week-old neurons were transfected with 4 µg of DNA with either a modified CaPO 4 (23) or LipofectAMINE 2000 (Invitrogen) according to the manufacturer’s instructions. Cells were mounted with Vectashield mounting media, which contained 4',6'-diamidino-2-phenylindole (DAPI) to visualize nuclei (Vector Labs, Burlingame, CA). Enhanced green fluorescent protein (eGFP) was visualized using an Axiovert 220M fluorescence microscope (Zeiss, Oberkochen, Germany).

Immunoblots—
Ten to 20 µg of PSD were loaded onto 4–12% gradient preformed criterion gels (Bio-Rad), transferred and blotted using a variety of antibodies generously donated by Catherine Chew (LASP-1) (24), Phillip J Coates (prohibitin and BAP37), Matt Welch (ARP2, ARP3, and ARP2/3sub5), Francis Castets (zinedin), Karl Matter (guanine exchange factor (GEF)-LFC), Katherine Wilson (barrier to autointegration-1), AB Reynolds (ARVCF and catenin p120), Gary Silverman (squamous cell carcinoma antigen-1), Michael Greenberg (ephexin N-GEF), Sharon Eden (NCK-associated protein 1-NAP125 and Wave1), Jordi Perez i Tur (LGN-1), Orly Reiner (doublecortin-like kinase), and Massimo Pietropaolo (islet cell autoantigen p69).

Bioinformatics—
Approximately 40,000 uninterpreted mass spectra were acquired and used for protein identification as follows. The raw MS data were processed using Micromass ProteinLynx 3.5 software. Sequential MS/MS scans with the same precursor ions were combined before charge state deconvolution by MaxEnt 3 software (start mass 700, peak width auto, 1 ensemble member, 20 iterations, data compressed). Background was subtracted (polynomial order 10, 10% below curve removed), peaks were smoothed (2 channel window, 1 smooth, Savitzky Golay model), and centroided (minimum peak width at half height 4, centroid top 80%). From these data, DTA files based on each MS/MS spectrum were produced, merged into a text file, and used for database searching.

Each query was searched using Mascot version 1.9.05 (25) (Matrix Science, London, United Kingdom) with permutations of the following search parameters: missed cleavages (0–3), modifications (unmodified, oxidation (M), deamidation (NQ), phosphorylation (ST), phosphorylation (Y), carbamylation (K), carbamylation (N-term)), taxonomy (mammalia, mus musculus, rattus), peptide tolerance in Daltons (0.25, 0.5, 1, 1.4), and MS/MS tolerance in Daltons (0.15, 0.25, 0.3, 0.4, 0.5). Unmodified parameters were as follows: peptide-charge (2+ and 3+), enzyme (trypsin), database (NCBI nonredundant August 16, 2003 with 1502194 sequences; 484011957 residues), instrument (ESI-QUAD-TOF), and monoisotopic masses were used. Peptides were searched with the highest number of potential modifications and with mass tolerances several times greater than the accuracy of the mass spectrometer so as to maximize the number of identified peptides. Peptides were then subjected to a series of increasingly stringent filters as variable modifications were reduced, and mass accuracy tolerances were reduced to approximate the accuracy of the mass spectrometer. The peptides were retained from the most stringent filter they passed in a manner similar to the "step analysis" algorithm of DTASelect and Contrast allows for Sequest search results (26). This analysis allowed for the optimal search condition to be retained for each peptide and overcomes the computational limitations of Mascot. This resulted in the maximum number of peptides to be identified from the 40,000 initial MS/MS spectra.

Determining the Probability of Correct Peptide Matches—
Values of p were calculated using the equation given by Mascot software to convert scores to probabilities for each query, x:

(Eq. 1)

where Score is the score given to the peptide by Mascot. This p value represents the probability that the sequence assignment is random, but does not reflect the database size, i.e. the number of potential matches for a given peptide m/z value within the peptide tolerance range. The integer Qmatch reflects the virtual database size within this range, and thus the overall probability this match is a false positive can be represented as follows:

(Eq. 2)

Determining the Probability of Correct Protein Identifications—
To assess the probability of correct protein identifications based on these peptide matches, the number of MS/MS spectra used in the search must be taken into account. To do this, each protein identifications is given an expectation value (E-value), E, dependent upon the number of queries (MS/MS spectra), q, and p'(x) for each unique matching peptide. For proteins with only one peptide match:

(Eq. 3)

and for proteins with y peptides where y > 1:

(Eq. 4)

which reflects the low probability of two or more peptides matching to one protein randomly (27).

Searches were integrated using custom software written in Java 1.3 (Sun Microsystems, Santa Clara, CA). Briefly, the Mascot search that yielded the lowest p'(x) for each peptide was used for all 40,000 queries. Peptides with p'(x) > 0.5 or Score < 15 or that were not at least 4 aa in sequence length were immediately discarded. Up to three top-scoring peptide matches were considered as long as each score was no less than 63% of the top-ranking peptide’s score, but only rarely did any second-ranking peptide pass these filters. The protein list was then constructed using a simple matrix matching proteins to any corresponding peptide identified by Mascot for all peptides that passed the filter. Any proteins without at least one peptide with p'(x) < 0.05, the threshold score determined by Mascot, were removed. All proteins were subsequently manually inspected for redundancy within clusters generated by grouping any proteins having at least 50% of the identified peptides in common. As a follow up, a second clustering was generated with NCBI BLASTCLUST (28) (minimum homology length = 0.0, percent identical residues = 80%) and manually inspected to remove orthologs that could not be clustered by common peptide analysis. The cutoff used for inclusion in the list was a maximum E-value of 1.0.

To test the validity of our statistical model for evaluating the validity of protein identifications, a random NCBI nonredundant database was generated to determine how well our E-value cutoff of 1 was able to prevent false positives. Each protein FASTA entry was rearranged such that the original sequence was destroyed, but the amino acid frequency and the protein length were unchanged for each entry.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
PSDs were purified from rat and mouse whole brains by density centrifugation using the "long" and "short" methods of Cohen et al. (11) and Carlin et al. (12), respectively. While subtle differences in staining intensities of individual proteins could be observed between Coomassie-stained SDS-PAGE gels of mouse and rat PSDs isolated by either method, the overall protein profiles appeared to be similar (Fig. 1). Each gel lane was subsequently cut into 25–50 gel slices, digested with trypsin, and analyzed for peptide content by LC-MS/MS. In total, ~40,000 MS spectra were obtained from ~120 separate LC-MS/MS experiments. These spectra were processed by Micromass MassLynx software and used to search the nonredundant NCBI database with in-house Mascot search software (25). The results from a variety of Mascot search conditions, including appropriate variable modifications, a range of tolerances, and multiple taxonomies, were integrated using custom software. This method allowed us to find the optimal search condition for each peptide in a manner similar to that of DTASelect software (26). The compiled results were then clustered by custom software and BLASTCLUST (28), which allowed orthologs and database redundancies to be collapsed manually from 4,418 initially identified proteins to ~750. The identified proteins have been reported with their calculated E-values, i.e. the number of times they would be expected to match any target randomly during a database search. We have determined an E-value cutoff mathematically (see "Experimental Procedures") and tested this value empirically by searching our entire query list against a randomized NCBI database using parameters similar to those used for our peptide identifications and integrated as described. The lowest E-value observed for a protein identification using the randomized database was 1.3, providing strong empirical corroboration for our mathematical E-value cutoff of 1. This result suggests, statistically, that we have made no false-positive protein identifications. Some proteins were identified on the basis of MS/MS spectra that appeared reasonable when inspected manually, but were excluded from our high-confidence list of 452 proteins (Table I) because they had E-values greater than 1. Moreover, several proteins that failed our rigorous statistical test have been shown by us (see below) and by others to localize to the PSD. Therefore, we have included all identified proteins in our supplemental data (Supplemental Table I). We have compared the proteins identified in this study to previously known and biochemically confirmed proteins of the PSD listed in several reviews (1–3, 29). We were unable to compare our set to the recent publication by Yoshimura et al. (17) as less than 30% of their set of identified proteins was published. Of the 452 proteins identified with high confidence, 145 were previously shown to localize to the PSD, 307 were novel to the PSD of which 75 were only characterized as cDNAs. These proteins fall into a variety of classes (see "Discussion") with likely both unique and overlapping functions with previously known components.



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  		FIG. 1. Rat and mouse PSDs do not exhibit gross differences after SDS-PAGE. Coomassie-stained SDS-PAGE gel lanes containing rat PSDs prepared by the "short" (rat PSD-s) and "long" (rat PSD-l) methods and mouse "short" (mouse PSD-s) PSDs showed that all major bands were observed in all preparations, although there were some quantitative differences in bands likely to be those identified by Walikonis et al. (13) as spectrin, tubulin, and VDAC-1 (arrows, from top to bottom).

 

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  		TABLE I Rodent postsynaptic density proteins identified with high confidence by LC-MS/MS

PSD constituents were grouped into functional families based on published reports. Novel proteins, which could be assigned to groups on the basis of predicted domains consistent with known functions, are denoted by (§). The "Pep" column refers to the number of unique (nonredundant) peptides found per protein, which were considered in the calculation of E-values, because some peptides were sequenced by MS/MS multiple times. When a single peptide was used to identify a protein not previously confirmed to be localized in the PSD, additional information is included in Supplemental Table 2. Included in "Notes/domains (predicted)" are homologs that were also found and in bold are the predicted domains for novel proteins. In the "Protein name" column, proteins that had not been previously localized to the PSD are shown in bold. For simplicity, in cases where no identified peptide could differentiate between closely related proteins, we only report those with the greatest number of peptides found; if the number of peptides is equal, proteins are listed in italics. An asterisk denotes proteins that are likely contaminants, such as GFAP and histones. We have removed keratins, cytokeratins, trypsin, albumin, and fibrinogen, as these are common contaminants in high-sensitivity MS protein identification experiments.

 
To estimate the probability that the uncharacterized proteins identified were contaminants, we cloned 16 proteins not previously found in the PSD and introduced eGFP-tagged constructs into dissociated cultures of primary cortical or hippocampal neurons. Some proteins were cloned by gene-specific PCR of cDNA reverse-transcribed from RNA isolated from rat brain or cDNA obtained from a cDNA repository (21). C-terminally tagged and N-terminally tagged eGFP constructs were transfected into neurons. All 16 proteins were enriched in dendritic spines compared with the negative control of eGFP (Fig. 2). Modifier of cell adhesion (MOCA) (Fig. 2E) and centaurin ß5 (Fig. 2H) were identified with E-values greater than 1, but exhibited strong enrichment in spines. This result supported their correct identification and demonstrated that our rigorous statistical filter, while guarding against false-positive identifications, resulted in at least some false-negative results. Only eGFP alone, the negative control, was diffusely localized throughout the neurons and did not exhibit this relative enrichment in spines (Fig. 2Q). Curiously, several proteins we identified exhibited putative nuclear localization signals (NLS), domains implicated in RNA or DNA binding, and general homology to known transcription factors or proteins that shuttle to the nucleus. Similar to MGC10772 was observed to be highly enriched in spines (Fig 2A). However, it contains the NLS (RRRLKRRQ) and was sometimes observed in nuclei when expressed in neurons (not shown). HnRNP A3-like protein contains the NLS (GGRGGYGGG), while proteins AIDA 1A and mKIAA0417 do not contain any known NLS, but were both predicted to be nuclear proteins using PSORTII and TargetP bioinformatics algorithms. When these proteins were expressed in cultured neurons, they displayed both a nuclear and spiny localization (Fig. 3). A pairwise BLAST comparison of the protein set identified in this study to a database of known NLS revealed that 11 proteins contained known NLS sequences.



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  		FIG. 2. Novel PSD members tagged with eGFP localize to spines in primary cultured neurons. A, XP_225171, similar to hypo MGC10772; B, AAH48407, Rpel1, KIAA 1733; C, XP_129322, mKIAA0417; D, NP_780693, coronin 2B/clipin C; E, NP_700462, KIAA0299, MOCA; F, BAB85526, KIAA1940; G, AAH58280, ProsapIP1; H, XP_233719, centaurin ß5; I, AAM73705, mKIAA0059; J, NP_775545, cylindromatosis turban tumor syndrome/mKIAA0849; K, NP_848893, Riken A930041I02; L, BAC27761, AIDA-1A; M, AAC52639, LASP-1; N, XP_228841, KIAA 0552-like ARF-GEP100; O, AAH46236, Riken 3110035P10; P, CAA76339, hnRNP A3; Q, GFP alone. Only GFP demonstrated a diffuse distribution, while the proteins in D, G, and K had variable distributions. Both centaurin ß5 (H) and MOCA (E) were proteins with E-values (1.4 and 25, respectively) that did not pass our statistical threshold; however, they localize strongly to dendritic spines.

 


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  		FIG. 3. Several novel proteins identified in the PSD fraction were observed in the nuclei as well as in spines. GFP-tagged proteins were transfected into primary dissociated hippocampal neurons. The mounting media included DAPI for nuclear staining. A, XP_129322, mKIAA0417; B, CAA76339, hnRNP A3; C, BAC27761, AIDA-1A.

 
Further biochemical validation of the PSD location of the identified proteins was sought by performing Western blots on different purified brain fractions. Antisera to a variety of proteins ("Experimental Procedures") were used to blot whole brain, synaptosomal, and PSD fractions (Fig. 4). The arm-domain containing catenin subfamily members p120 and armadillo repeat gene deleted in velo cardio facial syndrome, ARVCF, were observed to be highly enriched in the PSD fraction. These proteins have been implicated in cadherin turnover (30), which is a major component of PSDs (31) (Table I). The G-protein regulators GEF-LFC and ephexin-N also show substantial enrichment in the PSD fraction, with the latter showing a potentially posttranslationally modified ephexin-N being highly enriched there. Overall, the results show that a majority of the proteins probed demonstrate a clear enrichment in the PSD fraction.



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  		FIG. 4. Western blots of several novel members of the PSD. Ten to 20 µg of PSD protein were separated by SDS-PAGE and blotted using a variety of antibodies. W, whole brain extract; Syn, synaptosomal fraction; and PSD, postsynaptic density. Known molecular markers were also blotted to demonstrate the purity of the fractions used for MS/MS. ARP, actin-related protein; BAF-1, barrier to autointegration 1; BAP-37, B cell receptor-associated protein; ARVCF, armadillo-repeat-velo-cardio-facial syndrome protein; ARP2/3sub5, actin-related protein complex 2/3 subunit 5; SCCA-1, squamous cell carcinoma antigen-1, serpin3a; NAP-125, NCK-associated protein 1; LGN-1, leucine-rich repeat LGI family, member 1; DCLK, doublecortin-like kinase/mKIAA0369; ICAp69, islet cell autoantigen protein p69.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The PSD is the site at which the electrical inputs responsible for neural activity are received. In recent years, a model of the PSD as consisting of neurotransmitter receptors that mediate these electrical signals, associated downstream effectors, and an underlying matrix of cytoskeletal proteins has emerged. Our study suggests the PSD also contains a large number of other proteins involved in signal integration and anchoring and modification of other synaptic proteins. For example, we identified ~30 G-proteins and associated regulatory proteins, in addition to numerous novel serine/threonine kinases not previously shown to exist in the PSD including ELKL-motif kinase (MARK2), Traf2 and NCK-interacting kinase, and doublecortin-like kinase, the latter of which we show by Western blotting to be highly enriched in the PSD fraction (Fig. 4). We also found phosphatases and lipid modification proteins and direct regulators of these groups. Several proteins, such as diacylglycerol kinase and ABR-breakpoint cluster protein, contained calcium-binding sites in the forms of EF hands and C2 domains, which suggests they may be regulated by synaptic activity. Overall, the proteins identified illustrate a large capacity for downstream signal transduction, as well as synapse-to-nucleus signaling for regulation of gene expression and a capacity for protein turnover.

Ehlers (32) and others (9, 33) have shown that long-term changes in protein composition of the PSD and related proteins are associated with the ubiquitin degradation pathway. In particular, Ehlers’s data (32) suggests that the PSD contains ubiquitin ligase activity. We identified several proteins involved in the ubiquination pathway including the E3 ligase, cullin-5, which is ubiquitous in rat brain (34). We also identified cylindromatosis (CYLD), which has a functional ubiquitin carboxyl terminal hydrolase domain (35) and has been shown to negatively regulate NF-{kappa}B signaling by deubiquitination of proteins upstream of the I{kappa}B kinase, (36) is highly enriched in spines (Fig 2J).

In addition to protein degradation and removal, we find evidence for at least two mechanisms of accumulation of new proteins. First, remotely translated proteins may be shuttled to the PSD via a variety of proteins associated with vesicular trafficking identified here as PSD components. These include several molecular motors, ADP-ribosylation factors (ARFs) and their associated regulating proteins (37, 38), such as the putative ARF-GTPase-activating protein (GAP) centaurin ß5 (Fig 2H) and functional ARF-GEF, KIAA0763 (39) (Fig 2N) and phosphoinositide signaling proteins (40). Second and importantly, we find evidence for protein accumulation through local protein synthesis. mRNA trafficking proteins, such as pur {alpha} (41) and hnRNP A1/A3 (42), and protein translation machinery, such as ribosomal subunits and elongation factor-2, were found. Late-phase LTP is dependent on protein translation (9). Specifically, CaMKII{alpha} mRNA trafficking and translation are activity-regulated and are required for stabilization of synaptic modulations, such as LTP and memory tasks in vivo (43, 44). Therefore, our results suggest that protein turnover may be a local phenomenon that takes place proximal to the synapse, allowing for both rapid responses to synaptic activity and synapse-specific changes. That protein translation machinery itself can be locally translated in aplysia (45) suggests the possibility for a local positive feedback mechanism allowing for long-lasting synaptic modifications.

It is becoming increasingly clear that nuclear events are critical for memory formation and other high-level behavioral functions (46, 47). Signals from the synapse can be conveyed to the nucleus in several different ways, ranging from transient activity-dependent increases in nuclear Ca 2+ that may initiate nuclear events (48) to the translocation of transcriptional components such as the nuclear activator of T cells (NFAT) (49). We identified several proteins with characteristics consistent with a nuclear function. The proteins AIDA-1A, mKIAA0417, and hnRNP A3 were seen to localize to both the nucleus and to spines (Fig. 3). Moreover, we show by Western blots that several proteins known to localize to the nucleus and identified here are highly enriched in the PSD fraction (Fig. 4). Armadillo-repeat-velo-cardio-facial (ARVCF) and p120 catenin are members of the arm-repeat family and have been recently shown to regulate cadherin turnover (30). They have been shown to exist in the nucleus to varying extents (50, 51) and could function similarly to ß-catenin. RPEL-1 contains multiple RPEL domains as well as a basic region homologous to that found in proteins MAL and myocardin transcription factor, where it is known to mediate nuclear import (52, 53). The presence of multiple proteins with putative nuclear functions emphasizes the importance of synapse-to-nucleus signaling and suggests that the translocation of proteins to the nucleus represents an important pathway for transmitting activity-dependent signals to the nucleus.

Our approach identified 155 of the ~170 proteins previously confirmed (1–3, 29) independently to be located in the PSD, including 10 proteins identified with E-values that did not pass our stringent cutoff level for high-confidence protein identification. Some of the proteins missed are likely to be low-abundance and/or loosely associated proteins that may have become dislodged from the PSD during purification. For example, we detected a low-abundance protein, nNOS, in Western blots, but not by LC-MS/MS (data not shown), even though we attempted to maximize identification coverage by prefractionating PSD proteins by SDS-PAGE. For the most comprehensive analysis of the PSD that was practical, we combined the results from several purification strategies. We analyzed rat PSDs because of the historical importance of rats in electrophysiological studies, well-annotated database entries, and larger brain compared with that of mice. However, because the mouse genome is more thoroughly sequenced and given the close evolutionary relationship between rats and mice, we combined the data from both species and give the results for a composite rodent PSD. The combination of two species maximized the number of proteins identified by helping to overcome sequencing errors and non-silent, small nucleotide polymorphisms present in the NCBI database. We also applied two separate solubilization methods and combined the results to maximize identification of proteins inefficiently solubilized with either detergent set alone. Many proteins were identified based on peptides found in two or more experiments, which validates the integration of the data. The combined results identified a significantly more diverse group of proteins present in the PSD than previously reported (3, 13–16, 29, 54). A recent publication by Li et al. (16) identified over 100 proteins in the PSD by 2DE and MS. However their results encompass less than one-third of previously known PSD components and provide no independent verification of the novel identified proteins. A recent paper by Yoshimura et al. (17) reported the identification by MS of 492 proteins in PSDs derived from rat forebrain, though none of these identifications were confirmed by independent means. A comparison of their published list indicates that approximately a third of these proteins do not overlap with the proteins identified in our study. Some of the discrepancy is very likely due to differences in the sources (whole brain versus forebrain) and preparation of PSDs.

While the search of a randomized database using our MS data suggests our stringent statistical criteria for protein identification effectively guarded against false-positive protein identifications, it is possible that some of the proteins we identified are contaminants of the PSD preparations (11, 12). Many of these possible contaminants have also been identified in previous studies of the composition of the PSD (3). However, because statistical criteria for confident identification were met, these proteins were not excluded from the list. This is justified because many proteins have pleiotropic functions and by the possibility that a protein may function at low concentration in a given location (i.e. the PSD) despite high enrichment in other subcellular locations (55). We have also validated the location of many of the proteins identified that were novel to the PSD by immunofluorescence microscopy and by Western blotting. Some proteins contained a known NLS, such as similar to MGC10772 and RPEL1, and were located in the nucleus to some extent. However, in a majority of instances, these proteins were strongly enriched in spines (Fig. 2, A and B). Interestingly, our Western blot experiments shown in Fig. 4 suggest that specific forms of several proteins (for example LASP, BAP-37, ephexin-N, and ICAp69) may be enriched in the PSD based on the presence of higher or lower immunoreactive bands in PSD fractions compared with less-purified whole lysate or synaptosomal fractions.

The diverse cellular functions represented by the identified proteins suggest that some of the signal processing previously attributed to dendritic spines may be occurring in the PSD itself. For example, we found components of protein synthesis and degradation, elements of opposing processes for fine regulation through protein and phospholipid kinases and phosphatases, ubiquitination and deubiquitination enzymes, G-protein GEFs and GAPs, and proteins for bidirectional cytoskeletal modulation. That these cellular functions could be regulated at the surface of the PSD itself suggests that the PSD may have the principal role in directing the processes that regulate its own composition and ultimately synaptic strength. Localization of signaling processes at the PSD rather than more diffusely throughout the synaptic spine would also allow for more rapid biochemical responses to appropriate stimuli and increased biochemical independence between different synapses. The localization of a full complement of regulatory machinery at the PSD would reduce its reliance on the rest of the cell for fine-tuning synaptic strength. Furthermore, molecular markers of induced potentiation at the PSD (the "synaptic tag") may be the sum of the biochemical processes in the PSD rather than single molecules or small complexes. There are also likely many redundancies that allow for precise tuning of synaptic efficacy. However, because the identified proteins were from postsynaptic densities derived from the entire rat or mouse brain, it is unlikely that every PSD contains each of these protein components, especially considering the limited calculated volume of the PSD and the diverse plastic responses to identical stimuli observed among different brain areas (2, 56). Despite the evidence for increased biochemical autonomy, the ultimate function of the PSD is in its integration with the rest of the synaptic input. The output of the PSD to the whole cell is therefore not only electrical, which is summed to yield action potentials, but also chemical, which may modify genetic expression, as suggested by transcription-like factors and related proteins.


   ACKNOWLEDGMENTS
 
We thank Dr. Yuliang Ma and Dr. Vivekananda Shetty for expert help with Q-TOF LC-MS/MS.


   FOOTNOTES
 
Received, March 31, 2004, and in revised form, May 11, 2004.

Published, MCP Papers in Press, May 28, 2004, DOI 10.1074/mcp.M400045-MCP200

1 The abbreviations used are: NT, neurotransmitter; PSD, postsynaptic density; ARF, ADP-ribosylation factor; LTP, long-term potentiation; EM, electron microscopy; 2DE, two-dimensional electrophoresis; E-value, expectation value; NLS, nuclear localization signal; MOCA, modifier of cell adhesion; CYLD, cylindromatosis turban tumor syndrome protein; GEF, guanine exchange factor; GAP; GTPase-activating protein, ARVCF, armadillo-repeat-velo-cardio-facial protein; eGFP, enhanced green fluorescent protein. Back

* This work was supported by National Institutes of Health (NIH) Grant R21 NS44184-01 and Shared Instrumentation Grant S10 RR017990-01 (to T A N.) and NIH Grant R01 AG13620 (to E B Z.). B A J. and G G. were supported by NIH Training Grant T32 NS 07457-04. B D F. was supported by a National Science Foundation graduate research fellowship. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

S The on-line version of this manuscript (available at http://www.mcponline.org) contains supplemental material. Back

|| To whom correspondence should be addressed: Skirball Institute of Biomolecular Medicine Lab 5-18, New York University School of Medicine, New York, NY 10016. Tel.: 212-263-7265; Fax: 212-263-8214; E-mail: neubert{at}saturn.med.nyu.edu


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