O-GlcNAc is a widespread dynamic carbohydrate modification of cytosolic and nuclear proteins with features analogous to phosphorylation. O-GlcNAc acts critically in many cellular processes, including signal transduction, protein degradation, and regulation of gene expression. However, the study of its specific regulatory functions has been limited by difficulties in mapping sites of O-GlcNAc modification. We report methods for direct enrichment and identification of in vivo O-GlcNAc-modified peptides through lectin weak affinity chromatography (

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling.

The strength of synaptic transmission is controlled by the number and activity of neurotransmitter receptors. However, little is known about absolute numbers and densities of receptor and scaffold proteins and the stoichiometry of molecular interactions at synapses. Here, we conducted three-dimensional and quantitative nanoscopic imaging based on single-molecule detections to characterize the ultrastructure of inhibitory synapses and to count scaffold proteins and receptor binding sites. We observed a close correspondence between the spatial organization of gephyrin scaffolds and glycine receptors at spinal cord synapses. Endogenous gephyrin was clustered at densities of 5,000-10,000 molecules/μm(2). The stoichiometry between gephyrin molecules and receptor binding sites was approximately 1:1, consistent with a two-dimensional scaffold in which all gephyrin molecules can contribute to receptor binding. The competition of glycine and GABAA receptor complexes for synaptic binding sites highlights the potential of single-molecule imaging to quantify synaptic plasticity on the nanoscopic scale.

In the mammalian central nervous system, the structure known as the postsynaptic density (PSD) is a dense complex of proteins whose function is to detect and respond to neurotransmitter released from presynaptic axon terminals. Regulation of protein phosphorylation in this molecular machinery is critical to the activity of its components, which include neurotransmitter receptors, kinases/ phosphatases, scaffolding molecules, and proteins regulating cytoskeletal structure. To characterize the phosphorylation state of proteins in PSD samples, we combined strong cation exchange (SCX) chromatography with IMAC. Initially, tryptic peptides were separated by cation exchange and analyzed by reverse phase chromatography coupled to tandem mass spectrometry, which led to the identification of phosphopeptides in most SCX fractions. Because each of these individual fractions was too complex to characterize completely in single LC-MS/MS runs, we enriched for phosphopeptides by performing IMAC on each SCX fraction, yielding at least a 3-fold increase in identified phosphopeptides relative to either approach alone (SCX or IMAC). This enabled us to identify at least one site of phosphorylation on 23% (287 of 1,264) of all proteins found to be present in the postsynaptic density preparation. In total, we identified 998 unique phosphorylated peptides, mapping to 723 unique sites of phosphorylation. At least one exact site of phosphorylation was determined on 62% (621 of 998) of all phosphopeptides, and ϳ80% of identified phosphorylation sites are novel. Molecular & Cellular Proteomics 5: 914 -922, 2006.

The postsynaptic density (PSD) signaling machinery contains proteins with diverse functions. Brain region-specific variations in PSD components mediate distinct physiological responses to synaptic activation. We have developed mass spectrometry-based methods to comprehensively compare both relative protein expression and phosphorylation status from proteins present in biochemical preparations of postsynaptic density. Using these methods, we determined the relative expression of 2159 proteins and 1564 phosphorylation sites in PSD preparations from murine cortex, midbrain, cerebellum, and hippocampus. These experiments were conducted twice using independent biological replicates, which allowed us to assess the experimental and biological variability in this system. Concerning protein expression, cluster analysis revealed that known functionally associated proteins display coordinated synaptic expression. Therefore, proteins identified as co-clustering with known protein complexes are prime candidates for assignment as previously unrecognized components. Concerning degree of phosphorylation, we observed more extensive phosphorylation sites on N-methyl-D-aspartate (NMDA) receptors than ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, consistent with the central role of N-methyl-D-aspartate receptors in processing synaptic transmission patterns. Average kinase and phosphatase levels were highest in the hippocampus, correlating with a higher overall phosphopeptide abundance present in this brain region. These findings suggest that the hippocampus utilizes reversible protein phosphorylation to a greater extent than other brain regions when modifying synaptic strength. Molecular & Cellular Proteomics 7:684 -696, 2008.Synaptic transmission between neurons in the central nervous system involves the release of neurotransmitter from presynaptic neurons and its detection by specific ligand-gated ion channels in the surface membrane of postsynaptic neurons. These neurotransmitter receptors exist as part of a highly organized protein complex known as the postsynaptic density (PSD).1 In addition to neurotransmitter receptors, the PSD is composed of proteins of diverse function (1, 2). These functional groups include scaffolding molecules, kinases and phosphatases, G proteins and their effectors, and adhesion proteins.The coordinated functioning of the different PSD components regulates in part the strength of signaling between the pre-and postsynaptic neurons. At the molecular level, this regulation can be achieved by alterations in protein localization (3-5), reversible post-translational modifications (e.g. phosphorylation (6, 7) and O-GlcNAc glycosylation (8)), and changes in protein levels via local synthesis and degradation (9). Integration of these processes may be the basis of long term changes in synaptic efficacy thought to underlie higher cognitive processes such as learning and memory (10).Proteomics approaches aimed at identifying synaptic proteins (11-16) and their post-translational modificat...

Synaptic plasticity is dependent upon the differential sorting, delivery and retention of neurotransmitter receptors, yet the mechanisms underlying these processes are poorly understood. In the present study, we have found that differential sorting of glutamate receptor subtypes begins within the endoplasmic reticulum (ER) of rat hippocampal neurons. While AMPARs are trafficked to the plasma membrane via the conventional somatic Golgi network, NMDARs are diverted from the somatic ER into a specialized ER sub-compartment that bypasses somatic Golgi, merging instead with dendritic Golgi outposts. Intriguingly, this ER sub-compartment is composed of highly mobile vesicles containing the NMDAR subunits NR1 and NR2B, the microtubule-dependent motor protein KIF17, and the postsynaptic adaptor proteins CASK and SAP97. Furthermore, our data demonstrate that the retention and trafficking of NMDARs within this ER sub-compartment requires both CASK and SAP97. These data indicate that NMDARs are sorted away from AMPARs via a non-conventional secretory pathway that utilizes dendritic Golgi outposts.

High local concentrations of glycine receptors (GlyRs) at inhibitory postsynaptic sites are achieved through their binding to the scaffold protein gephyrin. The N-and C-terminal domains of gephyrin are believed to trimerize and dimerize, respectively, thus contributing to the formation of submembranous gephyrin clusters at synapses. GlyRs are associated with gephyrin also at extrasynaptic locations. We have investigated how gephyrin oligomerization influences GlyR dynamics and clustering in COS-7 cells and in cultured spinal cord neurons. To this aim, we have expressed isolated N-and C-terminal domains of gephyrin that interfere with the oligomerization of the full-length protein. We also studied the effect of an endogenous splice variant, ge(2,4,5), with a decreased propensity to trimerize. A reduction of the size and number of gephyrin-GlyR clusters was found in cells expressing the various interfering gephyrin constructs. Using fluorescence recovery after photobleaching, we studied the exchange kinetics of synaptic gephyrin clusters. Real-time singleparticle tracking was used to analyze the mobility of GlyRs. We found that all the tested constructs displayed faster rates of recovery than wild-type gephyrin and increased the mobility of extrasynaptic receptors, showing that gephyrin-gephyrin interactions modulate the lateral diffusion of GlyRs. Furthermore, we observed an inverse correlation between GlyR diffusion properties and gephyrin cluster size that depended on the number of binding sites blocked by the different constructs. Since alterations in the oligomerization properties of gephyrin are related to the dynamics of GlyRs, the gephyrin splice variant ge(2,4,5) may be implicated in the modulation of synaptic strength.

The synaptic insertion of GluR1-containing AMPA-type glutamate receptors (AMPARs) is critical for synaptic plasticity. However, mechanisms responsible for GluR1 insertion and retention at the synapse are unclear. The synapse-associated protein SAP97 directly binds GluR1 and participates in its forward trafficking from the Golgi network to the plasma membrane. Whether SAP97 also plays a role in scaffolding GluR1 at the postsynaptic membrane is controversial, due to its expression as a collection of alternatively spliced isoforms with ill-defined spatial and temporal distributions. In the present study, we have used live imaging and electrophysiology to demonstrate that two postsynaptic, N-terminal isoforms of SAP97 directly modulate the levels, dynamics, and function of synaptic GluR1-containing AMPARs. Specifically, the unique N-terminal domains confer distinct subsynaptic localizations onto SAP97, targeting the palmitoylated α-isoform to the postsynaptic density (PSD) and the L27 domain-containing β-isoform primarily to non-PSD, perisynaptic regions. Consequently, α- and βSAP97 differentially influence the subsynaptic localization and dynamics of AMPARs by creating binding sites for GluR1-containing receptors within their respective subdomains. These results indicate that N-terminal splicing of SAP97 can control synaptic strength by regulating the distribution of AMPARs, and hence their responsiveness to presynaptically released glutamate.