Dynamic regulation of AMPA-type glutamate receptors represents a primary mechanism for controlling synaptic strength, though mechanisms for this process are poorly understood. The palmitoylated postsynaptic density protein, PSD-95, regulates synaptic plasticity and associates with the AMPA receptor trafficking protein, stargazin. Here, we identify palmitate cycling on PSD-95 at the synapse and find that palmitate turnover on PSD-95 is regulated by glutamate receptor activity. Acutely blocking palmitoylation disperses synaptic clusters of PSD-95 and causes a selective loss of synaptic AMPA receptors. We also find that rapid glutamate-mediated AMPA receptor internalization requires depalmitoylation of PSD-95. In a nonneuronal model system, clustering of PSD-95, stargazin, and AMPA receptors is also regulated by ongoing palmitoylation of PSD-95 at the plasma membrane. These studies suggest that palmitate cycling on PSD-95 can regulate synaptic strength and regulates aspects of activity-dependent plasticity.

Streptavidin and avidin are used ubiquitously because of the remarkable affinity of their biotin binding, but they are tetramers, which disrupts many of their applications. Making either protein monomeric reduces affinity by at least 10(4)-fold because part of the binding site comes from a neighboring subunit. Here we engineered a streptavidin tetramer with only one functional biotin binding subunit that retained the affinity, off rate and thermostability of wild-type streptavidin. In denaturant, we mixed a streptavidin variant containing three mutations that block biotin binding with wild-type streptavidin in a 3:1 ratio. Then we generated monovalent streptavidin by refolding and nickel-affinity purification. Similarly, we purified defined tetramers with two or three biotin binding subunits. Labeling of site-specifically biotinylated neuroligin-1 with monovalent streptavidin allowed stable neuroligin-1 tracking without cross-linking, whereas wild-type streptavidin aggregated neuroligin-1 and disrupted presynaptic contacts. Monovalent streptavidin should find general application in biomolecule labeling, single-particle tracking and nanotechnology.

Factors that control differentiation of presynaptic and postsynaptic elements into excitatory or inhibitory synapses are poorly defined. Here we show that the postsynaptic density (PSD) proteins PSD-95 and neuroligin-1 (NLG) are critical for dictating the ratio of excitatory-to-inhibitory synaptic contacts. Exogenous NLG increased both excitatory and inhibitory presynaptic contacts and the frequency of miniature excitatory and inhibitory synaptic currents. In contrast, PSD-95 overexpression enhanced excitatory synapse size and miniature frequency, but reduced the number of inhibitory synaptic contacts. Introduction of PSD-95 with NLG augmented synaptic clustering of NLG and abolished NLG effects on inhibitory synapses. Interfering with endogenous PSD-95 expression alone was sufficient to reduce the ratio of excitatory-to-inhibitory synapses. These findings elucidate a mechanism by which the amounts of specific elements critical for synapse formation control the ratio of excitatory-to-inhibitory synaptic input.S ynapse formation involves stabilization of initial sites of contact between axons and dendrites, followed by recruitment of specific protein complexes to newly formed presynaptic and postsynaptic structures (1-4). Neuronal contact formation is spatially and temporally controlled by changes in protein content and shape at areas of contact (5, 6). The total number of synapses formed and ratio of excitatory-to-inhibitory synaptic inputs a neuron receives are factors critical for determining neuronal excitability. Appropriate synthesis and recruitment of specific factors important for building synaptic contacts are thought to power this process. However, the identity of molecules that dictate the balance between excitatory and inhibitory synaptic contacts remains elusive. Several lines of evidence indicate that the scaffolding postsynaptic density (PSD) protein, PSD-95, is involved in orchestrating excitatory synapse maturation and specificity (7). PSD-95 is exclusively localized to glutamatergic synapses (8). Moreover, PSD-95 expression correlates with the period of excitatory synapse maturation (7, 9-11). Augmentation of excitatory synapse activity and ion channel clustering is driven by PSD-95 but not by related proteins, including synapse-associated protein (SAP)-102 and , and PSD-95 regulates clustering and activity of the ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors through a direct interaction with stargazin (7,(13)(14)(15)(16)(17)(18)(19)(20). However, it is unknown how PSD-95 effects are translated into changes in apposing presynaptic terminals. A candidate molecule for mediating PSD-95 effects on presynaptic maturation is the cell adhesion molecule neuroligin (NLG). NLG is present at excitatory postsynaptic sites and associates with the third PSD-95͞Dlg͞ZO-1 homology (PDZ) domain of PSD-95 through its C-terminal PDZ-binding site (2,21,22).The postsynaptic PDZ protein S-SCAM, another known binding partner of NLG, is also possibly involved in modulating NLG effects on ...

During synaptic development, proteins aggregate at specialized pre- and postsynaptic structures. Mechanisms that mediate protein clustering at these sites remain unknown. To investigate this process, we analyzed synaptic targeting of a postsynaptic density protein, PSD-95, by expressing green fluorescent protein- (GFP-) tagged PSD-95 in cultured hippocampal neurons. We find that postsynaptic clustering relies on three elements of PSD-95: N-terminal palmitoylation, the first two PDZ domains, and a C-terminal targeting motif. In contrast, disruptions of PDZ3, SH3, or guanylate kinase (GK) domains do not affect synaptic targeting. Palmitoylation is sufficient to target the diffusely expressed SAP-97 to synapses, and palmitoylation cannot be replaced with alternative membrane association motifs, suggesting that a specialized synaptic lipid environment mediates postsynaptic clustering. The requirements for PDZ domains and a C-terminal domain of PSD-95 indicate that protein-protein interactions cooperate with lipid interactions in synaptic targeting.