Evidence that protein constituents of postsynaptic membrane specializations are locally synthesized: time course of appearance of recently synthesized proteins in synaptic junctions.

Previous studies have led to the hypothesis that some protein constituents of postsynaptic membrane specializations are locally synthesized near postsynaptic sites. The present study focuses on one prediction of this hypothesis, specifically, that if some proteins of the postsynaptic membrane specialization are locally synthesized, then the delay between synthesis and assembly into synaptic junctional membrane could be short. We evaluate the time course of appearance of recently synthesized protein in synaptic junctions by pulse-labeling hippocampal slices maintained in vitro with radiolabeled protein precursors, and then isolating subcellular fractions enriched in synaptic plasma membranes (SPM) and synaptic junctional complexes (SJC). We report that there is no evidence of a delay in the appearance of recently synthesized proteins in SPM and SJC fractions. Labeled proteins could be detected as early as 15 min after the initiation of the pulse-labeling period, and the extent of labeling increased monotonically thereafter. The labeling could not be accounted for by contamination of synaptic membrane fractions with other membranes, because the relative specific activity of the SPM and SJC fractions was the same or higher than that of the less pure fractions from which these synaptic fractions were derived. One-dimensional PAGE-fluorography was used to provide an initial characterization of which proteins were labeled in SJC fractions. We found that the most prominent labeled bands were at apparent molecular weights of approximately 43-44, 55-56, and 60 kd, with more lightly labeled bands at about 38 and 116 kd. In some preparations, there was a labeled doublet at about 36-38 kd. There were also other lightly labeled bands at other molecular weights. These bands were much less heavily labeled than the bands at 43-44, 55-56, and 60 kd, however. There was little labeling in the molecular weight range of the "major psd protein" (the alpha subunit of CAM-kinase), although there was diffuse labeling throughout the 45-52 kd region. These results are consistent with the hypothesis that some of the protein constituents of the postsynaptic junctional complex are synthesized by polyribosomes which are selectively localized beneath synaptic junctions.