Evidence that protein constituents of postsynaptic membrane specializations are locally synthesized: analysis of proteins synthesized within synaptosomes.
Previous studies have led to the hypothesis that some proteins of the postsynaptic membrane are locally synthesized at postsynaptic sites. To evaluate this hypothesis, synaptosome fractions that included fragments of dendrites were allowed to incorporate labeled amino acid into protein. The labeled synaptosomes were then subfractionated to the level of the synaptic plasma membrane (SPM) and then the synaptic junctional complex (SJC). The specific activity (cpm/microgram protein) of the synaptosome fraction and its subfractions was assessed by scintillation counting and protein assay, and labeled polypeptides were characterized by SDS-PAGE and fluorography. The contribution of mitochondrial and eucaryotic protein synthesis to the overall incorporation was evaluated using cycloheximide (CYC), a eucaryotic protein synthesis inhibitor, and chloramphenicol (CAP), a mitochondrial protein synthesis inhibitor. Both the SPM and the SJC subfractions obtained from labeled synaptosomes contained labeled polypeptides. The SPM from labeled synaptosomes had a specific activity approximately equal to that of other nonmitochondrial membrane components of the synaptosome. Thus, labeling of the SPM was not due to contamination by these other labeled membrane components. The mitochondrial fraction had the highest specific activity of the membrane components of the labeled synaptosome, but the specific activity was reduced by 47% in mitochondrial fractions from CAP-treated synaptosomes, while the specific activity of the SPM was not reduced by this treatment. Thus, SPM labeling is not due to mitochondrial contamination. The specific activity of the detergent-insoluble SJC was comparable to that of the SPM from which it was derived. The possibility of labeling of SPM and SJC by contamination with soluble proteins was assessed by adding labeled soluble proteins to a cold synaptosome preparation that was then subfractionated to obtain the SPM and SJC. There was no detectable binding of labeled soluble proteins to the SPM or SJC. These results support the hypothesis that some synaptic proteins are locally synthesized. Fluorographs of SDS gels of SPM from labeled synaptosomes revealed labeled bands at approximate molecular weights of 14, 18, 26, 28, 36, 38, 42, 45, 55, 60, and 116 kDa. Six of these labeled polypeptides at 38, 42, 45, 55, 60, and 116 kDa were still evident in fluorographs of the synaptic junctional complex from labeled synaptosomes. None of these labeled bands were seen in fluorographs of SPM and SJC from CYC-treated synaptosomes, whereas they were still present in fluorographs of CAP-treated synaptosomes. These labeled polypeptides are therefore produced by eucaryotic ribosomal systems.(ABSTRACT TRUNCATED AT 400 WORDS)
Rao
A
A
Department of Neuroscience, University of Virginia, Charlottesville 22908.
Steward
O
O
D000818:Animals; D002458:Cell Fractionation; D002701:Chloramphenicol / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D008565:Membrane Proteins / Q000096:biosynthesis*; D008854:Microscopy, Electron; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000096:biosynthesis*; D051381:Rats; D013570:Synaptic Membranes / Q000378:metabolism*; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D002701:Chloramphenicol; D003513:Cycloheximide