Rao A, Steward O
Department of Neuroscience, University of Virginia, Charlottesville 22908.
J Neurosci. 1991 Sep;11(9):2881-95. doi: 10.1523/JNEUROSCI.11-09-02881.1991.
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)
先前的研究提出了一个假说，即突触后膜的某些蛋白质是在突触后位点局部合成的。为了评估这一假说，将包含树突片段的突触体组分与标记氨基酸一起孵育，使其掺入蛋白质中。然后将标记的突触体进一步分级分离至突触质膜（SPM）水平，进而分离出突触连接复合体（SJC）。通过闪烁计数和蛋白质测定来评估突触体组分及其亚组分的比活性（每分钟计数/微克蛋白质），并用SDS - 聚丙烯酰胺凝胶电泳（SDS - PAGE）和荧光自显影对标记的多肽进行表征。使用真核生物蛋白质合成抑制剂环己酰亚胺（CYC）和线粒体蛋白质合成抑制剂氯霉素（CAP）来评估线粒体和真核生物蛋白质合成对总体掺入的贡献。从标记的突触体中获得的SPM和SJC亚组分均含有标记的多肽。来自标记突触体的SPM的比活性大约与突触体的其他非线粒体膜成分的比活性相等。因此，SPM的标记并非由于这些其他标记膜成分的污染。线粒体组分在标记的突触体的膜成分中具有最高的比活性，但在CAP处理的突触体的线粒体组分中，比活性降低了47%，而SPM的比活性并未因该处理而降低。因此，SPM标记并非由于线粒体污染。去污剂不溶性SJC的比活性与其来源的SPM相当。通过向冷突触体制备物中添加标记的可溶性蛋白质，然后分级分离以获得SPM和SJC，来评估可溶性蛋白质污染对SPM和SJC标记的可能性。未检测到标记的可溶性蛋白质与SPM或SJC的结合。这些结果支持了某些突触蛋白是局部合成的这一假说。来自标记突触体的SPM的SDS凝胶荧光自显影片显示，在大约14、18、26、28、36、38、42、45、55、60和116 kDa的分子量处有标记条带。在来自标记突触体的突触连接复合体的荧光自显影片中仍可明显看到这六种标记多肽，其分子量分别为38、42、45、55、60和116 kDa。在CYC处理的突触体的SPM和SJC的荧光自显影片中未见到这些标记条带，而在CAP处理的突触体的荧光自显影片中它们仍然存在。因此，这些标记多肽是由真核生物核糖体系统产生的。（摘要截断于400字）
