Cameron P L, Südhof T C, Jahn R, De Camilli P
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510.
J Cell Biol. 1991 Oct;115(1):151-64. doi: 10.1083/jcb.115.1.151.
We have reported previously that the synaptic vesicle (SV) protein synaptophysin, when expressed in fibroblastic CHO cells, accumulates in a population of recycling microvesicles. Based on preliminary immunofluorescence observations, we had suggested that synaptophysin is targeted to the preexisting population of microvesicles that recycle transferrin (Johnston, P. A., P. L. Cameron, H. Stukenbrok, R. Jahn, P. De Camilli, and T. C. Südhof. 1989. EMBO (Eur. Mol. Biol. Organ.) J. 8:2863-2872). In contrast to our results, another group reported that expression of synaptophysin in cells which normally do not express SV proteins results in the generation of a novel population of microvesicles (Leube, R. E., B. Wiedenmann, and W. W. Franke. 1989. Cell. 59:433-446). We report here a series of morphological and biochemical studies conclusively demonstrating that synaptophysin and transferrin receptors are indeed colocalized on the same vesicles in transfected CHO cells. These observations prompted us to investigate whether an overlap between the distribution of the two proteins also occurs in endocrine cell lines that endogenously express synaptophysin and other SV proteins. We have found that endocrine cell lines contain two pools of membranes positive for synaptophysin and other SV proteins. One of the two pools also contains transferrin receptors and migrates faster during velocity centrifugation. The other pool is devoid of transferrin receptors and corresponds to vesicles with the same sedimentation characteristics as SVs. These findings suggest that in transfected CHO cells and in endocrine cell lines, synaptophysin follows the same endocytic pathway as transferrin receptors but that in endocrine cells, at some point along this pathway, synaptophysin is sorted away from the recycling receptors into a specialized vesicle population. Finally, using immunofluorescent analyses, we found an overlap between the distribution of synaptophysin and transferrin receptors in the dendrites of hippocampal neurons in primary cultures before synapse formation. Axons were enriched in synaptophysin immunoreactivity but did not contain detectable levels of transferrin receptor immunoreactivity. These results suggest that SVs may have evolved from, as well as coexist with, a constitutively recycling vesicular organelle found in all cells.
我们之前报道过，突触囊泡（SV）蛋白突触素在成纤维细胞CHO细胞中表达时，会在一群循环微泡中积累。基于初步的免疫荧光观察，我们曾提出突触素靶向于预先存在的循环转铁蛋白的微泡群体（约翰斯顿，P.A.，P.L.卡梅伦，H.斯图肯布罗克，R.亚恩，P.德卡米利，和T.C.苏德霍夫。1989年。《欧洲分子生物学组织杂志》8:2863 - 2872）。与我们的结果相反，另一组报道称，突触素在正常不表达SV蛋白的细胞中表达会导致产生一群新的微泡（勒贝，R.E.，B.维登曼，和W.W.弗兰克。1989年。《细胞》59:433 - 446）。我们在此报告一系列形态学和生化研究，确凿地证明在转染的CHO细胞中，突触素和转铁蛋白受体确实共定位于同一囊泡上。这些观察结果促使我们研究这两种蛋白的分布是否也在内源表达突触素和其他SV蛋白的内分泌细胞系中存在重叠。我们发现内分泌细胞系含有两群对突触素和其他SV蛋白呈阳性的膜。这两群中的一群也含有转铁蛋白受体，并且在速度离心时迁移速度更快。另一群则没有转铁蛋白受体，对应于具有与SV相同沉降特性的囊泡。这些发现表明，在转染的CHO细胞和内分泌细胞系中，突触素与转铁蛋白受体遵循相同的内吞途径，但在内分泌细胞中，在这条途径的某个点上，突触素从循环受体中被分选到一个特殊的囊泡群体中。最后，通过免疫荧光分析，我们发现在原代培养的海马神经元突触形成前的树突中，突触素和转铁蛋白受体的分布存在重叠。轴突富含突触素免疫反应性，但不含有可检测水平的转铁蛋白受体免疫反应性。这些结果表明，SV可能是从所有细胞中都存在的一种组成型循环的囊泡细胞器进化而来，并与其共存。
