Howard Hughes Medical Institute, Department of Neuroscience, University of Wisconsin, Madison, Wisconsin 53706.
Howard Hughes Medical Institute, Department of Neuroscience, University of Wisconsin, Madison, Wisconsin 53706.
J Biol Chem. 2012 Oct 12;287(42):35658-35668. doi: 10.1074/jbc.M112.398883. Epub 2012 Aug 20.
Glycosylation is a major form of post-translational modification of synaptic vesicle membrane proteins. For example, the three major synaptic vesicle glycoproteins, synaptotagmin 1, synaptophysin, and SV2, represent ∼30% of the total copy number of vesicle proteins. Previous studies suggested that glycosylation is required for the vesicular targeting of synaptotagmin 1, but the role of glycosylation of synaptophysin and SV2 has not been explored in detail. In this study, we analyzed all glycosylation sites on synaptotagmin 1, synaptophysin, and SV2A via mutagenesis and optical imaging of pHluorin-tagged proteins in cultured neurons from knock-out mice lacking each protein. Surprisingly, these experiments revealed that glycosylation is completely dispensable for the sorting of synaptotagmin 1 to SVs whereas the N-glycans on SV2A are only partially dispensable. In contrast, N-glycan addition is essential for the synaptic localization and function of synaptophysin. Thus, glycosylation plays distinct roles in the trafficking of each of the three major synaptic vesicle glycoproteins.
糖基化是突触囊泡膜蛋白的一种主要翻译后修饰形式。例如，三种主要的突触囊泡糖蛋白，突触结合蛋白 1、突触小体相关蛋白和 SV2，代表囊泡蛋白总拷贝数的约 30%。先前的研究表明，糖基化对于突触结合蛋白 1 的囊泡靶向是必需的，但糖基化在突触小体相关蛋白和 SV2A 中的作用尚未详细探讨。在这项研究中，我们通过对敲除每种蛋白的基因敲除小鼠培养神经元中 pHluorin 标记蛋白的突变和光学成像，分析了突触结合蛋白 1、突触小体相关蛋白和 SV2A 上的所有糖基化位点。令人惊讶的是，这些实验表明，糖基化对于突触结合蛋白 1 到 SVs 的分拣是完全必需的，而 SV2A 上的 N-聚糖仅部分必需。相比之下，N-聚糖的添加对于突触小体相关蛋白的突触定位和功能是必需的。因此，糖基化在三种主要的突触囊泡糖蛋白的运输中发挥不同的作用。
