Leimer U, Franke W W, Leube R E
Division of Cell Biology, German Cancer Research Center, Im Neuenheimer Feld 280.
Exp Cell Res. 1996 Apr 10;224(1):88-95. doi: 10.1006/excr.1996.0114.
The N-glycosylated integral membrane protein synaptophysin is one of the major polypeptide components of small presynaptic transmitter-containing vesicles in neurons and of similar vesicles in neuroendocrine cells of mammals. Functional properties, including a possible participation in channel formation, have been investigated by integration of purified synaptophysin into planar lipid bilayers. To overcome some of the inherent limitations of such an in vitro approach we have overexpressed the rat synaptophysin cDNA in nonneuronal, non-neuroendocrine insect cells with the help of recombinant baculovirus. The complete polypeptide was produced in infected ovarian Sf9 cells at levels exceeding those observed in rat brain. The partially N-glycosylated molecules could be extracted from membranes with non-ionic detergents, most effectively with n-octyl-beta-D-glucopyranoside, and could be enriched on chromatofocusing columns. By immunoelectron microscopy synaptophysin was shown to be integrated in the correct orientation into the endoplasmic reticulum, various pleomorphic vesicles and the plasma membrane. Using cell fractionation, including density gradient centrifugation and immunoisolation, we characterized distinct synaptophysin-rich vesicles. These vesicles may help to understand molecular principles of vesicle biogenesis in general and the function of synaptophysin in particular.
N-糖基化整合膜蛋白突触素是神经元中含突触前递质的小囊泡以及哺乳动物神经内分泌细胞中类似囊泡的主要多肽成分之一。通过将纯化的突触素整合到平面脂质双分子层中，对其功能特性（包括可能参与通道形成）进行了研究。为了克服这种体外方法的一些固有局限性，我们借助重组杆状病毒在非神经元、非神经内分泌昆虫细胞中过表达大鼠突触素 cDNA。在感染的卵巢 Sf9 细胞中产生了完整的多肽，其水平超过了在大鼠脑中观察到的水平。部分 N-糖基化分子可用非离子去污剂从膜中提取，最有效的是用正辛基-β-D-吡喃葡萄糖苷，并且可以在色谱聚焦柱上富集。通过免疫电子显微镜显示，突触素以正确的方向整合到内质网、各种多形性囊泡和质膜中。使用细胞分级分离法，包括密度梯度离心和免疫分离，我们对富含突触素的不同囊泡进行了表征。这些囊泡可能有助于总体上理解囊泡生物发生的分子原理，特别是突触素的功能。
