Linstedt A D, Hauri H P
Department of Pharmacology, University of Basel, Switzerland.
Mol Biol Cell. 1993 Jul;4(7):679-93. doi: 10.1091/mbc.4.7.679.
The Golgi complex consists of a series of stacked cisternae in most eukaryotes. Morphological studies indicate the existence of intercisternal cross-bridge structures that may mediate stacking, but their identity is unknown. We have identified a 400-kDa protein, giantin, that is localized to the Golgi complex because its staining in double immunofluorescence experiments was coincident with that of galactosyltransferase, both in untreated cells and in cells treated with agents that disrupt Golgi structure. A monoclonal antibody against giantin yielded Golgi staining in one avian and all mammalian cell types tested, indicating that giantin is a conserved protein. Giantin exhibited reduced mobility on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was recovered in membrane fractions after differential centrifugation or sucrose flotation, and was not released from membranes by carbonate extraction. Thus, giantin appears to be an integral component of the Golgi membrane with a disulfide-linked lumenal domain. Strikingly, the majority of the polypeptide chain is cytoplasmically disposed, because large (up to 350 kDa) proteolytic fragments of giantin could be released from intact Golgi vesicles. This feature, a large contiguous cytoplasmic domain, is present in the calcium-release channel of muscle that cross-bridges the sarcoplasmic reticulum and transverse tubule membranes. Therefore, giantin's localization, conservation, and physical properties suggest that it may participate in forming the intercisternal cross-bridges of the Golgi complex.
在大多数真核生物中，高尔基体复合体由一系列堆叠的扁平囊组成。形态学研究表明存在可能介导堆叠的扁平囊间跨桥结构，但其身份尚不清楚。我们鉴定出一种400 kDa的蛋白质——巨蛋白，它定位于高尔基体复合体，因为在双免疫荧光实验中，无论在未处理的细胞还是用破坏高尔基体结构的试剂处理的细胞中，其染色都与半乳糖基转移酶的染色一致。针对巨蛋白的单克隆抗体在测试的一种禽类和所有哺乳动物细胞类型中均产生高尔基体染色，表明巨蛋白是一种保守蛋白。在非还原十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上，巨蛋白迁移率降低，经过差速离心或蔗糖密度梯度离心后，在膜组分中回收，并且不能通过碳酸盐抽提从膜上释放。因此，巨蛋白似乎是高尔基体膜的一个组成成分，具有一个通过二硫键连接的腔内结构域。引人注目的是，大多数多肽链位于细胞质中，因为巨蛋白的大（高达350 kDa）蛋白水解片段可以从完整的高尔基体小泡中释放出来。这一特征，即一个大的连续细胞质结构域，存在于肌肉的钙释放通道中，该通道横跨肌浆网和横管膜。因此，巨蛋白的定位、保守性和物理性质表明它可能参与形成高尔基体复合体的扁平囊间跨桥。
