Department of Plant Biology and Biotechnology, Faculty of Life Sciences, University of Copenhagen, Frederiksberg, Denmark.
PLoS One. 2012;7(2):e31324. doi: 10.1371/journal.pone.0031324. Epub 2012 Feb 21.
The Golgi apparatus is the main site of glycan biosynthesis in eukaryotes. Better understanding of the membrane topology of the proteins and enzymes involved can impart new mechanistic insights into these processes. Publically available bioinformatic tools provide highly variable predictions of membrane topologies for given proteins. Therefore we devised a non-invasive experimental method by which the membrane topologies of Golgi-resident proteins can be determined in the Golgi apparatus in living tissues. A Golgi marker was used to construct a series of reporters based on the principle of bimolecular fluorescence complementation. The reporters and proteins of interest were recombinantly fused to split halves of yellow fluorescent protein (YFP) and transiently co-expressed with the reporters in the Nicotiana benthamiana leaf tissue. Output signals were binary, showing either the presence or absence of fluorescence with signal morphologies characteristic of the Golgi apparatus and endoplasmic reticulum (ER). The method allows prompt and robust determinations of membrane topologies of Golgi-resident proteins and is termed GO-PROMTO (for GOlgi PROtein Membrane TOpology). We applied GO-PROMTO to examine the topologies of proteins involved in the biosynthesis of plant cell wall polysaccharides including xyloglucan and arabinan. The results suggest the existence of novel biosynthetic mechanisms involving transports of intermediates across Golgi membranes.
高尔基体是真核生物中糖基化生物合成的主要场所。更好地了解涉及的蛋白质和酶的膜拓扑结构，可以为这些过程提供新的机制见解。公开可用的生物信息学工具对给定蛋白质的膜拓扑结构提供了高度可变的预测。因此，我们设计了一种非侵入性的实验方法，可以在活组织中的高尔基体中确定高尔基体驻留蛋白的膜拓扑结构。高尔基体标记物用于根据双分子荧光互补原理构建一系列报告子。报告子和感兴趣的蛋白质被重组融合到黄色荧光蛋白（YFP）的分裂半部分，并与报告子在烟草叶组织中共表达。输出信号是二进制的，要么显示荧光，要么不显示荧光，其信号形态特征与高尔基体和内质网（ER）一致。该方法可以快速、稳健地确定高尔基体驻留蛋白的膜拓扑结构，称为 GO-PROMTO（用于高尔基体蛋白膜拓扑结构）。我们应用 GO-PROMTO 来研究参与植物细胞壁多糖（包括木葡聚糖和阿拉伯聚糖）生物合成的蛋白质的拓扑结构。结果表明存在涉及中间体跨高尔基体膜运输的新生物合成机制。
