Morel V, Poschet R, Traverso V, Deretic D
Department of Ophthalmology and Visual Sciences, University of Michigan, Ann Arbor 48105, USA.
Electrophoresis. 2000 Oct;21(16):3460-9. doi: 10.1002/1522-2683(20001001)21:16<3460::AID-ELPS3460>3.0.CO;2-I.
Polarized sorting of rhodopsin in retinal rod photoreceptor cells is mediated by post-Golgi carrier membranes that bud from the trans-Golgi network and fuse with the specialized domain of the plasma membrane in the rod inner segment. The identity of the majority of the resident proteins of this organelle still remains elusive, despite multifaceted approaches to study this compartment. In the present study we have taken a proteomic approach to the analysis of the post-Golgi carriers. First, we modified the previously established fractionation protocols in order to achieve greater purity of the isolated membranes. Specifically, the new fractionation scheme depleted the post-Golgi fraction of cytosolic proteins that were the most abundant contaminants complicating analysis of two-dimensional (2-D) gel profiles in our previous preparations. The isolated membranes were subjected to 2-D gel electrophoresis, immunoblotting and microsequencing. This analysis showed that the improved subcellular fractionation yielded a fraction highly enriched in rhodopsin-bearing post-Golgi carrier membranes. Two-dimensional mapping revealed 29 proteins that are preferentially found in this fraction and therefore represent candidates for post-Golgi membrane-specific proteins. This preparation of rhodopsin-bearing post-Golgi carriers is a first step towards the proteomics of this important organelle.
视紫红质在视网膜视杆光感受器细胞中的极化分选由高尔基体后载体膜介导，这些膜从反式高尔基体网络出芽，并与视杆内段质膜的特化区域融合。尽管采用了多方面的方法来研究这个区室，但该细胞器中大多数驻留蛋白的身份仍然难以捉摸。在本研究中，我们采用蛋白质组学方法分析高尔基体后载体。首先，我们修改了先前建立的分级分离方案，以提高分离膜的纯度。具体来说，新的分级分离方案去除了高尔基体后级分中的胞质蛋白，这些蛋白是最丰富的污染物，使我们之前制备物中二维（2-D）凝胶图谱的分析变得复杂。对分离的膜进行二维凝胶电泳、免疫印迹和微测序。该分析表明，改进的亚细胞分级分离产生了一个高度富集含视紫红质的高尔基体后载体膜的级分。二维图谱显示有29种蛋白质优先存在于该级分中，因此代表了高尔基体后膜特异性蛋白的候选物。这种含视紫红质的高尔基体后载体的制备是对这个重要细胞器进行蛋白质组学研究的第一步。
