Kerr Markus C, Bennetts Jennifer S, Simpson Fiona, Thomas Elaine C, Flegg Cameron, Gleeson Paul A, Wicking Carol, Teasdale Rohan D
Institute for Molecular Bioscience and ARC Centre in Bioinformatics, University of Queensland, St Lucia, Queensland 4072, Australia.
Traffic. 2005 Nov;6(11):991-1001. doi: 10.1111/j.1600-0854.2005.00328.x.
The mammalian retromer protein complex, which consists of three proteins--Vps26, Vps29, and Vps35--in association with members of the sorting nexin family of proteins, has been implicated in the trafficking of receptors and their ligands within the endosomal/lysosomal system of mammalian cells. A bioinformatic analysis of the mouse genome identified an additional transcribed paralog of the Vps26 retromer protein, which we termed Vps26B. No paralogs were identified for Vps29 and Vps35. Phylogenetic studies indicate that the two paralogs of Vps26 become evident after the evolution of the chordates. We propose that the chordate Vps26-like gene published previously be renamed Vps26A to differentiate it from Vps26B. As for Vps26A, biochemical characterization of Vps26B established that this novel 336 amino acid residue protein is a peripheral membrane protein. Vps26B co-precipitated with Vps35 from transfected cells and the direct interaction between these two proteins was confirmed by yeast 2-hybrid analysis, thereby establishing Vps26B as a subunit of the retromer complex. Within HeLa cells, Vps26B was found in the cytoplasm with low levels at the plasma membrane, while Vps26A was predominantly associated with endosomal membranes. Within A549 cells, both Vps26A and Vps26B co-localized with actin-rich lamellipodia at the cell surface. These structures also co-localized with Vps35. Total internal reflection fluorescence microscopy confirmed the association of Vps26B with the plasma membrane in a stable HEK293 cell line expressing cyan fluorescent protein (CFP)-Vps26B. Based on these observations, we propose that the mammalian retromer complex is located at both endosomes and the plasma membrane in some cell types.
哺乳动物的逆向转运蛋白复合物由三种蛋白质（Vps26、Vps29和Vps35）与分选连接蛋白家族的蛋白质成员结合而成，参与哺乳动物细胞内体/溶酶体系统中受体及其配体的运输。对小鼠基因组的生物信息学分析鉴定出Vps26逆向转运蛋白的另一个转录旁系同源物，我们将其命名为Vps26B。未鉴定出Vps29和Vps35的旁系同源物。系统发育研究表明，Vps26的两个旁系同源物在脊索动物进化后变得明显。我们建议将先前公布的脊索动物Vps26样基因重新命名为Vps26A，以将其与Vps26B区分开来。至于Vps26A，Vps26B的生化特性表明，这种新的336个氨基酸残基的蛋白质是一种外周膜蛋白。Vps26B与转染细胞中的Vps35共沉淀，酵母双杂交分析证实了这两种蛋白质之间的直接相互作用，从而确定Vps26B是逆向转运蛋白复合物的一个亚基。在HeLa细胞内，Vps26B存在于细胞质中，在质膜上水平较低，而Vps26A主要与内体膜相关。在A549细胞内，Vps26A和Vps26B都与细胞表面富含肌动蛋白的片状伪足共定位。这些结构也与Vps35共定位。全内反射荧光显微镜证实了Vps26B与表达青色荧光蛋白（CFP）-Vps26B的稳定HEK293细胞系中的质膜的关联。基于这些观察结果，我们认为哺乳动物的逆向转运蛋白复合物在某些细胞类型中位于内体和质膜上。
