Treyer Aleksandr, Pujato Mario, Pechuan Ximo, Müsch Anne
Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, NY 10461.
Department of Systems and Computational Biology, Albert Einstein College of Medicine, Bronx, NY 10461 Center for Autoimmune Genomics and Etiology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229.
Mol Biol Cell. 2016 Jul 15;27(14):2259-71. doi: 10.1091/mbc.E16-02-0096. Epub 2016 May 25.
For several decades, the trans-Golgi network (TGN) was considered the most distal stop and hence the ultimate protein-sorting station for distinct apical and basolateral transport carriers that reach their respective surface domains in the direct trafficking pathway. However, recent reports of apical and basolateral cargoes traversing post-Golgi compartments accessible to endocytic ligands before their arrival at the cell surface and the post-TGN breakup of large pleomorphic membrane fragments that exit the Golgi region toward the surface raised the possibility that compartments distal to the TGN mediate or contribute to biosynthetic sorting. Here we describe the development of a novel assay that quantitatively distinguishes different cargo pairs by their degree of colocalization at the TGN and by the evolution of colocalization during their TGN-to-surface transport. Keys to the high resolution of our approach are 1) conversion of perinuclear organelle clustering into a two-dimensional microsomal spread and 2) identification of TGN and post-TGN cargo without the need for a TGN marker that universally cosegregates with all cargo. Using our assay, we provide the first evidence that apical NTRp75 and basolateral VSVG in Madin-Darby canine kidney cells still undergo progressive sorting after they exit the TGN toward the cell surface.
几十年来，反式高尔基体网络（TGN）被认为是最末端的站点，因此是直接运输途径中分别到达各自表面结构域的不同顶端和基底外侧运输载体的最终蛋白质分选站。然而，最近有报道称，顶端和基底外侧货物在到达细胞表面之前会穿过内吞配体可进入的高尔基体后区室，并且离开高尔基体区域向表面移动的大型多形性膜片段会在TGN后解体，这就增加了TGN远端的区室介导或参与生物合成分选的可能性。在这里，我们描述了一种新型检测方法的开发，该方法通过不同货物对在TGN处的共定位程度以及它们在从TGN到表面运输过程中共定位的演变来定量区分不同的货物对。我们方法实现高分辨率的关键在于：1）将核周细胞器聚集转化为二维微粒体铺展；2）无需与所有货物普遍共分离的TGN标记物即可识别TGN和TGN后的货物。使用我们的检测方法，我们首次提供了证据，表明Madin-Darby犬肾细胞中的顶端NTRp75和基底外侧VSVG在离开TGN向细胞表面移动后仍会进行渐进性分选。
