Johnson A O, Ghosh R N, Dunn K W, Garippa R, Park J, Mayor S, Maxfield F R, McGraw T E
Department of Pathology, Columbia University, New York 10032, USA.
J Cell Biol. 1996 Dec;135(6 Pt 2):1749-62. doi: 10.1083/jcb.135.6.1749.
The SDYQRL motif of the cytoplasmic domain of TGN38 is involved in targeting TGN38 from endosomes to the TGN. To create a system for studying this pathway, we replaced the native transferrin receptor (TR) internalization motif (YTRF) with the SDYQRL TGN-targeting motif. The advantages of using TR as a reporter molecule include the ability to monitor trafficking, in both biochemical and microscopy experiments, using the natural ligand transferrin. When expressed in CHO cells, the SDYQRL-TR construct accumulated in juxtanuclear tubules and vesicles that are in the vicinity of the TGN. The SDYQRL-TR-containing structures, however, do not colocalize with TGN markers (e.g., NBD ceramide), and therefore the SDYQRL motif is not sufficient to target the TR to the TGN. The morphology of the SDYQRL-TR-containing juxtanuclear structures is different from the recycling compartment found in cells expressing the wild-type TR. In addition, the SDYQRL-TR-containing juxtanuclear compartment is more acidic than the recycling compartment in cells expressing the wild-type TR. The juxtanuclear compartment, however, is a bona fide recycling compartment since SDYQRL-TR was recycled back to the cell surface at a rate comparable to the wild-type TR, and sphingomyelin and cellubrevin, both of which label all compartments of the endocytic recycling pathway, colocalize with SDYQRL-TR in the juxtanuclear structures. These findings demonstrate that expression of the SDYQRL-TR construct alters the morphology and pH of endocytic recycling compartments rather than selectively affecting the intracellular trafficking pathway of the SDYQRL-TR construct. Therefore, the SDYQRL trafficking motif is not simply a molecular address that targets proteins to the TGN, but it can play an active role in determining the physical characteristics of endosomal compartments.
TGN38细胞质结构域的SDYQRL基序参与将TGN38从内体靶向至反式高尔基体网络（TGN）。为创建一个研究该途径的系统，我们用SDYQRL TGN靶向基序替换了天然转铁蛋白受体（TR）内化基序（YTRF）。使用TR作为报告分子的优势包括在生化和显微镜实验中利用天然配体转铁蛋白监测运输的能力。当在CHO细胞中表达时，SDYQRL-TR构建体聚集在TGN附近的近核小管和囊泡中。然而，含有SDYQRL-TR的结构并不与TGN标志物（如NBD神经酰胺）共定位，因此SDYQRL基序不足以将TR靶向至TGN。含有SDYQRL-TR的近核结构的形态与表达野生型TR的细胞中发现的再循环区室不同。此外，含有SDYQRL-TR的近核区室比表达野生型TR的细胞中的再循环区室酸性更强。然而，近核区室是一个真正的再循环区室，因为SDYQRL-TR以与野生型TR相当的速率循环回到细胞表面，并且鞘磷脂和细胞ubrevin（两者均标记内吞再循环途径的所有区室）在近核结构中与SDYQRL-TR共定位。这些发现表明，SDYQRL-TR构建体的表达改变了内吞再循环区室的形态和pH，而不是选择性地影响SDYQRL-TR构建体的细胞内运输途径。因此，SDYQRL运输基序不仅仅是将蛋白质靶向至TGN的分子地址，但它可以在决定内体区室的物理特征方面发挥积极作用。
