Leitinger B, Hille-Rehfeld A, Spiess M
Department of Biochemistry, Biozentrum, University of Basel, Switzerland.
Proc Natl Acad Sci U S A. 1995 Oct 24;92(22):10109-13. doi: 10.1073/pnas.92.22.10109.
Signals for endocytosis and for basolateral and lysosomal sorting are closely related in a number of membrane proteins, suggesting similar sorting mechanisms at the plasma membrane and in the trans-Golgi network (TGN). We tested the hypothesis that basolateral membrane proteins are transported to the cell surface via endosomes for the asialoglycoprotein receptor H1. This protein was tagged with a tyrosine sulfation site (H1TS) to allow specific labeling with [35S]sulfate in the TGN. Madin-Darby canine kidney cells expressing H1TS were pulse-labeled and chased for a period of time insufficient for labeled H1TS to reach the cell surface. Upon homogenization and gradient centrifugation, fractions devoid of TGN were subjected to immunoisolation of compartments containing mannose 6-phosphate receptor, which served as an endosomal marker. H1TS in transit to the cell surface was efficiently coisolated, whereas a labeled secretory protein and free glycosaminoglycan chains were not. This indicates an indirect pathway for the asialoglycoprotein receptor to the plasma membrane via endosomes and has important implications for protein sorting in the TGN and endosomes.
在许多膜蛋白中，内吞作用信号以及基底外侧和溶酶体分选信号密切相关，这表明在质膜和反式高尔基体网络（TGN）中存在相似的分选机制。我们对以下假说进行了验证：基底外侧膜蛋白通过内体转运至细胞表面，以运走脱唾液酸糖蛋白受体H1。该蛋白被标记了一个酪氨酸硫酸化位点（H1TS），以便在TGN中用[35S]硫酸盐进行特异性标记。对表达H1TS的Madin-Darby犬肾细胞进行脉冲标记并追踪一段时间，这段时间不足以使标记的H1TS到达细胞表面。匀浆和梯度离心后，对不含TGN的组分进行免疫分离，分离出含有甘露糖6-磷酸受体的区室，该受体作为内体标记物。正在转运至细胞表面的H1TS能被有效地共同分离出来，而标记的分泌蛋白和游离糖胺聚糖链则不能。这表明脱唾液酸糖蛋白受体通过内体间接转运至质膜，这对TGN和内体中的蛋白质分选具有重要意义。
