Volz B, Orberger G, Porwoll S, Hauri H P, Tauber R
Institut für Klinische Chemie und Biochemie, Universitätsklinikum Rudolf-Virchow, Freie Universität Berlin, Germany.
J Cell Biol. 1995 Aug;130(3):537-51. doi: 10.1083/jcb.130.3.537.
Return of cell surface glycoproteins to compartments of the secretory pathway has been examined in HepG2 cells comparing return to the trans-Golgi network (TGN), the trans/medial- and cis-Golgi. Transport to these sites was studied by example of the transferrin receptor (TfR) and the serine peptidase dipeptidylpeptidase IV (DPPIV) after labeling these proteins with the N-hydroxysulfosuccinimide ester of biotin on the cell surface. This experimental design allowed to distinguish between glycoproteins that return to these biosynthetic compartments from the cell surface and newly synthesized glycoproteins that pass these compartments during biosynthesis en route to the surface. Reentry to the TGN was measured in that surface glycoproteins were desialylated with neuraminidase and were monitored for resialylation during recycling. Return to the trans-Golgi was traced measuring the transfer of [3H]fucose residues to recycling surface proteins by fucosyltransferases. To study return to the cis-Golgi, surface proteins were metabolically labeled in the presence of the mannosidase I inhibitor deoxymannojirimycin (dMM). As a result surface proteins retained N-glycans of the oligomannosidic type. Return to the site of mannosidase I in the medial/cis-Golgi was measured monitoring conversion of these glycans to those of the complex type after washout of dMM. Our data demonstrate that DPPIV does return from the cell surface not only to the TGN, but also to the trans-Golgi thus linking the endocytic to the secretory pathway. In contrast, no reentry to sites of mannosidase I could be detected indicating that the early secretory pathway is not or is only at insignificant rates accessible to recycling DPPIV. In contrast to DPPIV, TfR was very efficiently sorted from endosomes to the cell surface and did not return to the TGN or to other biosynthetic compartments in detectable amounts, indicating that individual surface proteins are subject to different sorting mechanisms or sorting efficiencies during recycling.
在HepG2细胞中，研究了细胞表面糖蛋白返回分泌途径各区室的情况，比较了其返回反式高尔基体网络（TGN）、反式/中间高尔基体和顺式高尔基体的情况。在用生物素的N-羟基琥珀酰亚胺酯在细胞表面标记转铁蛋白受体（TfR）和丝氨酸肽酶二肽基肽酶IV（DPPIV）后，以这两种蛋白为例研究了向这些位点的转运。这种实验设计能够区分从细胞表面返回这些生物合成区室的糖蛋白和在生物合成过程中经过这些区室并最终到达细胞表面的新合成糖蛋白。通过用神经氨酸酶去除表面糖蛋白的唾液酸，并在循环过程中监测其重新唾液酸化来测量返回TGN的情况。通过测量岩藻糖基转移酶将[3H]岩藻糖残基转移到循环表面蛋白上的情况来追踪返回反式高尔基体的过程。为了研究返回顺式高尔基体的情况，在甘露糖苷酶I抑制剂脱氧甘露基野尻霉素（dMM）存在的情况下对表面蛋白进行代谢标记。结果，表面蛋白保留了寡甘露糖型的N-聚糖。在去除dMM后，通过监测这些聚糖向复合型聚糖的转化来测量返回中间/顺式高尔基体中甘露糖苷酶I位点的情况。我们的数据表明，DPPIV不仅从细胞表面返回TGN，还返回反式高尔基体，从而将内吞途径与分泌途径联系起来。相比之下，未检测到其返回甘露糖苷酶I位点的情况，这表明早期分泌途径对循环的DPPIV来说不可进入或仅以极低的速率可进入。与DPPIV不同，TfR非常有效地从内体分选到细胞表面，并且没有以可检测到的量返回TGN或其他生物合成区室，这表明在循环过程中，单个表面蛋白受到不同的分选机制或分选效率的影响。
