Stoorvogel W, Geuze H J, Griffith J M, Strous G J
Laboratory of Cell Biology, University of Utrecht, Medical School, The Netherlands.
J Cell Biol. 1988 Jun;106(6):1821-9. doi: 10.1083/jcb.106.6.1821.
We used a conjugate of transferrin and horseradish peroxidase (Tf/HRP) to label the intracellular transferrin receptor route in the human hepatoma cell line HepG2. The recycling kinetics of [125I]Tf/HRP were similar to those of unmodified [125I]Tf, implying identical routes for both ligands. 3,3'Diaminobenzidine (DAB)-cytochemistry was performed on post-nuclear supernatants of homogenates of cells which were incubated with both Tf/HRP and [125I]Tf, and caused two different effects: (a) the equilibrium density of [125I]Tf containing microsomes in a Percoll density gradient was increased, and (b) the amount of immunoprecipitable [125I]Tf from density-shifted lysed microsomes was only 20% of that of nonDAB treated microsomes. The whole biosynthetic route of alpha 1-antitrypsin (AT), a typical secretory glycoprotein in HepG2 cells, was labeled during a 60-min incubation with [35S]methionine. DAB cytochemistry was performed on post-nuclear supernatants of homogenates of cells which were also incubated with Tf/HRP. DAB cytochemistry caused approximately 40% of microsome-associated "complex" glycosylated [35S]alpha 1-antitrypsin ([35S]c-AT) to shift in a Percoll density gradient. Only part of the density shifted [35S]c-AT could be recovered by immunoprecipitation. A maximum effect was measured already after 10 min of Tf/HRP uptake. The density distribution of the "high mannose" glycosylated form of 35S-alpha 1-anti-trypsin [( 35S]hm-AT) was not affected by Tf/HRP. If in addition to Tf/HRP also an excess of non-conjugated transferrin was present in the medium, [35S]c-AT was not accessible for Tf/HRP, showing the involvement of the transferrin receptor (TfR) in the process. Furthermore, we show that if Tf/HRP and [35S]c-AT were located in different vesicles, the density distribution of [35S]c-AT was not affected by DAB-cytochemistry. Pulse-labeling with [35S]methionine was used to show that [35S]c-AT became accessible to endocytosed Tf/HRP minutes after acquirement of the complex configuration. A common intracellular localization of endocytosed Tf/HRP and secretory protein could be confirmed by immuno-electron microscopy: cryosections labeled with anti-albumin (protein A-colloidal gold) as well as DAB reaction product showed double-labeling in the trans-Golgi reticulum.
我们使用转铁蛋白与辣根过氧化物酶的共轭物（Tf/HRP）标记人肝癌细胞系HepG2中的细胞内转铁蛋白受体途径。[125I]Tf/HRP的循环动力学与未修饰的[125I]Tf相似，这意味着两种配体的途径相同。对同时用Tf/HRP和[125I]Tf孵育的细胞匀浆的核后上清液进行3,3'-二氨基联苯胺（DAB）细胞化学分析，产生了两种不同的效应：（a）在Percoll密度梯度中，含[125I]Tf的微粒体的平衡密度增加；（b）密度转移的裂解微粒体中可免疫沉淀的[125I]Tf的量仅为未用DAB处理的微粒体的20%。在与[35S]甲硫氨酸孵育60分钟期间，标记了HepG2细胞中典型的分泌性糖蛋白α1-抗胰蛋白酶（AT）的整个生物合成途径。对也用Tf/HRP孵育的细胞匀浆的核后上清液进行DAB细胞化学分析。DAB细胞化学导致约40%的微粒体相关“复杂”糖基化的[35S]α1-抗胰蛋白酶（[35S]c-AT）在Percoll密度梯度中发生转移。只有部分密度转移的[35S]c-AT可通过免疫沉淀回收。在摄取Tf/HRP 10分钟后就已测得最大效应。35S-α1-抗胰蛋白酶（[35S]hm-AT）的“高甘露糖”糖基化形式的密度分布不受Tf/HRP影响。如果培养基中除了Tf/HRP还存在过量的非共轭转铁蛋白，[35S]c-AT就无法被Tf/HRP作用，这表明转铁蛋白受体（TfR）参与了该过程。此外，我们表明，如果Tf/HRP和[35S]c-AT位于不同的囊泡中，[35S]c-AT的密度分布不受DAB细胞化学影响。用[35S]甲硫氨酸进行脉冲标记表明，[35S]c-AT在获得复杂构型几分钟后就可被内吞的Tf/HRP作用。通过免疫电子显微镜可以证实内吞的Tf/HRP和分泌蛋白在细胞内的共同定位：用抗白蛋白（蛋白A-胶体金）标记的冷冻切片以及DAB反应产物在反式高尔基体网状结构中显示出双重标记。
