Stockert Richard J, Potvin Barry, Nath Sangeeta, Wolkoff Allan W, Stanley Pamela
The Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Biochim Biophys Acta. 2007 Jul;1768(7):1741-9. doi: 10.1016/j.bbamem.2007.04.004. Epub 2007 Apr 14.
To isolate mutant liver cells defective in the endocytic pathway, a selection strategy using toxic ligands for two distinct membrane receptors was utilized. Rare survivors termed trafficking mutants (Trf2-Trf7) were stable and more resistant than the parental HuH-7 cells to both toxin conjugates. They differed from the previously isolated Trf1 HuH-7 mutant as they expressed casein kinase 2 alpha'' (CK2alpha'') which is missing from Trf1 cells and which corrects the Trf1 trafficking phenotype. Binding of (125)I-asialoorosomucoid (ASOR) and cell surface expression of asialoglycoprotein receptor (ASGPR) were reduced approximately 20%-60% in Trf2-Trf7 cells compared to parental HuH-7, without a reduction in total cellular ASGPR. Based on (125)I-transferrin binding, cell surface transferrin receptor activity was reduced between 13% and 88% in the various mutant cell lines. Distinctive phenotypic traits were identified in the differential resistance of Trf2-Trf7 to a panel of lectins and toxins and to UV light-induced cell death. By following the endocytic uptake and trafficking of Alexa(488)-ASOR, significant differences in endosomal fusion between parental HuH-7 and the Trf mutants became apparent. Unlike parental HuH-7 cells in which the fusion of endosomes into larger vesicles was evident as early as 20 min, ASOR endocytosed into the Trf mutants remained within small vesicles for up to 60 min. Identifying the biochemical and genetic mechanisms underlying these phenotypes should uncover novel and unpredicted protein-protein or protein-lipid interactions that orchestrate specific steps in membrane protein trafficking.
为了分离内吞途径有缺陷的突变肝细胞，采用了一种针对两种不同膜受体使用毒性配体的筛选策略。被称为转运突变体（Trf2 - Trf7）的罕见存活细胞比亲代HuH - 7细胞更稳定，且对两种毒素偶联物具有更强的抗性。它们与先前分离的Trf1 HuH - 7突变体不同，因为它们表达酪蛋白激酶2α''（CK2α''），而Trf1细胞中缺失该蛋白，并且该蛋白可纠正Trf1转运表型。与亲代HuH - 7相比，Trf2 - Trf7细胞中（125）I - 去唾液酸糖蛋白（ASOR）的结合以及去唾液酸糖蛋白受体（ASGPR）的细胞表面表达降低了约20% - 60%，而细胞内总ASGPR没有减少。基于（125）I - 转铁蛋白结合情况，各种突变细胞系中细胞表面转铁蛋白受体活性降低了13%至88%。在Trf2 - Trf7对一组凝集素、毒素以及紫外线诱导的细胞死亡的差异抗性中鉴定出了独特的表型特征。通过追踪Alexa(488) - ASOR的内吞摄取和转运，亲代HuH - 7与Trf突变体之间在内涵体融合方面的显著差异变得明显。与亲代HuH - 7细胞不同，在亲代HuH - 7细胞中，内涵体融合形成较大囊泡早在20分钟时就很明显，而内吞进入Trf突变体的ASOR在小囊泡中保留长达60分钟。确定这些表型背后的生化和遗传机制应该能够揭示协调膜蛋白转运特定步骤的新的和意想不到的蛋白质 - 蛋白质或蛋白质 - 脂质相互作用。
