Jalal F, Dehbi M, Berteloot A, Crine P
Groupe de Recherche en Transport Membranaire, Faculté de Médecine, Université de Montréal, Quebec, Canada.
Biochem J. 1994 Sep 15;302 ( Pt 3)(Pt 3):669-74. doi: 10.1042/bj3020669.
When cultured in defined medium, kidney proximal convoluted tubule (PCT) cells form a homogeneous population and retain a number of differentiated functions. To characterize this cell system further as a functional model of epithelial polarity, we investigated the biogenic pathway of neutral endopeptidase (NEP), one of the most abundant microvillar membrane proteins in intestinal and kidney cells. We showed that, in contrast with some tumoral cell lines, RNA extracted from PCT cells shows the presence of a single mRNA species encoding NEP. Pulse-chase studies followed by selective immunoprecipitation of NEP molecules present either at the cell surface or in intracellular cell compartments showed that newly synthesized NEP molecules reached the cell surface as early as 30 min after the beginning of the chase with maximum cell surface expression at 60 min. When grown on semipermeable supports, PCT cells were found to target NEP exclusively to the apical plasma membrane. Similar results have been described using MDCK cells to study targeting of recombinant NEP. Thus primary cultures of PCT cells represent a new model with which to investigate the biogenic pathway of endogenous proteins in native epithelial cells.
在限定培养基中培养时，肾近端曲管（PCT）细胞形成同质群体并保留多种分化功能。为了将该细胞系统进一步表征为上皮极性的功能模型，我们研究了中性内肽酶（NEP）的生物合成途径，NEP是肠和肾细胞中最丰富的微绒毛膜蛋白之一。我们发现，与一些肿瘤细胞系不同，从PCT细胞中提取的RNA显示存在一种编码NEP的单一mRNA种类。脉冲追踪研究以及对存在于细胞表面或细胞内区室中的NEP分子进行选择性免疫沉淀表明，新合成的NEP分子在追踪开始后30分钟就最早到达细胞表面，在60分钟时细胞表面表达达到最大值。当在半透性支持物上生长时，发现PCT细胞将NEP专门靶向顶端质膜。使用MDCK细胞研究重组NEP的靶向时也描述了类似结果。因此，PCT细胞的原代培养代表了一种新模型，可用于研究天然上皮细胞中内源性蛋白质的生物合成途径。
