Chillarón J, Estévez R, Samarzija I, Waldegger S, Testar X, Lang F, Zorzano A, Busch A, Palacín M
Department of Biochemistry and Molecular Biology, Faculty of Biology, Universitat de Barcelona, Avda. Diagonal 645, Barcelona 08028, Spain.
J Biol Chem. 1997 Apr 4;272(14):9543-9. doi: 10.1074/jbc.272.14.9543.
The human rBAT protein elicits sodium-independent, high affinity obligatory exchange of cystine, dibasic amino acids, and some neutral amino acids in Xenopus oocytes (Chillarón, J., Estévez, R., Mora, C., Wagner, C. A., Suessbrich, H., Lang, F., Gelpí, J. L., Testar, X., Busch, A. E., Zorzano, A., and Palacín, M. (1996) J. Biol. Chem. 271, 17761-17770). Mutations in rBAT have been found to cause cystinuria (Calonge, M. J., Gasparini, P., Chillarón, J., Chillón, M., Galluci, M., Rousaud, F., Zelante, L., Testar, X., Dallapiccola, B., Di Silverio, F., Barceló, P., Estivill, X., Zorzano, A., Nunes, V., and Palacín, M. (1994) Nat. Genet. 6, 420-426). We have performed functional studies with the most common point mutation, M467T, and its relative, M467K, using the oocyte system. The Km and the voltage dependence for transport of the different substrates were the same in both M467T and wild type-injected oocytes. However, the time course of transport was delayed in the M467T mutant: maximal activity was accomplished 3-4 days later than in the wild type. This delay was cRNA dose-dependent: at cRNA levels below 0.5 ng the M467T failed to achieve the wild type transport level. The M467K mutant displayed a normal Km, but the Vmax was between 5 and 35% of the wild type. The amount of rBAT protein was similar in normal and mutant-injected oocytes. In contrast to the wild type, the mutant proteins remained endoglycosidase H-sensitive, suggesting a longer residence time in the endoplasmic reticulum. We quantified the amount of rBAT protein in the plasma membrane by surface labeling with biotin 2 and 6 days after injection. Most of the M467T and M467K protein was located in an intracellular compartment. The converse situation was found in the wild type. Despite the low amount of M467T protein reaching the plasma membrane, the transport activity at 6 days was the same as in the wild type-injected oocytes. The increase in plasma membrane rBAT protein between 2 and 6 days was completely dissociated from the rise in transport activity. These data indicate impaired maturation and transport to the plasma membrane of the M467T and M467K mutant, and suggest that rBAT alone is unable to support the transport function.
人rBAT蛋白在非洲爪蟾卵母细胞中引发不依赖钠的、高亲和力的胱氨酸、二碱基氨基酸和一些中性氨基酸的强制性交换（奇亚隆，J.，埃斯特韦斯，R.，莫拉，C.，瓦格纳，C.A.，苏斯布里希，H.，朗，F.，赫尔皮，J.L.，泰斯塔，X.，布施，A.E.，佐尔扎诺，A.，和帕拉辛，M.（1996年）《生物化学杂志》271卷，17761 - 17770页）。已发现rBAT中的突变会导致胱氨酸尿症（卡隆格，M.J.，加斯帕里尼，P.，奇亚隆，J.，奇隆，M.，加卢奇，M.，鲁索，F.，泽兰特，L.，泰斯塔，X.，达拉皮科拉，B.，迪西尔维奥，F.，巴塞洛，P.，埃斯蒂维尔，X.，佐尔扎诺，A.，努涅斯，V.，和帕拉辛，M.（1994年）《自然遗传学》6卷，420 - 426页）。我们使用卵母细胞系统对最常见的点突变M467T及其相关突变M467K进行了功能研究。在注射了M467T和野生型的卵母细胞中，不同底物转运的米氏常数（Km）和电压依赖性是相同的。然而，M467T突变体的转运时间进程延迟：最大活性比野生型晚3 - 4天实现。这种延迟是依赖于cRNA剂量的：在cRNA水平低于0.5纳克时，M467T无法达到野生型的转运水平。M467K突变体显示出正常的Km，但最大反应速度（Vmax）是野生型的5%至35%。正常注射和突变体注射的卵母细胞中rBAT蛋白的量相似。与野生型不同，突变蛋白对内切糖苷酶H仍敏感，这表明在内质网中的停留时间更长。我们在注射后2天和6天通过用生物素进行表面标记来定量质膜中rBAT蛋白的量。大多数M467T和M467K蛋白位于细胞内区室。在野生型中发现的情况则相反。尽管到达质膜的M467T蛋白量很少，但6天时的转运活性与注射野生型的卵母细胞相同。质膜rBAT蛋白在2天至6天之间的增加与转运活性的增加完全不相关。这些数据表明M467T和M467K突变体的成熟和向质膜的转运受损，并表明单独的rBAT无法支持转运功能。
