Bosshart H, Humphrey J, Deignan E, Davidson J, Drazba J, Yuan L, Oorschot V, Peters P J, Bonifacino J S
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
J Cell Biol. 1994 Sep;126(5):1157-72. doi: 10.1083/jcb.126.5.1157.
To investigate the mechanisms of membrane protein localization to the Golgi complex, we have examined the intracellular trafficking of epitope-tagged forms of the mammalian endopeptidase, furin, in stably transformed rat basophilic leukemia cells. Our studies show that furin is predominantly localized to the trans-Golgi network (TGN) at steady state, with smaller amounts present in intracellular vesicles. Biochemical and morphological analyses reveal that furin is progressively delivered to a lysosomal compartment, where it is degraded. Analyses of furin deletion mutants and chimeric proteins show that the cytoplasmic domain is both necessary and sufficient for localization to the TGN in various cell types. Interestingly, deletion of most of the cytoplasmic domain of furin results in a molecule that is predominantly localized to intracellular vesicles, some of which display characteristics of lysosomes. To a lesser extent, the cytoplasmically deleted molecule is also localized to the plasma membrane. These observations suggest the existence of an additional determinant for targeting to the endosomal/lysosomal system within the lumenal and/or transmembrane domains of furin. Thus, the overall pattern of trafficking and steady state localization of furin are determined by targeting information contained within more than one region of the molecule.
为了研究膜蛋白定位于高尔基体复合体的机制，我们检测了稳定转化的大鼠嗜碱性白血病细胞中，带有表位标签的哺乳动物内肽酶弗林蛋白酶的细胞内运输情况。我们的研究表明，在稳态时，弗林蛋白酶主要定位于反式高尔基体网络（TGN），细胞内囊泡中存在少量该酶。生化和形态学分析表明，弗林蛋白酶会逐渐被转运至溶酶体区室并在那里被降解。对弗林蛋白酶缺失突变体和嵌合蛋白的分析表明，在各种细胞类型中，细胞质结构域对于定位于TGN既必要又充分。有趣的是，缺失弗林蛋白酶大部分细胞质结构域会产生一种主要定位于细胞内囊泡的分子，其中一些囊泡具有溶酶体的特征。在较小程度上，细胞质缺失的分子也定位于质膜。这些观察结果表明，在弗林蛋白酶的腔和/或跨膜结构域内存在靶向内体/溶酶体系统的另一个决定因素。因此，弗林蛋白酶运输和稳态定位的总体模式是由分子多个区域中包含的靶向信息决定的。
