San Millan J L, Boyd D, Dalbey R, Wickner W, Beckwith J
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115.
J Bacteriol. 1989 Oct;171(10):5536-41. doi: 10.1128/jb.171.10.5536-5541.1989.
A topology of the Escherichia coli leader peptidase has been previously proposed on the basis of proteolytic studies. Here, a collection of alkaline phosphatase fusions to leader peptidase is described. Fusions to the periplasmic domain of this protein exhibit high alkaline phosphatase activity, while fusions to the cytoplasmic domain exhibit low activity. Elements within the cytoplasmic domain are necessary to stably anchor alkaline phosphatase in the cytoplasm. The amino-terminal hydrophobic segment of leader peptidase acts as a weak export signal for alkaline phosphatase. However, when this segment is preceded by four lysines, it acts as a highly efficient export signal. The coherence of in vitro studies with alkaline phosphatase fusion analysis of the topology of leader peptidase further indicates the utility of this genetic approach to membrane protein structure and insertion.
先前已基于蛋白水解研究提出了大肠杆菌前导肽酶的一种拓扑结构。在此，描述了一系列与前导肽酶的碱性磷酸酶融合体。与该蛋白周质结构域的融合体表现出高碱性磷酸酶活性，而与细胞质结构域的融合体表现出低活性。细胞质结构域内的元件对于将碱性磷酸酶稳定锚定在细胞质中是必需的。前导肽酶的氨基末端疏水片段作为碱性磷酸酶的弱输出信号。然而，当该片段之前有四个赖氨酸时，它就作为高效的输出信号。体外研究与前导肽酶拓扑结构的碱性磷酸酶融合分析的一致性进一步表明了这种遗传方法在膜蛋白结构和插入研究中的实用性。
