Kim D W, Matsuzawa H
Department of Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113, Japan.
Biochem Biophys Res Commun. 2000 Oct 14;277(1):216-20. doi: 10.1006/bbrc.2000.3657.
Aqualysin I from Thermus aquaticus YT-1 is an extracellular subtilisin-type serine protease. The protease is synthesized as a distinct precursor composed of four functional domains: an N-terminal signal sequence, an N-terminal pro-sequence, a protease domain, and a C-terminal pro-sequence. The N-terminal pro-sequence is essential for the production of active aqualysin I while the C-terminal pro-sequence is required for extracellular secretion of aqualysin I. In an E. coli expression system, the function of C-terminal pro-sequence in the translocation of aqualysin I across the cytoplasmic membrane was investigated. More than 60-70% of the total activity was detected in the cytoplasmic fraction in the deletion mutations of the C-terminal pro-sequence while less than 30% was found in this fraction in wild type. In addition, in vitro processing of aqualysin I precursors with these mutations to a mature form promptly occurred and the folding into active aqualysin I was rapid. These results suggest that the C-terminal pro-sequence, probably in conjunction with the signal sequence, facilitates the translocation of the precursor across the cytoplasmic membrane by preventing the precursor from taking on an active conformation.
嗜热水生栖热菌YT-1来源的嗜热栖热菌蛋白酶I是一种细胞外枯草杆菌蛋白酶型丝氨酸蛋白酶。该蛋白酶作为一种由四个功能域组成的独特前体进行合成：一个N端信号序列、一个N端前序列、一个蛋白酶结构域和一个C端前序列。N端前序列对于活性嗜热栖热菌蛋白酶I的产生至关重要，而C端前序列是嗜热栖热菌蛋白酶I细胞外分泌所必需的。在大肠杆菌表达系统中，研究了C端前序列在嗜热栖热菌蛋白酶I跨细胞质膜转运中的功能。在C端前序列缺失突变体中，超过60 - 70%的总活性在细胞质部分被检测到，而在野生型中该部分的活性不到30%。此外，具有这些突变的嗜热栖热菌蛋白酶I前体迅速进行体外加工形成成熟形式，并且快速折叠成活性嗜热栖热菌蛋白酶I。这些结果表明，C端前序列可能与信号序列一起，通过防止前体呈现活性构象来促进前体跨细胞质膜的转运。
