Fujishige A, Smith K R, Silen J L, Agard D A
Department of Biochemistry, University of California, San Francisco 94143-0448.
J Cell Biol. 1992 Jul;118(1):33-42. doi: 10.1083/jcb.118.1.33.
alpha-Lytic protease is a bacterial serine protease of the trypsin family that is synthesized as a 39-kD preproenzyme (Silen, J. L., C. N. McGrath, K. R. Smith, and D. A. Agard. 1988. Gene (Amst.). 69: 237-244). The 198-amino acid mature protease is secreted into the culture medium by the native host, Lysobacter enzymogenes (Whitaker, D. R. 1970. Methods Enzymol. 19:599-613). Expression experiments in Escherichia coli revealed that the 166-amino acid pro region is transiently required either in cis (Silen, J. L., D. Frank, A. Fujishige, R. Bone, and D. A. Agard. 1989. J. Bacteriol. 171:1320-1325) or in trans (Silen, J. L., and D. A. Agard. 1989. Nature (Lond.). 341:462-464) for the proper folding and extracellular accumulation of the enzyme. The maturation process is temperature sensitive in E. coli; unprocessed precursor accumulates in the cells at temperatures above 30 degrees C (Silen, J. L., D. Frank, A. Fujishige, R. Bone, and D. A. Agard. 1989. J. Bacteriol. 171:1320-1325). Here we show that full-length precursor produced at nonpermissive temperatures is tightly associated with the E. coli outer membrane. The active site mutant Ser 195----Ala (SA195), which is incapable of self-processing, also accumulates as a precursor in the outer membrane, even when expressed at permissive temperatures. When the protease domain is expressed in the absence of the pro region, the misfolded, inactive protease also cofractionates with the outer membrane. However, when the folding requirement for either wild-type or mutant protease domains is provided by expressing the pro region in trans, both are efficiently secreted into the extracellular medium. Attempts to separate folding and secretion functions by extensive deletion mutagenesis within the pro region were unsuccessful. Taken together, these results suggest that only properly folded and processed forms of alpha-lytic protease are efficiently transported to the medium.
α-溶胞素蛋白酶是胰蛋白酶家族的一种细菌丝氨酸蛋白酶，它最初以39kD的前体酶原形式合成（西伦，J.L.，C.N.麦格拉思，K.R.史密斯，和D.A.阿加德。1988年。《基因》（阿姆斯特丹）。69:237 - 244）。由天然宿主溶杆菌属（惠特克，D.R.1970年。《酶学方法》。19:599 - 613）将198个氨基酸的成熟蛋白酶分泌到培养基中。在大肠杆菌中的表达实验表明，166个氨基酸的前肽区域对于该酶的正确折叠和细胞外积累在顺式（西伦，J.L.，D.弗兰克，A.藤岛，R.博恩，和D.A.阿加德。1989年。《细菌学杂志》。171:1320 - 1325）或反式（西伦，J.L.，和D.A.阿加德。1989年。《自然》（伦敦）。341:462 - 464）情况下都是暂时需要的。在大肠杆菌中成熟过程对温度敏感；在30摄氏度以上的温度下，未加工的前体在细胞中积累（西伦，J.L.，D.弗兰克，A.藤岛，R.博恩，和D.A.阿加德。1989年。《细菌学杂志》。171:1320 - 1325）。在这里我们表明，在非允许温度下产生的全长前体与大肠杆菌外膜紧密结合。活性位点突变体Ser195→Ala（SA195），它不能自我加工，即使在允许温度下表达时也以前体形式在外膜中积累。当蛋白酶结构域在没有前肽区域的情况下表达时，错误折叠的、无活性的蛋白酶也与外膜共分级分离。然而，当通过反式表达前肽区域为野生型或突变型蛋白酶结构域提供折叠所需条件时，两者都能有效地分泌到细胞外培养基中。通过在前肽区域内进行广泛的缺失诱变来分离折叠和分泌功能的尝试未成功。综上所述，这些结果表明只有正确折叠和加工的α-溶胞素蛋白酶形式才能有效地转运到培养基中。
