Gennity J M, Inouye M
Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine & Dentistry of New Jersey, Piscataway 08854-5635.
J Biol Chem. 1991 Sep 5;266(25):16458-64.
Structural information defining an N-terminal sequence required for the membrane sorting of bacterial lipoproteins has been previously garnered through the study of a hybrid outer membrane (OM) lipo-beta-lactamase (LL) (Ghrayeb and Inouye (1984) J. Biol. Chem. 259, 463-467). Introduction of an aspartate as the second residue of mature LL (D2 mutant) causes an inner membrane (IM) localization of this protein (Yamaguchi, K., Yu, F., and Inouye, M. (1988) Cell 53, 423-432). Introduction of as aspartate at the third residue of mature LL (D3) causes a weaker IM sorting signal and when present as the fourth residue (D4), normal OM sorting occurs. A positively charged residue at the second position (K2) has no effect on OM localization. Remarkably, glutamate substitution at either the second (E2) or third (E3) position does not interfere with OM sorting. Sorting of the mutant D2 LL can be partially suppressed by introduction of a positively charged histidine (D2H3) or lysine (D2K3) at residue 3 of the mature protein. These results indicate that both the negative charge of the aspartate residue and some structural feature not present in a glutamate residue are required for sorting to the IM. The suppression of IM localization of the D2H3 LL double mutant can be eliminated by growing Escherichia coli at pH 8.4 to reduce the histidine partial positive charge. This result supports the essentiality of a negative charge in IM localization and indicates that the committed step in lipoprotein sorting is made in a cellular compartment, the periplasm, at equilibrium with the external pH.
通过对杂交外膜（OM）脂β-内酰胺酶（LL）的研究，先前已获得了定义细菌脂蛋白膜分选所需N端序列的结构信息（Ghrayeb和Inouye，（1984年）《生物化学杂志》259卷，463 - 467页）。在成熟LL的第二个残基处引入天冬氨酸（D2突变体）会导致该蛋白定位于内膜（IM）（Yamaguchi，K.，Yu，F.和Inouye，M.（1988年）《细胞》53卷，423 - 432页）。在成熟LL的第三个残基处引入天冬氨酸（D3）会产生较弱的IM分选信号，而当作为第四个残基存在时（D4），则会发生正常的OM分选。第二个位置的带正电荷残基（K2）对OM定位没有影响。值得注意的是，在第二个（E2）或第三个（E3）位置的谷氨酸替代并不干扰OM分选。通过在成熟蛋白的第3位残基处引入带正电荷的组氨酸（D2H3）或赖氨酸（D2K3），可以部分抑制突变体D2 LL的分选。这些结果表明，天冬氨酸残基的负电荷和谷氨酸残基中不存在的某些结构特征对于分选到IM都是必需的。通过在pH 8.4下培养大肠杆菌以降低组氨酸的部分正电荷，可以消除D2H3 LL双突变体的IM定位抑制。这一结果支持了IM定位中负电荷的必要性，并表明脂蛋白分选的关键步骤是在与外部pH平衡的细胞区室周质中进行的。
