Kim H, Paul S, Gennity J, Jennity J [corrected to Gennity J ], Inouye M
Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854-5635.
Mol Microbiol. 1994 Mar;11(5):819-31. doi: 10.1111/j.1365-2958.1994.tb00360.x.
Hybrid genes were constructed to express bifunctional hybrid proteins in which staphyloccal nuclease A with or without an amino-terminal OmpA signal sequence was fused with TEM beta-lactamase (at the carboxyl terminal side) using the signal peptide of the major outer membrane lipoprotein of Escherichia coli as an internal linker. The hybrid proteins were found to be inserted in the membrane. Orientation of the hybrid protein with the OmpA signal peptide showed that the nuclease was translocated into the periplasm and the beta-lactamase remained in the cytoplasm. This indicates that the cleavable OmpA signal peptide served as a secretory signal for nuclease and the internal lipoprotein signal served as the transmembrane anchor. In the absence of the OmpA signal sequence the topology of the hybrid protein was reversed indicating that the internal lipoprotein signal peptide initially served as the signal peptide for the secretion of the carboxy terminal beta-lactamase domain across the membrane and subsequently as a membrane anchoring signal. The role of charged amino acids in the translocation and transmembrane orientation of membrane proteins was also analysed by introducing charged amino acids to either or both sides of the internal lipoprotein signal sequence in the bifunctional hybrid proteins in the absence of the amino-terminal signal sequence. Introduction of two lysine residues at the carboxy-terminal side of the internal signal sequence reversed the topology of the transmembrane protein by translocating the amino-terminal nuclease domain across the membrane, leaving the carboxyl terminal beta-lactamase domain in the cytoplasm. When three more lysine residues were added to the amino-terminal side of the internal signal sequence of the same construct the membrane topology flipped back to the original orientation. A similar reversion of the topology could be obtained by introducing negatively charged residues at the amino-terminal side of the internal signal sequence. Present results demonstrate for the first time that a bifunctional transmembrane protein can be engineered to assume either of the two opposite orientations and that charge balance around the transmembrane domain is a major factor in controlling the topology of a transmembrane protein.
构建了杂合基因以表达双功能杂合蛋白，其中带有或不带有氨基末端OmpA信号序列的葡萄球菌核酸酶A与TEMβ-内酰胺酶（在羧基末端一侧）融合，使用大肠杆菌主要外膜脂蛋白的信号肽作为内部连接子。发现杂合蛋白插入到膜中。带有OmpA信号肽的杂合蛋白的定位表明，核酸酶被转运到周质中，而β-内酰胺酶保留在细胞质中。这表明可裂解的OmpA信号肽作为核酸酶的分泌信号，而内部脂蛋白信号作为跨膜锚定信号。在没有OmpA信号序列的情况下，杂合蛋白的拓扑结构发生了反转，这表明内部脂蛋白信号肽最初作为羧基末端β-内酰胺酶结构域跨膜分泌的信号肽，随后作为膜锚定信号。还通过在没有氨基末端信号序列的双功能杂合蛋白的内部脂蛋白信号序列的一侧或两侧引入带电荷的氨基酸，分析了带电荷的氨基酸在膜蛋白转运和跨膜定位中的作用。在内部信号序列的羧基末端一侧引入两个赖氨酸残基，通过使氨基末端核酸酶结构域跨膜转运，使跨膜蛋白的拓扑结构发生反转，从而使羧基末端β-内酰胺酶结构域留在细胞质中。当在同一构建体的内部信号序列的氨基末端一侧再添加三个赖氨酸残基时，膜拓扑结构又翻转回原来的方向。通过在内部信号序列的氨基末端一侧引入带负电荷的残基，也可以获得类似的拓扑结构反转。目前的结果首次证明，可以设计一种双功能跨膜蛋白使其呈现两种相反方向中的任何一种，并且跨膜结构域周围的电荷平衡是控制跨膜蛋白拓扑结构的主要因素。
