Miller K W, Jewell J E
Department of Molecular Biology, University of Wyoming, Laramie 82071, USA.
Arch Biochem Biophys. 1995 Oct 1;322(2):445-52. doi: 10.1006/abbi.1995.1487.
Two N-terminal fusion proteins combining Escherichia coli maltose-binding protein (MBP) and the 12-transmembrane-segment pBR322 tetracycline resistance protein (Tet) have been constructed to determine the strength and location of topology control signals within the N-terminal portion of the Tet protein. The fusions contain either a secretable (wild-type) or a nonsecretable (MBP delta 2-26) MBP domain joined to the normally cytoplasmic N-terminus of the Tet protein. The effects of MBP targeting on Tet topology were investigated by analyzing the susceptibility of fusion strains to tetracycline and by proteolysis of the fusion proteins in inverted membrane vesicles and spheroplasts. The fusion protein containing MBP delta 2-26 conferred tetracycline resistance to the host strain and gave a normal pattern of Tet digestion fragments, indicating that its Tet domain is oriented and folded properly in the membrane. In contrast, the fusion containing secretable MBP was catalytically inactive apparently due to transfer of the Tet N-terminus to the periplasm with MBP. However, protease treatment of this fusion revealed that MBP secretion seems to affect only the topology of segments 1 and/or 2 of the Tet domain. Therefore, a strong topology control sequence appears to be located in the first cytoplasmic loop of the protein.
构建了两种N端融合蛋白，它们将大肠杆菌麦芽糖结合蛋白（MBP）与含12个跨膜片段的pBR322四环素抗性蛋白（Tet）相结合，以确定Tet蛋白N端部分拓扑控制信号的强度和位置。这些融合蛋白包含一个可分泌的（野生型）或不可分泌的（MBPδ2 - 26）MBP结构域，该结构域与Tet蛋白正常位于细胞质的N端相连。通过分析融合菌株对四环素的敏感性以及在反相膜泡和原生质球中对融合蛋白进行蛋白酶解，研究了MBP靶向对Tet拓扑结构的影响。含有MBPδ2 - 26的融合蛋白赋予宿主菌株四环素抗性，并产生正常模式的Tet消化片段，表明其Tet结构域在膜中定向和折叠正确。相比之下，含有可分泌MBP的融合蛋白显然由于Tet N端与MBP一起转移到周质而无催化活性。然而，对该融合蛋白进行蛋白酶处理表明，MBP分泌似乎仅影响Tet结构域第1和/或2片段的拓扑结构。因此，一个强大的拓扑控制序列似乎位于该蛋白的第一个细胞质环中。
