Identification of a topology control domain in the tetracycline resistance protein.

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.