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.
Miller
K W
KW
Department of Molecular Biology, University of Wyoming, Laramie 82071, USA.
Jewell
J E
JE
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001692:Biological Transport; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics; D002462:Cell Membrane / Q000737:chemistry; D016764:Cell Polarity; D004926:Escherichia coli / Q000502:physiology; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013752:Tetracycline / Q000378:metabolism*; D013753:Tetracycline Resistance
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C461599:maltose transport system, E coli; C034811:tetracycline resistance-encoding transposon repressor protein; D013752:Tetracycline