Membrane topology of the ArsB protein, the membrane subunit of an anion-translocating ATPase.
The ars operon of the conjugative R-factor R773 encodes an oxyanion pump that catalyzes extrusion of arsenicals from cells of Escherichia coli. The oxyanion translocation ATPase is composed of two polypeptides, the catalytic ArsA protein and the intrinsic membrane protein, ArsB. The topology of regions of the ArsB protein in the inner membrane was determined using a variety of gene fusions. Random gene fusions with lacZ and phoA were generated using transposon mutagenesis. A series of gene fusions with blaM were constructed in vitro using a beta-lactamase fusion vector. To localize individual segments of the ArsB protein, a ternary fusion method was developed, where portions of the arsB gene were inserted in-frame between the coding regions for two heterologous proteins, in this case a portion of a newly identified arsD gene and the blaM sequence encoding the mature beta-lactamase. The location of a periplasmic loop was determined from V8 protease digestion of an ArsA-ArsB chimera. From analysis of data from 26 fusions, a topological model of the ArsB protein with 12 membrane-spanning regions is proposed.
Wu
J
J
Department of Biochemistry, School of Medicine, Wayne State University, Detroit, Michigan 48201.
Tisa
L S
LS
Rosen
B P
BP
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D053501:Arsenite Transporting ATPases; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics; D016623:Ion Pumps; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
D004269:DNA, Bacterial; D016623:Ion Pumps; D008565:Membrane Proteins; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase; D000251:Adenosine Triphosphatases; D053501:Arsenite Transporting ATPases