Antonie van Leeuwenhoek 65: 359-368, 1994. 359 © 1994 Kluwer Academic Publishers. Printed in the Netherlands. A functional chimeric membrane subunit of an ion-translocating ATPase Dexian Dou, Saibal Dey & Barry E Rosen Department of Biochemistry, Wayne State University, School of Medicine, Detroit, M148201, USA Key words: Anion pump, antimony, arsenic, ion-translocating atpase, membrane protein, plasmid resistance Abstract A chimeric transport protein was made by expression of a fusion of the arsB genes from Escherichia coli plasmid R773 and Staphylococcus aureus plasmid pi258. The two genes were fused to encode a functional protein with first eight membrane spanning c~-helices of the S. aureus and the last four helices of the E. coli protein. The hybrid protein provided arsenite resistance and transport. When an arsA gene was expressed in trans with the ArsB proteins encoded by the R773, pi258 and fusion genes, arsenite efflux was dependent on chemical but not electrochemical energy. The Ars system is hypothesized to be a novel transport system that functions as a primary ATP-driven pump or a secondary carrier, depending on the subunit composition of the complex. Introduction Resistance to arsenite and antimonite in Escherichia coli is mediated by the arsenical resistance ars oper- on encoded by plasmid R773 (Kaur & Rosen 1992). The operon consists of five genes, arsRDABC (Fig. 1) (Chen et al. 1986; San Francisco et al. 1990; Wu and Rosen 1993). The products of the arsA and arsB genes are sufficient to confer arsenite resistance by forming a membrane-bound oxyanion translocating ATPase that catalyzes extrusion of the toxic oxyanions arsenite and antimonite from the cell (Fig. 2) (Kaur & Rosen 1992). The ArsA protein is the catalytic subunit of the pump (Hsu & Rosen 1989; Rosen et al. 1988) and is bound to the inner membrane of E. coli by interaction with the ArsB protein, an intrinsic membrane protein (Tisa & Rosen 1991). The ArsB protein is also most likely the anion conducting pathway. In addition to the arsA and arsB genes, the arsC gene is required for arsenate resistance (Chen et al. 1985). The 16 kDa ArsC pro- tein is an arsenate reductase that transforms arsenate to the pump substrate arsenite, perhaps by channeling the arsenite directly into the active site of the pump (Fig. 2) (Oden et al. 1994). A similar operon on plasmids of Gram-positive bac- teria provides arsenical resistance via an efflux mecha- nism (Silver & Keach 1982). The closely related oper- ons from the staphylococcal plasmids pi258 (S. aureus) (Ji & Silver 1992) and pSX267 (S. xylosus) (Rosenstein et al. 1992) each consist of only three genes, arsR- BC. Since the gram positive operon lacks the gene for the catalytic subunit of the pump, the question arises whether the mechanism of resistance at the molecular level is the same in gram positive and gram negative organisms or whether the two operons function by dif- ferent mechanisms. The amino acid sequence of the two ArsBs proteins exhibit 58% homology with the ArsB~ protein (Fig. 3). (For simplicity the staphyloc- cal plasmid encoded Ars homologues are designated Arss, and the E. coli plasmid encoded proteins des- ignated Ars~.) The S. aureus ars8 operon conferred resistance to both arsenite and antimonite in E. coli (Ji & Silver 1992). The level of resistance increased when the arsA gene was expressed in trans, suggesting that the ArsBs protein might be able to interact with the ArsA protein in vivo (Br6er et al. 1993). Although the similarity of the two 429 residue ArsB proteins was only 58% at the level of primary sequence, the hydropathic profiles of the two are more similar (Fig. 4), suggesting that the topological structure in the membrane and the quaternary structure of the fold- ed proteins might be basically the same, in which case their catalytic properties might be expected to be simi- lar as well. The ArsB~ protein has been shown to span