We report the identification of the proteins encoded by the mttABC operon (formerly yigTUW), which mediate a novel Sec-independent membrane targeting and translocation system in Escherichia coli that interacts with cofactor-containing redox proteins having a S/TRRXFLK "twin arginine" leader motif. A pleiotropic-negative mutant in mttA prevents the periplasmic localization of twin arginine redox enzymes, including nitrate reductase (NapA) and trimethylamine N-oxide reductase (TorA). The mutation also prevents the correct localization of the integral membrane molybdoenzyme dimethylsulfoxide reductase (DmsABC). The DmsA subunit has a twin arginine leader. Proteins with a Sec-dependent leader or which assemble spontaneously in the membrane are not affected by this mutation. MttA, B, and C are members of a large family of related sequences extending from archaebacteria to higher eukaryotes.

Dimethyl sulfoxide (Me 2 SO) reductase of Escherichia coli is a terminal electron transport chain enzyme that is expressed under anaerobic growth conditions and is required for anaerobic growth with Me 2 SO as the terminal electron acceptor. The trimeric enzyme is composed of a membrane extrinsic catalytic dimer (DmsAB) and a membrane intrinsic anchor (DmsC). The amino terminus of DmsA has a leader sequence with a twin arginine motif that targets DmsAB to the membrane via a novel Sec-independent mechanism termed MTT for membrane targeting and translocation. We demonstrate that the Met-1 present upstream of the twin arginine motif serves as the correct translational start site. The leader is essential for the expression of DmsA, stability of the DmsAB dimer, and membrane targeting of the reductase holoenzyme. Mutation of arginine 17 to aspartate abolished membrane targeting. The reductase was labile in the leader sequence mutants. These mutants failed to support growth on glycerol-Me 2 SO minimal medium. Replacing the DmsA leader with the TorA leader of trimethylamine N-oxide reductase produced a membranebound DmsABC with greatly reduced enzyme activity and inefficient anaerobic respiration indicating that the twin arginine leaders may play specific roles in the assembly of redox enzymes.

The terminal electron-transfer enzyme fumarate reductase ofEscherichia coli is a complex iron-sulfur flavoenzyme composed of four nonidentical subunits organized into two domains: FrdA and -B (a membrane-extrinsic catalytic domain) and FrdC and -D (a transmembrane anchor domain). We have identified a mutation within the membrane-intrinsic domain that alters the electron transfer properties of the iron-sulfur and flavin redox centers of the catalytic domain. Functional electron flow from the quinone analog 2,3-dimethyl-1,4-naphthoquinone or from the electron transport chain is impaired. However, the mutant enzyme can be reduced normally by single-electron donors such as the dye benzyl viologen.The mutant phenotype results from a single A -* G transition changing His-82, within the second transmembrane a-helix of the FrdC anchor sequence, to an arginine. The mutation, physically located within the anchor domain, is manifested by altered catalytic properties, indicating that the intrinsic and extrinsic domains are conformationally connected. These resuilts confirm the important role of the anchor subunits in functional electron transport and have implications for communication between intrinsic and extrinsic domains of membrane proteins.Fumarate reductase is a complex, membrane-bound iron-sulfur flavoenzyme that serves as the terminal electrontransfer enzyme when Escherichia coli is grown anaerobically with fumarate as the electron acceptor (1). The enzyme is composed of two distinct domains: a membrane-extrinsic catalytic domain comprised of the FrdA and -B polypeptides of 69 and 27 kDa, respectively, and a membrane-intrinsic domain consisting of the FrdC and -D subunits of 15 and 13 kDa, respectively (2). Two forms of the enzyme can be isolated: a tetrameric holoenzyme, composed of equimolar amounts of each subunit, and a catalytic dimer, composed of the FrdA and -B subunits (3, 4). By analogy with the E. coli succinate dehydrogenase, the active site of fumarate reductase is located in FrdA, the flavin-containing subunit (5). The iron-sulfur centers are located in the FrdB subunit (6) and the FrdC and -D polypeptides do not contain any known redox centers. The FrdC and -D polypeptides not only anchor the catalytic subunits to the membrane surface (4) but also induce an optimal conformation in the catalytic dimer as reflected by increased stability and modulated turnover ofthe holoenzyme (7). The enzyme is believed to accept reducing equivalents (electrons) from a reduced b-type cytochrome buried within the membrane. However, the pathways for electrons within the electron transport chain and within the enzyme itself remain to be firmly established.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.Previously characterized mutants ofthefrd operon have been localized to thefrdA gene (8). We now report the isolation and analysis of a mutant within the Fr...

Dimethyl sulfoxide reductase is a heterotrimeric enzyme (DmsABC) localized to the cytoplasmic surface of the inner membrane. Targeting of the DmsA and DmsB catalytic subunits to the membrane requires the membrane targeting and translocation (Mtt) system. The DmsAB dimer is a member of a family of extrinsic, cytoplasmic facing membrane subunits that require Mtt in order to assemble on the membrane. We show that the MttA 2 , MttB, and presumably MttA 1 but not the MttC proteins are required for targeting DmsAB to the membrane. Unlike other Mtt substrates such as trimethylamine N-oxide reductase, the soluble cytoplasmic DmsAB dimer that accumulates in the mtt deletions is very labile. Deletion of the mttA 2 or mttB genes also prevents anaerobic growth on fumarate even though fumarate reductase does not require Mtt for assembly. This was due to the lethality of membrane insertion of DmsC in the absence of the DmsAB subunits. In the absence of DmsC, DmsAB accumulates in the cytoplasm. A 45-amino acid leader on DmsA is removed during assembly. Processing does not require DmsC but does require Mtt. Translocation of DmsAB to the periplasm is not required for processing. The leader may be cleaved by a novel leader peptidase, or the long DmsA leader may traverse the membrane through the Mtt system resulting in cleavage by the periplasmic leader peptidase I followed by release of DmsA into the cytoplasm.

We have generated a chromosomal mutant of moeB (moeBA228T) that demonstrates limited molybdenum cofactor (molybdo-bis(molybdopterin guanine dinucleotide) (Mo-bisMGD)) availability in Escherichia coli and have characterized its effect on the maturation and physiological function of two well-characterized respiratory molybdoenzymes: the membrane-bound dimethylsulfoxide (DMSO) reductase (DmsABC) and the membrane-bound nitrate reductase A (NarGHI). In the moeBA228T mutant strain, E. coli F36, anaerobic respiratory growth is possible on nitrate but not on DMSO, indicating that cofactor insertion occurs into NarGHI but not into DmsABC. Fluorescence analyses of cofactor availability indicate little detectable cofactor in the moeBA228T mutant compared with the wild-type, suggesting that NarGHI is able to scavenge limiting cofactor, whereas DmsABC is not. MoeB functions to sulfurylate MoaD, and in the structure of the MoeB-MoaD complex, Ala-228 is located in the interface region between the two proteins. This suggests that the moeBA228T mutation disrupts the interaction between MoeB and MoaD. In the case of DmsABC, despite the absence of cofactor, the twin-arginine signal sequence of DmsA is cleaved in the moeBA228T mutant, indicating that maturation of the holoenzyme is not cofactor-insertion dependent.