The Topology of the l -Arginine Exporter ArgO Conforms to an N _in -C _out Configuration in Escherichia coli: Requirement for the Cytoplasmic N-Terminal Domain, Functional Helical Interactions, and an Aspartate Pair for ArgO Function

Abstract

ArgO and LysE are members of the LysE family of exporter proteins and ordinarily mediate the export of L-arginine (Arg) in Escherichia coli and L-lysine (Lys) and Arg in Corynebacterium glutamicum, respectively. Under certain conditions, ArgO also mediates Lys export. To delineate the arrangement of ArgO in the cytoplasmic membrane of E. coli, we have employed a combination of cysteine accessibility in situ, alkaline phosphatase fusion reporters, and protein modeling to arrive at a topological model of ArgO. Our studies indicate that ArgO assumes an N in -C out configuration, potentially forming a five-transmembrane helix bundle flanked by a cytoplasmic N-terminal domain (NTD) comprising roughly its first 38 to 43 amino acyl residues and a short periplasmic C-terminal region (CTR). Mutagenesis studies indicate that the CTR, but not the NTD, is dispensable for ArgO function in vivo and that a pair of conserved aspartate residues, located near the opposing edges of the cytoplasmic membrane, may play a pivotal role in facilitating transmembrane Arg flux. Additional studies on amino acid substitutions that impair ArgO function in vivo and their derivatives bearing compensatory amino acid alterations indicate a role for intramolecular interactions in the Arg export mechanism, and some interactions are corroborated by normal-mode analyses. Lastly, our studies suggest that ArgO may exist as a monomer in vivo, thus highlighting the requirement for intramolecular interactions in ArgO, as opposed to interactions across multiple ArgO monomers, in the formation of an Arg-translocating conduit. IMPORTANCEThe orthologous proteins LysE of C. glutamicum and ArgO of E. coli function as exporters of the basic amino acids L-arginine and L-lysine and the basic amino acid L-arginine, respectively, and LysE can functionally substitute for ArgO when expressed in E. coli. Notwithstanding this functional equivalence, studies reported here show that ArgO possesses a membrane topology that is distinct from that reported for LysE, with substantial variation in the topological arrangement of the proximal one-third portions of the two exporters. Additional genetic and in silico studies reveal the importance of (i) the cytoplasmic N-terminal domain, (ii) a pair of conserved aspartate residues, and (iii) potential intramolecular interactions in ArgO function and indicate that an Arg-translocating conduit is formed by a monomer of ArgO. In growing bacteria, the steady-state intracellular level of an amino acid is determined by the balance of its biosynthesis, transmembrane (TM) transport, utilization for anabolic processes such as protein synthesis, and conversion to other metabolites. Whereas amino acid transport is usually associated with its uptake from the medium, the phenomenon of amino acid export has only recently been recognized (reviewed in references 1 and 2). The physiological basis of the existence of exporters of amino acids that are essential cellular metabolites is enigmatic. It is thought that exporters may function as o...

Metabolism of long-chain fatty acids affects disulfide bond formation in Escherichia coli and activates envelope stress response pathways as a combat strategy

“…AP activity was determined as described in [ 71 ], with slight modifications. Freshly prepared iodoacetamide (1 mM) was added directly to the cultures.…”

Topological analyses of the L-lysine exporter LysO reveal a critical role for a conserved pair of intramembrane solvent-exposed acidic residues

“…Toward determining the oligomeric state of LysO in vivo , we treated crude membrane preparations from the Δ lysO mutant, GJ9026, overexpressing LysO N-HA with the primary amine cross-linker DSS and with the DSS solvent DMSO. Crude membrane preparations from the Δ mscL ::Kan mutant, GJ16372, overexpressing a C-terminally HA epitope tagged version of the mechanosensitive channel MscL ( 39 ) were also treated as above. Following cross-linking and immunoblotting, distinct oligomeric species of MscL C-HA were detected, as reported earlier (( 40 ) and Fig.…”

“…The cultures were processed for PhoA assay as described in Pathania et al. ( 39 ). Values of PhoA units represent mean ± SD of two independent measurements performed in duplicate.…”

“…For these studies, J o u r n a l P r e -p r o o f a derivative of UTL2, GJ16286 was employed. Method 2 was performed as described earlier (39), with minor modifications that are described in the legend for figure S2.…”

Metabolism of long-chain fatty acids affects disulfide bond formation inEscherichia coliand activates envelope stress response pathways as a combat strategy

“…AP activity was determined as described in [69], with slight modifications. Freshly prepared iodoacetamide (1 mM) was added directly to the cultures.…”