Mutational analyses define helix organization and key residues of a bacterial membrane energy-transducing complex.
In Gram-negative bacteria, many biological processes are coupled to inner membrane ion gradients. Ions transit at the interface of helices of integral membrane proteins, generating mechanical energy to drive energetic processes. To better understand how ions transit through these channels, we used a model system involved in two different processes, one of which depends on inner membrane energy. The Tol machinery of the Escherichia coli cell envelope is dedicated to maintaining outer membrane stability, a process driven by the proton-motive force. The Tol system is parasitized by bacterial toxins called colicins, which are imported through the outer membrane using an energy-independent process. Herein, we mutated TolQ and TolR transmembrane residues, and we analyzed the mutants for outer membrane stability, colicin import and protein complex formation. We identified residues involved in the assembly of the complex, and a new class of discriminative mutations that conferred outer membrane destabilization identical to a tol deletion mutant, but which remained fully sensitive to colicins. Further genetic approaches revealed transmembrane helix interactions and organization in the bilayer, and suggested that most of the discriminative residues are located in a putative aqueous ion channel. We discuss a model for the function of related bacterial molecular motors.
Goemaere
Emilie L
EL
Laboratoire d'Ingénierie des Systèmes Macromoléculaires, Institut de Biologie Structurale et Microbiologie, CNRS-UPR 9027, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
Cascales
Eric
E
Lloubès
Roland
R
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003087:Colicins / Q000378:metabolism / Q000494:pharmacology; D016384:Consensus Sequence; D017124:Conserved Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry; D004252:DNA Mutational Analysis; D048168:Escherichia coli K12 / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D005557:Formaldehyde / Q000737:chemistry; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011392:Proline / Q000378:metabolism; D017433:Protein Structure, Secondary; D018892:Proton-Motive Force / Q000502:physiology*; D013489:Suppression, Genetic
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D003087:Colicins; D003432:Cross-Linking Reagents; D005557:Formaldehyde; D011392:Proline