title: Topology and boundaries of the aerotaxis receptor Aer in the membrane of Escherichia coli.
abstract: Escherichia coli chemoreceptors are type I membrane receptors that have a periplasmic sensing domain, a cytosolic signaling domain, and two transmembrane segments. The aerotaxis receptor, Aer, is different in that both its sensing and signaling regions are proposed to be cytosolic. This receptor has a 38-residue hydrophobic segment that is thought to form a membrane anchor. Most transmembrane prediction programs predict a single transmembrane-spanning segment, but such a topology is inconsistent with recent studies indicating that there is direct communication between the membrane flanking PAS and HAMP domains. We studied the overall topology and membrane boundaries of the Aer membrane anchor by a cysteine-scanning approach. The proximity of 48 cognate cysteine replacements in Aer dimers was determined in vivo by measuring the rate and extent of disulfide cross-linking after adding the oxidant copper phenanthroline, both at room temperature and to decrease lateral diffusion in the membrane, at 4 degrees C. Membrane boundaries were identified in membrane vesicles using 5-iodoacetamidofluorescein and methoxy polyethylene glycol 5000 (mPEG). To map periplasmic residues, accessible cysteines were blocked in whole cells by pretreatment with 4-acetamido-4'-maleimidylstilbene-2, 2' disulfonic acid before the cells were lysed in the presence of mPEG. The data were consistent with two membrane-spanning segments, separated by a short periplasmic loop. Although the membrane anchor contains a central proline residue that reaches the periplasm, its position was permissive to several amino acid and peptide replacements.
authors.affiliation: Division of Microbiology and Molecular Genetics, Loma Linda University, Loma Linda, CA 92350, USA.
authors.forename: Divya N
authors.initials: DN
authors.lastname: Amin
authors.forename: Barry L
authors.initials: BL
authors.lastname: Taylor
authors.forename: Mark S
authors.initials: MS
authors.lastname: Johnson
mesh_terms: D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002633:Chemotaxis / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D015398:Signal Transduction / Q000502:physiology*
chemical_list: C108679:Aer protein, E coli; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins