title: Site-directed fluorescence labeling reveals a revised N-terminal membrane topology and functional periplasmic residues in the Escherichia coli cell division protein FtsK.
abstract: In Escherichia coli, FtsK is a large integral membrane protein that coordinates chromosome segregation and cell division. The N-terminal domain of FtsK (FtsKN) is essential for division, and the C terminus (FtsKC) is a well characterized DNA translocase. Although the function of FtsKN is unknown, it is suggested that FtsK acts as a checkpoint to ensure DNA is properly segregated before septation. This may occur through modulation of protein interactions between FtsKN and other division proteins in both the periplasm and cytoplasm; thus, a clear understanding of how FtsKN is positioned in the membrane is required to characterize these interactions. The membrane topology of FtsKN was initially determined using site-directed reporter fusions; however, questions regarding this topology persist. Here, we report a revised membrane topology generated by site-directed fluorescence labeling. The revised topology confirms the presence of four transmembrane segments and reveals a newly identified periplasmic loop between the third and fourth transmembrane domains. Within this loop, four residues were identified that, when mutated, resulted in the appearance of cellular voids. High resolution transmission electron microscopy of these voids showed asymmetric division of the cytoplasm in the absence of outer membrane invagination or visible cell wall ingrowth. This uncoupling reveals a novel role for FtsK in linking cell envelope septation events and yields further evidence for FtsK as a critical checkpoint of cell division. The revised topology of FtsKN also provides an important platform for future studies on essential interactions required for this process.
authors.affiliation: From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
authors.forename: Alison M
authors.initials: AM
authors.lastname: Berezuk
authors.affiliation: From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
authors.forename: Mara
authors.initials: M
authors.lastname: Goodyear
authors.affiliation: From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada ckhursig@uoguelph.ca.
authors.forename: Cezar M
authors.initials: CM
authors.lastname: Khursigara
mesh_terms: D001483:Base Sequence; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D017931:DNA Primers; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D019897:Periplasm / Q000378:metabolism*; D016133:Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; C096452:FtsK protein, E coli; D008565:Membrane Proteins; D003545:Cysteine
keywords: Cell Division; Electron Microscopy (EM); Escherichia coli (E. coli); FtsK; Membrane Protein; Membrane Topology; Site-directed Mutagenesis