Topological characterization of the essential Escherichia coli cell division protein FtsN.
Genetic and biochemical approaches were used to analyze a topological model for FtsN, a 36-kDa protein with a putative transmembrane segment near the N terminus, and to ascertain the requirements of the putative cytoplasmic and membrane-spanning domains for the function of this protein. Analysis of FtsN-PhoA fusions revealed that the putative transmembrane segment of FtsN could act as a translocation signal. Protease accessibility studies of FtsN in spheroblasts and inverted membrane vesicles confirmed that FtsN had a simple bitopic topology with a short cytoplasmic amino terminus, a single membrane-spanning domain, and a large periplasmic carboxy terminus. To ascertain the functional requirements of the N-terminal segments of FtsN, various constructs were made. Deletion of the N-terminal cytoplasmic and membrane-spanning domains led to intracellular localization of the carboxy domain, instability,and loss of function. Replacement of the N-terminal cytoplasmic and membrane-spanning domains with a membrane-spanning domain from MalG restored subcellular localization and function. These N-terminal domains of FtsN could also be replaced by the cleavable MalE signal sequence with restoration of subcellular localization and function. It is concluded that the N-terminal, cytoplasmic, and transmembrane domains of FtsN are not required for function of the carboxy domain other than to transport it to the periplasm. FtsQ and FtsI were also analyzed.
Dai
K
K
Department of Microbiology, Molecular Genetics, and Immunology, University of Kansas Medical Center, Kansas City 66160, USA.
Xu
Y
Y
Lutkenhaus
J
J
D018528:ATP-Binding Cassette Transporters; D000469:Alkaline Phosphatase / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002455:Cell Division; D019286:Endopeptidase K; D004926:Escherichia coli / Q000254:growth & development*; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D010449:Peptide Mapping; D033902:Periplasmic Binding Proteins; D011487:Protein Conformation; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000648:ultrastructure; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000378:metabolism; D013104:Spheroplasts
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C098711:FtsN protein, E coli; C461619:MalE protein, E coli; C079123:MalG protein, E coli; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D000469:Alkaline Phosphatase; D012697:Serine Endopeptidases; D019286:Endopeptidase K