Probing the domain structure of FtsZ by random truncation and insertion of GFP.
Random transposon-mediated mutagenesis has been used to create truncations and insertions of green fluorescent protein (GFP), and Venus-yellow fluorescent protein (YFP), in Escherichia coli FtsZ. Sixteen unique insertions were obtained, and one of them, in the poorly conserved C-terminal spacer, was functional for cell division with the Venus-YFP insert. The insertion of enhanced GFP (eGFP) at this same site was not functional; Venus-YFP was found to be superior to eGFP in other respects too. Testing the constructs for dominant negative effects led to the following general conclusion. The N-terminal domain, aa 1-195, is an independently folding domain that can poison Z-ring function when expressed without a functional C-terminal domain. The effects were weak, requiring expression of the mutant at 3-5 times the level of wild-type FtsZ. The C-terminal domain, aa 195-383, was also independently folding, but had no activity in vivo. The differential activity of the N- and C-terminal domains suggests that FtsZ protofilament assembly is directional, with subunits adding primarily at the bottom of the protofilament. Directional assembly could occur by either a treadmilling or a dynamic instability mechanism.
Osawa
Masaki
M
Dept of Cell Biology, 3709 Duke University Medical Center, Durham, NC 27710, USA.
Erickson
Harold P
HP
Dept of Cell Biology, 3709 Duke University Medical Center, Durham, NC 27710, USA.
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry*; D017353:Gene Deletion; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry
D001426:Bacterial Proteins; D003598:Cytoskeletal Proteins; D004251:DNA Transposable Elements; D029968:Escherichia coli Proteins; C071525:FtsZ protein, Bacteria; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins