Structural organization of the protein-tyrosine autokinase Wzc within Escherichia coli cells.
Protein Wzc from Escherichia coli is a member of a newly defined family of protein-tyrosine autokinases that are essential for surface polysaccharide production in both Gram-negative and Gram-positive bacteria. Although the catalytic mechanism of the autophosphorylation of Wzc was recently described, the in vivo structural organization of this protein remained unclear. Here, we have determined the membrane topology of Wzc by performing translational fusions of lacZ and phoA reporter genes to the wzc gene. It has been shown that Wzc consists of two main structural domains: an N-terminal domain, bordered by two transmembrane helices, which is located in the periplasm of cells, and a C-terminal domain, harboring all phosphorylation sites of the protein, which is located in the cytoplasm. In addition, it has been demonstrated for the first time that Wzc can oligomerize in vivo to form essentially trimers and hexamers. Cross-linking experiments performed on strains expressing various domains of Wzc have shown that the cytoplasmic C-terminal domain is sufficient to generate oligomerization of Wzc. Mutant proteins, modified in either the ATP-binding site or the different phosphorylation sites, i.e. rendered unable to undergo autophosphorylation, have appeared to oligomerize into high molecular mass species identical to those formed by the wild-type protein. It was concluded that phosphorylation of Wzc is not essential to its oligomerization. These data, connected with the phosphorylation mechanism of Wzc, may be of biological significance in the regulatory role played by this kinase in polysaccharide synthesis.
Institut de Biologie et Chimie des Protéines, Centre National de la Recherche Scientifique, Université de Lyon, 69367 Lyon cedex 07, France.
Patricia
P
Doublet
Christophe
C
Grangeasse
Brice
B
Obadia
Elisabeth
E
Vaganay
Alain J
AJ
Cozzone
D000469:Alkaline Phosphatase; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain; D002462:Cell Membrane / Q000201:enzymology; D018844:Cyclin-Dependent Kinases / Q000737:chemistry / Q000235:genetics; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005838:Genotype; D008565:Membrane Proteins; D008958:Models, Molecular; D019897:Periplasm / Q000201:enzymology; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011487:Protein Conformation; D021122:Protein Subunits; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; C417168:WZC protein, Bacteria; D011505:Protein-Tyrosine Kinases; C499615:wzc protein, E coli; D018844:Cyclin-Dependent Kinases; D000469:Alkaline Phosphatase; C114089:phoA protein, E coli