Site-specific mutagenesis of residues in the Escherichia coli mannitol permease that have been suggested to be important for its phosphorylation and chemoreception functions.
The Escherichia coli mannitol permease is an integral membrane protein that catalyzes the concomitant transport and phosphorylation of D-mannitol and also acts as the chemoreceptor for chemotaxis of E. coli to this hexitol. At least 4 aminoacyl residues in this protein have been suggested to be important in these activities: His-195, His-256, Cys-384, and His-554. Previous evidence has implicated His-554 and Cys-384 as residues that are covalently phosphorylated, in sequence, as intermediates in phosphotransfer to mannitol. We have constructed a number of site-specific mutants of the mannitol permease at these positions. The properties of proteins in which His-554 or Cys-384 has been changed are consistent with their essential roles in phosphorylation. We also used these mutants to show that intermolecular phosphotransfer between His-554 and Cys-384 can occur in vivo in membrane-bound heterodimers consisting of different mutant subunits. The properties of proteins with mutations at position 195 suggest an important role for this residue involving hydrogen bonding, while His-256 performs no significant function in the mannitol permease. Finally, the phosphorylation and chemoreception activities for each mutant protein were each roughly in the same proportion to these activities in the wild-type protein, showing that these functions of the mannitol permease are tightly coupled under normal physiological conditions.
Weng
Q P
QP
Department of Biology, Boston University, Massachusetts 02215.
Elder
J
J
Jacobson
G R
GR
D001426:Bacterial Proteins; D001483:Base Sequence; D002628:Chemoreceptor Cells / Q000737:chemistry / Q000378:metabolism*; D002633:Chemotaxis; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D046911:Macromolecular Substances; D008353:Mannitol / Q000378:metabolism*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D015398:Signal Transduction; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D009004:Monosaccharide Transport Proteins; D009838:Oligodeoxyribonucleotides; D008353:Mannitol; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli