Hydrophilic C-terminal domain of the Escherichia coli mannitol permease: phosphorylation, functional independence, and evidence for intersubunit phosphotransfer.
The mannitol-specific enzyme II (mannitol permease) of the Escherichia coli phosphotransferase system (PTS) catalyzes the concomitant transport and phosphorylation of D-mannitol. Previous studies have shown that the mannitol permease (637 amino acid residues) consists of 2 structural domains of roughly equal size: an N-terminal, hydrophobic, membrane-bound domain and a C-terminal, hydrophilic, cytoplasmic domain. The C-terminal domain can be released from the membrane by mild proteolysis of everted membrane vesicles [Stephan, M.M., & Jacobson, G.R. (1986) Biochemistry 25, 8230-8234]. In this report, we show that phosphorylation of the intact permease by [32P]HPr (a general phosphocarrier protein of the PTS) followed by tryptic separation of the two domains resulted in labeling of only the C-terminal domain. Phosphorylation of the C-terminal domain occurred even in the complete absence of the N-terminal domain, showing that the former contains most, if not all, of the critical residues comprising the interaction site for phospho-HPr. The phosphorylated C-terminal domain, however, could not transfer its phospho group to mannitol, suggesting that the N-terminal domain is necessary for mannitol binding and/or phosphotransfer from the enzyme to the sugar. The elution profile of the C-terminal domain after molecular sieve chromatography showed that the isolated domain is monomeric, unlike the native permease which is likely a dimer in the membrane. Experiments employing a deletion mutation of the mtlA gene, which encodes a protein lacking the first phosphorylation site in the C-terminal domain (His-554) but retaining the second phosphorylation site (Cys-384), demonstrated that a phospho group could be transferred from phospho-HPr to Cys-384 of the deletion protein, and then to mannitol, only in the presence of the full-length permease.(ABSTRACT TRUNCATED AT 250 WORDS)
Stephan
M M
MM
Department of Biology, Boston University, Massachusetts 02215.
Khandekar
S S
SS
Jacobson
G R
GR
D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments / Q000302:isolation & purification; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids; D011487:Protein Conformation; D012995:Solubility
D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli