Structure/function relationships in the Escherichia coli mannitol permease: identification of regions important for membrane insertion, substrate binding and oligomerization

• Published in: Research in microbiology Vol. 143; no. 2; pp. 139 - 149
• Main Authors: Briggs, C.E, Khandekar, S.S, Jacobson, G.R
• Format: Journal Article
• Language: English
• Published: Paris Elsevier SAS 1992; Elsevier
• Subjects: Autoradiography; Bacteriology; Binding Sites - physiology; Biological and medical sciences; Biological Transport, Active; Chromosome Deletion; E. coli, Transport, Deletion mutations; E. coli, Transport, Mutations de délétion; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli Proteins; Fundamental and applied biological sciences. Psychology; In Vitro Techniques; mannitol; Mannitol - pharmacokinetics; mannitol permease; Mannitol, PEP, PTS, Insertion de la membrane, Oligomérisation, Perméase; Mannitol, PEP, PTS, Membrane insertion, Oligomerization, Permease; membrane proteins; Metabolism. Enzymes; Microbiology; Monosaccharide Transport Proteins; permease; Phosphoenolpyruvate Sugar Phosphotransferase System - chemistry; Phosphoenolpyruvate Sugar Phosphotransferase System - metabolism; structure; Structure-Activity Relationship; E. coli, Transport, Deletion mutations; Mannitol, PEP, PTS, Membrane insertion, Oligomerization, Permease; Mannitol, PEP, PTS, Insertion de la membrane, Oligomérisation, Perméase; E. coli, Transport, Mutations de délétion; Oligomerization; Enzyme; Escherichia coli; Molecular interaction; Topology; Structure function relationship; Substrate; Mannitol; Deletion; Bacteria; Phosphohistidinoprotein-hexose phosphotransferase; Mutation; Enterobacteriaceae
• ISSN: 0923-2508; 1769-7123
• DOI: 10.1016/0923-2508(92)90003-7

The Escherichia coli mannitol permease (EII Mtl) of the phosphoenolpyruvate-dependent phosphotransferase system is a 68-kDa membrane protein that carries out thatconcomitant transport and phosphorylation of D-mannitol. Previous studies indicated that there are ca. 6 membrane-spanning helices within the N-terminal half of the protein, while the hydrophilic C-terminal half was shown to be exposed in the cytoplasm. In the present study, an analysis of C-terminally truncated EII Mtl mutants showed that proteins from which only the cytoplasmic domain has been deleted were present in the membrane at ⩾ 50 % the amount of the intact protein. However, deletion proteins smaller than ca. 34 kDa were present in the membrane at only about 20 % the amount of the intact protein. We also constructed a plasmid that encodes the first 43 amino acid residues of EII Mtl fused to residues 378 to 637 (the C-terminal domain). The corresponding protein was associated with the cytoplasmic membrane. These results show that the first 43 amino acid residues of the N terminus are sufficient for membrane localization, although the region comprising the last 2 membrane-spanning helices appears to be important for maximum stability and/or efficient membrane insertion of the complete N-terminal domain. Further studies of these deletion proteins showed that binding of mannitol to the permease occurs even if the entire cytoplasmic domain is absent, but is abolished if the last putative membrane-spanning region is removed. Finally, regions of the protein within the membrane-bound domain were identified that influence the oligomerization state of the protein. These results further define domains of this multifunctional transport protein that are important for membrane insertion, stability, substrate binding and oligomerization. Des mutants de délétion de la mannitol-perméase de Escherichia coli ont été étudiés pour leurs propriétés fonctionnelles. Les résultats montrent que diverses fonctions de la perméase correspondent aux séquences d'acides aminés dans l'ordre suivant: insertion de la membrane, résidus 1–43; oligomérisation, résidus 43–290; stabilité de la liaison de la membrane et du mannitol, résidus 290–380; dimérisation, résidus 378–465 (chiffres ronds). Ces données confirment et renforcent les observations sur les relations structure/ fonction de la mannitol-perméase.