Domain complementation studies reveal residues critical for the activity of the mannitol permease from Escherichia coli

Abstract

This paper presents domain complementation studies in the mannitol transporter, EII mtl, from Escherichia coli. EII mtl is responsible for the transport and concomitant phosphorylation of mannitol over the cytoplasmic membrane. By using tryptophan-less EII mtl as a basis, each of the four phenylalanines located in the cytoplasmic loop between putative transmembrane helices II and III in the membrane-embedded C domain were replaced by tryptophan, yielding the mutants W97, W114, W126, and W133. Except for W97, these single-tryptophan mutants exhibited a high, wild-type-like, binding affinity for mannitol. Of the four mutants, only W114 showed a high mannitol phosphorylation activity. EII mtl is functional as a dimer and the effect of these mutations on the oligomeric activity was investigated via heterodimer formation (C/C domain complementation studies). The low phosphorylation activities of W126 and W133 could be increased 7–28 fold by forming heterodimers with either the C domain of W97 (IIC mtlW97) or the inactive EII mtl mutant G196D. W126 and W133, on the other hand, did not complement each other. This study points towards a role of positions 97, 126 and 133 in the oligomeric activation of EII mtl. The involvement of specific residue positions in the oligomeric functioning of a sugar-translocating EII protein has not been presented before.