Membrane topology analysis of Escherichia coli mannitol permease by using a nested-deletion method to create mtlA-phoA fusions.
The Escherichia coli mannitol permease catalyzes the concomitant transport and phosphorylation of D-mannitol. This 68-kDa protein consists of a membrane-bound, N-terminal domain involved in mannitol binding and translocation and a C-terminal, cytoplasmic domain responsible for mannitol phosphorylation. Secondary-structure prediction methods suggest that the N-terminal half of the permease spans the membrane approximately seven times in alpha-helical segments, but these data cannot conclusively predict the structure. We have used gene fusions between mtlA (encoding the permease) and 'phoA (encoding alkaline phosphatase lacking its signal sequence) to further investigate the topology of the mannitol permease. Initially, fusions were constructed by using a lambda TnphoA vector and in vitro cloning of 'phoA into naturally occurring restriction sites in mtlA. However, the former method gave severe problems with insertion "hot-spots" in our vector systems, and the latter method was limited by the number of useful restriction sites. Therefore, we developed a nested-deletion method for creating mtlA-phoA fusions. 'phoA was first cloned downstream from the part of mtlA encoding the membrane-bound half of the permease. This construct was then treated with the appropriate restriction enzymes and with exonuclease III to create random fusions. An analysis of greater than 40 different fusion clones constructed by these methods provides strong evidence for six membrane-spanning regions in the mannitol permease with three relatively short periplasmic loops and two large cytoplasmic loops in the membrane-bound half of the protein.
Sugiyama
J E
JE
Department of Biology, Boston University, MA 02215.
Mahmoodian
S
S
Jacobson
G R
GR
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000648:ultrastructure; D002872:Chromosome Deletion; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000648:ultrastructure*; D029968:Escherichia coli Proteins; D008565:Membrane Proteins / Q000235:genetics / Q000648:ultrastructure; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics / Q000648:ultrastructure*; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli; D000469:Alkaline Phosphatase