The glucose transporter of the Escherichia coli phosphotransferase system:: Linker insertion mutants and split variants

The IICBGlc subunit of the glucose transporter acts by a mechanism which couples vectorial translocation with phosphorylation of the substrate. It contains 8 transmembrane segments connected by 4 periplasmic, 2 short, 1 long (80 residues), cytoplasmic loops and an independently folding cytoplasmic domain at the C-terminus, Random DNase I cleavage, EcoRI linker insertion, and screening for transport-active mutants afforded 12 variants with between 46% and 116% of wild-type sugar phosphorylation activity. They carried inserts of up to 29 residues and short deletions in periplasmic loops 1, 2, and 3, in the long cytoplasmic loop 3, and in the linker region between the membrane spanning IICGlc and the cytoplasmic IIBGlc domains. Disruption of the gene at the sites of linker insertion decreased the expression level and diminished phosphotransferase activity to between 7% and 32%. IICBGlc with a discontinuity in the cytoplasmic loop was purified to homogeneity as a stable complex. It was active only if encoded by a dicistronic operon but not if encoded by two genes on two different replicons, suggesting that spatial proximity of the nascent polypeptide chains is important for folding and membrane assembly.