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 co uples vectorial translocation with phosphorylation of the substrate. It con tains 8 transmembrane segments connected by 4 periplasmic, 2 short, 1 long (80 residues), cytoplasmic loops and an independently folding cytoplasmic d omain at the C-terminus, Random DNase I cleavage, EcoRI linker insertion, a nd screening for transport-active mutants afforded 12 variants with between 46% and 116% of wild-type sugar phosphorylation activity. They carried ins erts of up to 29 residues and short deletions in periplasmic loops 1, 2, an d 3, in the long cytoplasmic loop 3, and in the linker region between the m embrane spanning IICGlc and the cytoplasmic IIBGlc domains. Disruption of t he 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 s table complex. It was active only if encoded by a dicistronic operon but no t if encoded by two genes on two different replicons, suggesting that spati al proximity of the nascent polypeptide chains is important for folding and membrane assembly.