R. Beutler et al., The glucose transporter of the Escherichia coli phosphotransferase system:Linker insertion mutants and split variants, BIOCHEM, 39(13), 2000, pp. 3745-3750
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.