Folding and activity of circularly permuted forms of a polytopic membrane protein

The transmembrane subunit of the Glc transporter (IICBGlc), which mediates uptake and concomitant phosphorylation of glucose, spans the membrane eight times. Variants of IICBGlc with the native N and C termini joined and new N and C termini in the periplasmic and cytoplasmic surface loops were expre ssed in Escherichia coil. In vivo transport/in vitro phosphotransferase act ivities of the circularly permuted variants with the termini in the peripla smic loops 1 to 4 were 35/58, 32/37, 0/3, and 0/0% of wild type, respective ly. The activities of the variants with the termini in the cytoplasmic loop s 1 to 3 were 0/25, 0/4 and 24/70, respectively. Fusion of alkaline phospha tase to the periplasmic C termini stabilized membrane integration and incre ased uptake and/or phosphorylation activities. These results suggest that i nternal signal anchor and stop transfer sequences can function as N-termina l signal sequences in a circularly permuted cu-helical bundle protein and t hat the orientation of transmembrane segments is determined by the amino ac id sequence and not by the sequential appearance during translation, Of the four IICBGlc variants with new termini in periplasmic loops, only the one with the discontinuity in loop 4 is inactive. The sequences of loop 4 and o f the adjacent TM7 and TM8 are conserved in all phosphoenolpyruvate-depende nt carbohydrate:phosphotransferase system transporters of the glucose famil y.