Folding and activity of circularly permuted forms of a polytopic membrane protein.
The transmembrane subunit of the Glc transporter (IICB(Glc)), which mediates uptake and concomitant phosphorylation of glucose, spans the membrane eight times. Variants of IICB(Glc) with the native N and C termini joined and new N and C termini in the periplasmic and cytoplasmic surface loops were expressed in Escherichia coli. In vivo transport/in vitro phosphotransferase activities of the circularly permuted variants with the termini in the periplasmic loops 1 to 4 were 35/58, 32/37, 0/3, and 0/0% of wild type, respectively. The activities of the variants with the termini in the cytoplasmic loops 1 to 3 were 0/25, 0/4 and 24/70, respectively. Fusion of alkaline phosphatase to the periplasmic C termini stabilized membrane integration and increased uptake and/or phosphorylation activities. These results suggest that internal signal anchor and stop transfer sequences can function as N-terminal signal sequences in a circularly permuted alpha-helical bundle protein and that the orientation of transmembrane segments is determined by the amino acid sequence and not by the sequential appearance during translation. Of the four IICB(Glc) variants with new termini in periplasmic loops, only the one with the discontinuity in loop 4 is inactive. The sequences of loop 4 and of the adjacent TM7 and TM8 are conserved in all phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system transporters of the glucose family.
Beutler
R
R
Departement für Chemie und Biochemie, Universität Bern, Freiestrasse 3, CH-3012 Bern, Switzerland.
Ruggiero
F
F
Erni
B
B
D000469:Alkaline Phosphatase / Q000235:genetics; D000595:Amino Acid Sequence; D001692:Biological Transport; D002942:Circular Dichroism; D004926:Escherichia coli / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D008757:Methylglucosides; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D008565:Membrane Proteins; D008757:Methylglucosides; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; C027020:methylglucoside; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D000469:Alkaline Phosphatase