Carboxyl-terminal truncations of the melibiose carrier of Escherichia coli.
The melibiose carrier of Escherichia coli is predicted to possess a short NH2 terminus, 11 transmembrane segments joined by short hydrophilic regions, and a 40-residue hydrophilic carboxyl terminus of unknown function. This paper describes truncations of the carboxyl terminus at eight locations using site-specific mutagenesis to introduce stop codons. Measurement of sugar transport and cation-coupling characteristics indicate that the carboxyl tail plays no direct role in substrate recognition or energy transduction. Thirty-six amino acids could be removed from the hydrophilic carboxyl domain without the loss of sugar specificity, facilitated diffusion, uphill transport, H+-coupling or Na+-coupling characteristics. These results are consistent with the hypothesis that the sugar/cation binding site is formed by the interaction of the transmembrane helices 3, 4, 6, 9, and 10 and does not involve the carboxyl-terminal portion of the protein. When truncations were made within the hydrophobic domain of transmembrane helix 11 (truncations of 41 or more residues), the carrier was no longer found in the membrane. This suggests that the carboxyl terminus may be involved in the membrane insertion process, stabilization of the carrier within the membrane following insertion, or protection of the inserted carrier from proteolytic scavenging. A new plasmid that expresses the temperature-resistant isoform of the melibiose carrier under inducible control of a tac promoter, designated pKKMB, is also described.
Botfield
M C
MC
Department of Cellular and Molecular Physiology, Harvard Medical School, Boston, Massachusetts 02115.
Wilson
T H
TH
D001665:Binding Sites; D001692:Biological Transport; D003062:Codon; D004926:Escherichia coli / Q000201:enzymology*; D005786:Gene Expression Regulation; D006859:Hydrogen / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D010957:Plasmids; D011487:Protein Conformation; D011887:Raffinose / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D027981:Symporters; D013696:Temperature
D003062:Codon; D026901:Membrane Transport Proteins; D027981:Symporters; D006859:Hydrogen; C021548:melibiose permease; D012964:Sodium; D011887:Raffinose