The N-terminal 22 amino acid residues in the lactose permease of Escherichia coli are not obligatory for membrane insertion or transport activity.

Abstract

When the lactose (lac) permease of Escherchia coli is expressed from the lac promoter at relatively low rates, deletion of amino acid residues 2-8 (AT) or 2-9 (A8) from the hydrophilic N terminus has a relatively minor effect on the ability of the permease to catalyze active lactose transport. Activity is essentially abolished, however, and the permease is hardly detected in the membrane when two additional amino acid residues are deleted (A4O), and mutants deleted ofresidues 2-23 (A22) or 2-39 (A38) also exhibit no activity and are not inserted into the membrane. Dramatically, when the defective deletion mutants are overexpressed at high rates via the T7 promoter, A10 and A22 are inserted into the membrane in a stabIeforjnand catalyze active lactose transport in a highly significant manner, whereas A38 is hardly detected in the membrane and exhibits no activity. Interestingly, a fusion protein consisting of A38 and the ompA leader peptide is inserted into the membrane but exhibits no transport activity. The results indicate that the N-terminal hydrophilic domain of lac permease and the N-terminal half of the first putative transmembrane a-helix are not mandatory for either membrane insertion or transport activity.The lactose (lac) permease of Escherichia coli is a hydrophobic, polytopic cytoplasmic membrane protein that. catalyzes the coupled translocation ofp-galactosides and H' with a 1:1 stoichiometry (i.e., symport or cotransport). Encoded by the lacY gene, the permease has been solubilized from the membrane, purified, reconstituted into proteoliposomes, and shown to be solely responsible for f3-galactoside transport (1-3). Based on circular dichroic studies and hydropathy analysis (4), a secondary structure was proposed in which the permease has a short hydrophilic N terminus, 12 transmembrane hydrophobic domains in a-helical configuration connected by hydrophilic loops, and a 17-residue hydrophilic C-terminal tail. Evidence confirming some of the general features of the model and demonstrating that the N and C termini are on the cytoplasmic face ofthe membrane has been obtained from laser Raman (5) and Fourier transform infrared

Truncation of MalF Results in Lactose Transport via the Maltose Transport System of Escherichia coli

“…Very similar results have been observed with LacY. A large portion (N-terminal 22 amino acids) of the first transmembrane helix of LacY was found not to be obligatory for lactose transport activity (46). However, a deletion construct of LacY, which contains only the first N-terminal transmembrane helix and the last six C-terminal transmembrane helices, was found to transport lactose efficiently (ϳ80% of wild type) and specifically (47).…”

Structural cues involved in endoplasmic reticulum degradation of G85E and G91R mutant cystic fibrosis transmembrane conductance regulator.

“…Significant evidence indicates that transmembrane topology of polytopic proteins is directed through the action of discrete sequence determinants (i.e., topogenic sequences [25]) encoded within the nascent chain (26)(27)(28)(29)(30). These determinants are comprised of hydrophobic transmembrane segments along with their flanking sequences (31), and function to direct translocation of specific peptide regions across the ER membrane.…”

Involvement of the central loop of the lactose permease of Escherichia coli in its allosteric regulation by the glucose-specific enzyme IIA of the phosphoenolpyruvate-dependent phosphotransferase system

“…The experiments presented here provide both in vivo and in vitro evidence that the binding site in the lactose permease for IIA Glc (1,12,13). These plasmids are described in Table 1.…”