Impact of charged amino acid substitution in the transmembrane domain of l-alanine exporter, AlaE, of Escherichia coli on the l-alanine export

Abstract

The Escherichia coli alaE gene encodes the L-alanine exporter, AlaE, that catalyzes active export of L-alanine using proton electrochemical potential. The transporter comprises only 149 amino acid residues and four predicted transmembrane domains (TMs), which contain three charged amino acid residues. The AlaE-deficient L-alanine non-metabolizing cells (ΔalaE cells) appeared hypersusceptible to L-alanyl-L-alanine showing a minimum inhibitory concentration (MIC) of 2.5 µg/ml for the dipeptide due to a toxic accumulation of L-alanine. To elucidate the mechanism by which AlaE exports L-alanine, we replaced charged amino acid residues in the TMs, glutamic acid-30 (TM-I), arginine-45 (TM-II), and aspartic acid-84 (TM-III) with their respective charge-conserved amino acid or a net neutral cysteine. The ΔalaE cells producing R45K or R45C appeared hypersusceptible to the dipeptide, indicating that arginine-45 is essential for AlaE activity. MIC of the dipeptide in the ΔalaE cells expressing E30D and E30C was 156 µg/ml and >10,000 µg/ml, respectively, thereby suggesting that a negative charge at this position is not essential. The ΔalaE cells expressing D84E or D84C showed an MIC >10,000 and 78 µg/ml, respectively, implying that a negative charge is required at this position. These results were generally consistent with that of the L-alanine accumulation experiments in intact cells. We therefore concluded that charged amino acid residues (R45 and D84) in the AlaE transmembrane domain play a pivotal role in L-alanine export. Replacement of three cysteine residues at C22, C28 (both in TM-I), and C135 (C-terminal region) with alanine showed only a marginal effect on L-alanine export.

Expansion of the Transporter-Opsin-G protein-coupled receptor superfamily with five new protein families

“…Thus, YgaW is likely to be the physiologically most relevant exporter for L-alanine in E. coli. More recently, two charged residues (R45 and D84) were found to be essential for AlaE efflux activity [19].…”

Importance of transmembrane helix 4 of l-alanine exporter AlaE in oligomer formation and substrate export activity in Escherichia coli

“…These results indicated that the C-terminus of AlaE is localized in the cytoplasm of the cells. Next, we used a cysteine-less AlaE variant, which retained l -alanine export activity [20], to construct single Cys variants (V12C, Q40C, S41C, S76C, W107C and A149C); the amino acid residues involved in these substitutions were predicted to be localized in the aqueous phase of the cytoplasm and periplasm. When cells are first treated with MTSET, a non-fluorescent reagent, followed by fluorescent OGM treatment as described in the Methods, cysteine residues present in the cytoplasm are supposed to be labelled with OGM.…”

“…Certain transmembranes of AlaE were predicted to be involved in the oligomerization step. In our earlier random mutagenesis study, four AlaE missense mutations (S34F, S116L, G125E and G129D) were identified in the TM helices, each of which leads to the dysfunction of AlaE [20]. Interestingly, three (S116L, G125E and G129D) amino acid changes among them were present in the TM4, implying that TM4 plays an important role in AlaE function.…”

“…In our previous study, we isolated 22 E. coli mutants that were unable to export l -alanine out of their cells and found that four mutants had a missense point mutation in the alaE gene (S34F, S116L, G125E and G129D) [19, 20]. Three mutations (S116L, G125E and G129D) were predicted to be present in the fourth transmembrane (TM4) region of AlaE.…”

“…Three mutations (S116L, G125E and G129D) were predicted to be present in the fourth transmembrane (TM4) region of AlaE. Interestingly, two glycine mutations (G125 and G129) matched a GxxxG motif [19,20] that is known to be involved in transmembrane helix interactions and plays an important role in oligomer formation [21,22]. To further understand the molecular mechanism of AlaE export activity, we biochemically analysed the membrane topology of AlaE and addressed the question of whether AlaE could form oligomers through the GxxxG motif.…”