The prokaryotic aspartate transporter, Glt_Ph, is sensitive to lipid bilayer composition when it is reconstituted into liposomes. We explored the influence of phospholipid headgroup chemistry by reconstituting purified protein into liposomes composed primarily of phosphatidylethanolamine (PE) or its mono- (MM), di- (DM), or tri- (TM) methyl derivatives.

In PE liposomes, transport rates of aspartate were robust. These rates were reduced by approximately 50% in MM and DM liposomes, and rates displayed a further 25% reduction in TM liposomes.

Utilising a thiol-modification assay¹, we have determined the transmembrane orientation of Glt_Ph and shown that PE is able to orient Glt_Ph in a mostly right-side out orientation, however previous work in our laboratory has shown that both inside-out and right-side out orientation are indistinguishable in their ability to transport aspartate.  We have determined that in the absence of appreciable influence on binding kinetics²  or consequence in the transmembrane orientation of Glt_Ph, that there exists a site-specific lipid-protein interaction that reports the headgroup composition of the lipid bilayer.

Analysis of the available crystal structures^3,4,5 of Glt_Ph reveal a residue (Tyrosine-33) that we hypothesise make direct interactions with phospholipid headgroups and define the ability of the transporter to undergo conformational transitions within the lipid bilayer. The Y33F mutant displays WT rates of transport; however in the Y33S mutant, rates of transport are doubled. We hypothesise that a cation-pi interaction between Tyrosine-33 and the phospholipid bilayer confers favourability to enter structural- and rate-determining intermediates of the transport cycle⁵  .