Abstract
Transport of lipids across membranes is fundamental for diverse biological pathways in cells. Multiple ion-coupled transporters take part in lipid translocation, but their mechanisms remain largely unknown. Major facilitator superfamily (MFS) lipid transporters play central roles in cell wall synthesis, brain development and function, lipids recycling, and cell signaling. Recent structures of MFS lipid transporters revealed overlapping architectural features pointing towards a common mechanism. Here we used cysteine disulfide trapping, molecular dynamics simulations, mutagenesis analysis, and transport assays in vitro and in vivo, to investigate the mechanism of LtaA, a proton-dependent MFS lipid transporter essential for lipoteichoic acid synthesis in the pathogen Staphylococcus aureus. We reveal that LtaA displays asymmetric lateral openings with distinct functional relevance and that cycling through outward- and inward-facing conformations is essential for transport activity. We demonstrate that while the entire amphipathic central cavity of LtaA contributes to lipid binding, its hydrophilic pocket dictates substrate specificity. We propose that LtaA catalyzes lipid translocation by a ‘trap-and-flip’ mechanism that might be shared among MFS lipid transporters.
Highlights
Transport of lipids across membranes is fundamental for diverse biological pathways in cells
Some examples include the bacterial lysophospholipid transporter LplT, involved in lipids recycling in Gram-negative bacteria[7,18]; the human transporter MFSD2A, expressed at the blood–brain and blood–retinal barrier, contributing to major uptake of docosahexaenoic acid (DHA)[5,6,19–22]; the human transporters Spns[223,24], and MFSD2B25, which contribute to transport of sphingosine 1-phosphate (S1P) in endothelial cells and erythrocytes; and the gentiobiosyl-diacylglycerol transporter LtaA, involved in cell wall synthesis in Staphylococcus aureus[26,27]
We used cysteine disulfide trapping of outward- and inward-facing LtaA, in combination with molecular dynamics simulations, mutagenesis analysis, and transport assays in vitro and in vivo, and showed that cycling through outward- and inward-facing conformations is essential for LtaA activity
Summary
Transport of lipids across membranes is fundamental for diverse biological pathways in cells. Some examples include the bacterial lysophospholipid transporter LplT, involved in lipids recycling in Gram-negative bacteria[7,18]; the human transporter MFSD2A, expressed at the blood–brain and blood–retinal barrier, contributing to major uptake of docosahexaenoic acid (DHA)[5,6,19–22]; the human transporters Spns[223,24], and MFSD2B25, which contribute to transport of sphingosine 1-phosphate (S1P) in endothelial cells and erythrocytes; and the gentiobiosyl-diacylglycerol transporter LtaA, involved in cell wall synthesis in Staphylococcus aureus[26,27]. Despite their well-described cellular roles, the mechanisms of MFS lipid transporters remain insufficiently understood. We describe critical mechanistic elements revealing that LtaA adopts a “trapand-flip” mechanism that might be shared among MFS lipid transporters
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