Yeast and human P4-ATPases transport glycosphingolipids using conserved structural motifs.
Lipid transport is an essential process with manifest importance to human health and disease. Phospholipid flippases (P4-ATPases) transport lipids across the membrane bilayer and are involved in signal transduction, cell division, and vesicular transport. Mutations in flippase genes cause or contribute to a host of diseases, such as cholestasis, neurological deficits, immunological dysfunction, and metabolic disorders. Genome-wide association studies have shown that  ATP10A  and  ATP10D  variants are associated with an increased risk of diabetes, obesity, myocardial infarction, and atherosclerosis. Moreover,  ATP10D  SNPs are associated with elevated levels of glucosylceramide (GlcCer) in plasma from diverse European populations. Although sphingolipids strongly contribute to metabolic disease, little is known about how GlcCer is transported across cell membranes. Here, we identify a conserved clade of P4-ATPases from  Saccharomyces cerevisiae  (Dnf1, Dnf2),  Schizosaccharomyces pombe  (Dnf2), and  Homo sapiens  (ATP10A, ATP10D) that transport GlcCer bearing an  sn2  acyl-linked fluorescent tag. Further, we establish structural determinants necessary for recognition of this sphingolipid substrate. Using enzyme chimeras and site-directed mutagenesis, we observed that residues in transmembrane (TM) segments 1, 4, and 6 contribute to GlcCer selection, with a conserved glutamine in the center of TM4 playing an essential role. Our molecular observations help refine models for substrate translocation by P4-ATPases, clarify the relationship between these flippases and human disease, and have fundamental implications for membrane organization and sphingolipid homeostasis.
Roland BP, Naito T, Best JT, Arnaiz-Yépez C, Takatsu H, Yu RJ, Shin HW, Graham TR