Abstract
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.
Highlights
Lipid transport is an essential process with manifest importance to human health and disease
We broadly examined the requirement for Dnf1 and Dnf2 in sphingolipid transport relative to known substrates using WT; dnf1⌬; dnf2⌬; and dnf1,2⌬ knockout (KO) strains
We tested the role of lem3 in glycosphingolipid transport and found that it recapitulated the transport defects of the dnf1,2⌬ strain but did not enhance this defect in a dnf1,2⌬ lem3⌬ strain (Fig. S2C)
Summary
Dnf and Dnf are two related P4-ATPases that localize to the plasma membrane of Saccharomyces cerevisiae and transport phosphatidylcholine (PC) and phosphatidylethanolamine (PE). ATP10A preferred NBD-PC as previously reported (Fig. 2C) [30, 31], but measuring the kinetics of GlcCer uptake relative to parental (Ϫ) and ATP10DE215Q controls revealed that ATP10A was capable of transporting NBDGlcCer, at half the rate of ATP10D (Fig. 2, D and E). Dnf chimeras bearing Drs TM segments 1– 4 displayed a significant reduction in NBD-GlcCer transport and 2-fold changes to the ratio of GlcCer to PC (a measure of substrate preference) (Fig. S6). Single-position mutations in the WVAV motif of Dnf did not alter GlcCer preference or selection (Fig. S8, C and D), suggesting that the general integrity of this region may be more critical to lipid transport than individual amino acid side chains. Altering the central glutamine (Dnf2Q655A/N) elicited the strongest perturbation of GlcCer preference and
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