RRC ID 54533
Author Roland BP, Naito T, Best JT, Arnaiz-Yépez C, Takatsu H, Yu RJ, Shin HW, Graham TR.
Title Yeast and human P4-ATPases transport glycosphingolipids using conserved structural motifs.
Journal J Biol Chem
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.
Volume 294(6)
Pages 1794-1806
Published 2019-2-8
DOI 10.1074/jbc.RA118.005876
PII RA118.005876
PMID 30530492
PMC PMC6369285
MeSH ATP-Binding Cassette Transporters / chemistry* ATP-Binding Cassette Transporters / genetics ATP-Binding Cassette Transporters / metabolism Adenosine Triphosphatases / chemistry* Adenosine Triphosphatases / genetics Adenosine Triphosphatases / metabolism Biological Transport, Active Glucosylceramides / chemistry Glucosylceramides / metabolism HeLa Cells Humans Membrane Transport Proteins / chemistry* Membrane Transport Proteins / genetics Membrane Transport Proteins / metabolism Models, Molecular* Mutagenesis, Site-Directed Protein Domains Saccharomyces cerevisiae / enzymology* Saccharomyces cerevisiae Proteins / chemistry* Saccharomyces cerevisiae Proteins / genetics Saccharomyces cerevisiae Proteins / metabolism Schizosaccharomyces / enzymology* Schizosaccharomyces pombe Proteins / chemistry* Schizosaccharomyces pombe Proteins / metabolism
IF 4.106
Times Cited 15
Resource
DNA material pCMV-VSV-G-RSV-Rev (RDB04393)