Yeast and human P4-ATPases transport glycosphingolipids using conserved structural motifs

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.

Keywords: P4-ATPase; cerebroside; enzyme mechanism; flippase; glucosylceramide; glycolipid; glycosphingolipids; lipid transport; membrane asymmetry; membrane bilayer; membrane biology; sphingolipid.

Figure 1.

S. cerevisiae NBD-monosaccharide glycosphingolipid uptake requires plasma membrane P4-ATPases, Dnf1 and Dnf2. A, NBD-lipid uptake was measured in WT (BY4741) and P4-ATPase knockout strains and presented as raw, arbitrary fluorescent units (A.F.U.) (n ≥ 9) ± S.D. (error bars). B, upon Dnf1,2-dependent uptake, NBD-GlcCer localized to mitochondria (mt-RFP). C and D, kinetics assessments of NBD-PC, NBD-GlcCer, and NBD-GalCer uptake in dnf1,2Δ cells expressing pRS313-DNF1 (C) or pRS313-DNF2 (D), normalized to empty vector controls (n = 6) ± S.D. E, velocities of substrate transport from linear regression fits of data in C and D, ± S.E. F, NBD-lipid uptake was measured in S. cerevisiae WT, S. pombe WT, and S. pombe dnf2Δ strains. Asterisks in F indicate differences in S. pombe KO strains from S. pombe WT. A one-way ANOVA was performed to assess variance in A and F, and comparisons were made with Tukey's post hoc analysis. A two-way repeated measures ANOVA was used to assess variance in the kinetic data, and comparisons with NBD-PC were made with Tukey's post hoc analysis: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 2.

H. sapiens ATP10A and -10D translocate NBD-GlcCer at the plasma membrane. A–C, parental HeLa cells (−) and cells stably expressing human P4-ATPases and mutants were incubated with the indicated NBD-lipids at 15 °C for 15 min. After extraction with fatty acid–free BSA, the residual fluorescence intensity associated with the cells was determined by flow cytometry. Graphs display averages from 3–4 independent experiments ± S.D. (error bars). -Fold increase of NBD-lipid uptake compared with parental cells (−) is shown. A one-way ANOVA was performed to assess variance in A–C, and comparisons with parental cells (−) were made with Tukey's post hoc analysis. D, HeLa cells were incubated with NBD-GlcCer at 15 °C for the indicated times (x axis). E, velocities of GlcCer transport from linear regression fits of data in D (AU, arbitrary units), ± S.D. Graphs display averages from three independent experiments ± S.D. A two-way repeated measures ANOVA was used to assess variance in the kinetic data, and comparisons with parental cells (−) were made with Tukey's post hoc analysis: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 3.

The exofacial TM1 “GA motif” facilitates Dnf2 selection and preference for GlcCer. A, a sequence logo was created from an alignment of TM1 of P4-ATPases from different organisms, with letter size representing residue frequency and color denoting chemical differences. Hydrophilic residues are shown in green and purple, acidic residues in red, and hydrophobic residues in black. B, a focused alignment comparing a region of TM1 from S. cerevisiae, H. sapiens, and S. pombe, highlighting the GA motif. C and D, dissecting the first and second positions of the GA motif reveals that substitutions in both positions can reduce GlcCer preference (C) but do not alter PC or PE recognition (D). E and F, double substitutions were created to examine S. pombe and H. sapiens sequences in the context of the S. cerevisiae Dnf2. These compound mutations reduced GlcCer preference (E) and selection (F) without altering the known glycerophospholipid substrates (F). Variance was assessed among data sets using one-way ANOVAs, and comparisons with WT were made with Tukey's post hoc analysis. Although first- and second-position GA analyses are presented in different panels (C), their statistical variance was tested together. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Error bars, S.D.

Figure 4.

A Pro−4 glutamine in TM4 of Dnf2 is required for GlcCer transport. A, a sequence logo was created from an alignment of TM4 of P4-ATPases from different organisms, with letter size representing residue frequency and color denoting chemical characteristics. Hydrophilic residues are shown in green and purple, acidic residues in red, and hydrophobic residues in black. B, a focused alignment comparing a region of TM4 from S. cerevisiae, H. sapiens, and S. pombe, highlighting the YQS motif that was previously altered in S. cerevisiae Dnf1 (Fig. S7). C and D, the three YQS positions were tested for their influence on GlcCer preference (C) and selection (D), revealing that the central glutamine was the strongest determinant of GlcCer transport. E, homology model of Dnf1 with TM1–6 shown as pink cylinders and the rest of the protein colored green. GA, YQS, and WVAV motifs are represented by spheres and colored by element. PM boundaries are indicated. Variance was assessed with one-way ANOVAs, and comparisons with WT were made with Tukey's post hoc analysis. Although the YQS positions in C are presented in separate panels, their statistical variance was tested together. n ≥ 9, ± S.D. (error bars). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 5.

Residues modeled in proximity to the Pro−4 glutamine influence GlcCer transport. A, homology model of Dnf1 (PDB code 3W5D) with TM1–6 shown as pink cylinders, the rest of the protein colored green, and the YQS motif represented by spheres and colored by element; PM boundaries are indicated. B, a 90° rotated and enhanced view of the peri-Gln⁶⁵⁵ region formed by TM2, -4, and -6. C, enhanced view of TM1, -2, -3, and -4, which surround Gln⁶⁵⁵. Residues were selected for mutagenesis by identifying positions that were predicated to be planar with the YQS motif and are shown in sticks and colored by element. D and E, GlcCer preference (D) and selection (E) were examined for all positions, revealing that TM1, TM2, and TM6 positions were capable of altering GlcCer transport. One position required chemical specificity to alter GlcCer transport (Leu²⁶⁴). Variance was assessed among data sets using one-way ANOVAs, and comparisons with WT were made with Tukey's post hoc analysis. n ≥ 9, ± S.D. (error bars). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 6.

Structure–function analysis of the H. sapiens ATP10D substrate pathway demonstrates primary structural conservation of TM4 Pro−4 position in GlcCer transport. A, sequence alignments of TM1, TM4, and TM6 of P4-ATPases are shown. Hydrophilic residues are indicated in green, red (negatively charged), and blue (positively charged), and hydrophobic residues are indicated in black. Three motifs, which were required for GlcCer preference in Dnf2, are underlined. The arrowheads indicate amino acids that were critical for ATP10D to transport GlcCer. B–H, NBD-lipid uptake was measured in HeLa cells stably expressing C-terminally HA-tagged ATP10A (WT), ATP10D (WT), each mutant (indicated), and parental cells (−); TM is numbered in parenthesis. B–D, the glutamine at TM4 Pro−4 was critical for ATP10D to transport GlcCer but was dispensable for ATP10A to transport PC. E and F, the motif in TM6 was critical for GlcCer transport of ATP10D but was dispensable for PC transport of ATP10A. The experiments of panels E were performed together with panels C, and thus graphs (−) and WT of E are equivalent to those of C. Graphs display averages from three independent experiments ± S.D. (error bars). -Fold increase of NBD-lipid uptake compared with parental cells (−) is shown. A one-way ANOVA was performed to assess variance in B–H, and comparisons with WT were made with Tukey's post hoc analysis. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. I, TM1, -2, -3, -4, -5, and -6 are indicated in red, orange, pink, magenta, purple, and blue, respectively, and others are indicated in gray. Critical residues for GlcCer transport are indicated by green sticks.

Figure 7.

Yeast Dnf1,2 phylogenically clusters with the human ATP10D family members. An unrooted phylogenic tree of S. cerevisiae, S. pombe, and H. sapiens P4-ATPases with branch length indicating character change. A new clade of GlcCer P4-ATPases is indicated. Protein accession numbers and tools used for analysis are found under “Experimental procedures.”