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Coupling between endocytosis and sphingosine kinase 1 recruitment.

Shen H, Giordano F, Wu Y, Chan J, Zhu C, Milosevic I, Wu X, Yao K, Chen B, Baumgart T, Sieburth D, De Camilli P - Nat. Cell Biol. (2014)

Bottom Line: Membrane recruitment of SPHK1 involves a direct, curvature-sensitive interaction with the lipid bilayer mediated by a hydrophobic patch on the enzyme's surface.The knockdown of SPHKs results in endocytic recycling defects, and a mutation that disrupts the hydrophobic patch of Caenorhabditis elegans SPHK fails to rescue the neurotransmission defects in loss-of-function mutants of this enzyme.Our studies support a role for sphingosine phosphorylation in endocytic membrane trafficking beyond the established function of sphingosine-1-phosphate in intercellular signalling.

View Article: PubMed Central - PubMed

Affiliation: 1] Howard Hughes Medical Institute, Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, Connecticut 06510, USA [2] Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA [3].

ABSTRACT
Genetic studies have suggested a functional link between cholesterol/sphingolipid metabolism and endocytic membrane traffic. Here we show that perturbing the cholesterol/sphingomyelin balance in the plasma membrane results in the massive formation of clusters of narrow endocytic tubular invaginations positive for N-BAR proteins. These tubules are intensely positive for sphingosine kinase 1 (SPHK1). SPHK1 is also targeted to physiologically occurring early endocytic intermediates, and is highly enriched in nerve terminals, which are cellular compartments specialized for exo/endocytosis. Membrane recruitment of SPHK1 involves a direct, curvature-sensitive interaction with the lipid bilayer mediated by a hydrophobic patch on the enzyme's surface. The knockdown of SPHKs results in endocytic recycling defects, and a mutation that disrupts the hydrophobic patch of Caenorhabditis elegans SPHK fails to rescue the neurotransmission defects in loss-of-function mutants of this enzyme. Our studies support a role for sphingosine phosphorylation in endocytic membrane trafficking beyond the established function of sphingosine-1-phosphate in intercellular signalling.

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Related in: MedlinePlus

SPHK1 directly binds to negatively charged membranes with a preference for high positive curvaturea. Liposome flotation assay demonstrating that purified SPHK1 does not bind neutral liposomes (control, ctrl)[PE:PC:chol = 45:50:5], but does bind negatively charged liposomes that contain PS (PS) [PE:PC:PS:chol = 45:10:45:5] or a mixture of acidic phospholipids (mix)[PE:PC:PS:PA:PI(4)P:PI(4,5)P2:chol = 25:10:30:10:10:10:5]. Arrowhead indicates SPHK1-FLAG and arrow indicates fluorescent lipids. b. Curvature-dependent sorting of SPHK1-GFP on an artificial lipid bilayer. SPHK1-GFP is enriched relative to a membrane lipid (red, Texas red-DHPE) on a membrane tether pulled from a low curvature giant unilamellar vesicle (GUV). c. Mean ratio between green (SPHK1-GFP) and red (Texas red-DHPE) fluorescence intensities from the cross-sectional area of the tether, normalized to the two fluorescence intensities on the GUVs, for varying membrane tension (and thus membrane curvature). n = 6 pulled tubules. Each tubule represents one independent experiment, and data from 6 measurements were pooled. Error bars: standard error of the mean. d. Crystal structure of human SPHK1 (PDB ID 3VZB). Ribbon representation (left), surface hydrophobicity (middle) and electrostatic surface (right) are shown. In the ribbon representation, the N-terminal lobe is shown in blue and the C-terminal lobe in magenta. The bound substrate and ADP (superposed from PDB ID 3VZD) are also shown. e. Zoomed-in view of the framed region in d, highlighting the three mutated, surface-exposed and hydrophobic residues, L194, F197 and L198 (green), which are key components of the hydrophobic patch shown in the middle panel of d. f. Coomassie blue-stained SDS-PAGE showing purified SPHK1-FLAG (WT), SPHK1L194Q-FLAG (L194Q) and SPHK1F197A/L198Q-FLAG (F197A/L198Q). g. CD spectrum of purified SPHK1-FLAG (WT), SPHK1L194Q-FLAG (L194Q) and SPHK1F197A/L198Q-FLAG (F197A/L198Q) showing correct folding of the mutant fusion proteins. h. SPHK1L194Q-FLAG (L194Q) and SPHK1F197A/L198Q-FLAG (F197A/L198Q) do not bind either neutral liposomes (ctrl) or negatively charged liposomes (PS and mix). i. Confocal images of cells co-expressing endophilin 2-Ruby (bottom) and either SPHK1WT-GFP, SPHK1L194Q-GFP or SPHK1F197A/L198Q –GFP as indicated, after MβCD treatment. Scale bar: 1 µm in b and 10µm in h.
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Figure 5: SPHK1 directly binds to negatively charged membranes with a preference for high positive curvaturea. Liposome flotation assay demonstrating that purified SPHK1 does not bind neutral liposomes (control, ctrl)[PE:PC:chol = 45:50:5], but does bind negatively charged liposomes that contain PS (PS) [PE:PC:PS:chol = 45:10:45:5] or a mixture of acidic phospholipids (mix)[PE:PC:PS:PA:PI(4)P:PI(4,5)P2:chol = 25:10:30:10:10:10:5]. Arrowhead indicates SPHK1-FLAG and arrow indicates fluorescent lipids. b. Curvature-dependent sorting of SPHK1-GFP on an artificial lipid bilayer. SPHK1-GFP is enriched relative to a membrane lipid (red, Texas red-DHPE) on a membrane tether pulled from a low curvature giant unilamellar vesicle (GUV). c. Mean ratio between green (SPHK1-GFP) and red (Texas red-DHPE) fluorescence intensities from the cross-sectional area of the tether, normalized to the two fluorescence intensities on the GUVs, for varying membrane tension (and thus membrane curvature). n = 6 pulled tubules. Each tubule represents one independent experiment, and data from 6 measurements were pooled. Error bars: standard error of the mean. d. Crystal structure of human SPHK1 (PDB ID 3VZB). Ribbon representation (left), surface hydrophobicity (middle) and electrostatic surface (right) are shown. In the ribbon representation, the N-terminal lobe is shown in blue and the C-terminal lobe in magenta. The bound substrate and ADP (superposed from PDB ID 3VZD) are also shown. e. Zoomed-in view of the framed region in d, highlighting the three mutated, surface-exposed and hydrophobic residues, L194, F197 and L198 (green), which are key components of the hydrophobic patch shown in the middle panel of d. f. Coomassie blue-stained SDS-PAGE showing purified SPHK1-FLAG (WT), SPHK1L194Q-FLAG (L194Q) and SPHK1F197A/L198Q-FLAG (F197A/L198Q). g. CD spectrum of purified SPHK1-FLAG (WT), SPHK1L194Q-FLAG (L194Q) and SPHK1F197A/L198Q-FLAG (F197A/L198Q) showing correct folding of the mutant fusion proteins. h. SPHK1L194Q-FLAG (L194Q) and SPHK1F197A/L198Q-FLAG (F197A/L198Q) do not bind either neutral liposomes (ctrl) or negatively charged liposomes (PS and mix). i. Confocal images of cells co-expressing endophilin 2-Ruby (bottom) and either SPHK1WT-GFP, SPHK1L194Q-GFP or SPHK1F197A/L198Q –GFP as indicated, after MβCD treatment. Scale bar: 1 µm in b and 10µm in h.

Mentions: C-terminally tagged human SPHK1 (either SPHK1-FLAG or SPHK1-GFP-FLAG) was purified to near homogeneity from extracts of transfected Expi293T cells by anti-FLAG affinity chromatography followed by size-exclusion chromatography (Fig. 5f and Supplementary Fig. 5a). The purified protein was enzymatically active as shown by an in vitro kinase assay involving sphingosine and 32P-ATP as substrates, which revealed an activity of approximately 106 pmol/mg/min (Supplementary Fig. 5b), consistent with previous reports45. SPHK1-FLAG bound to liposomes containing negatively charged lipids (PS or an acidic lipids mixture), but not to liposomes containing neutral lipids only (Fig. 5a). Moreover, when SPHK1-GFP-FLAG was tested for binding to giant unilamellar vesicles (GUVs labeled by a lipid dye) from which narrow tubules had been pulled, it became selectively enriched relative to lipids on the narrow tubules (Fig. 5b), although it did not change the membrane curvature (Supplementary Fig. 5c), suggesting that SPHK1 senses, but does not generate high bilayer curvature (Fig. 5c) under the experimental conditions tested.


Coupling between endocytosis and sphingosine kinase 1 recruitment.

Shen H, Giordano F, Wu Y, Chan J, Zhu C, Milosevic I, Wu X, Yao K, Chen B, Baumgart T, Sieburth D, De Camilli P - Nat. Cell Biol. (2014)

SPHK1 directly binds to negatively charged membranes with a preference for high positive curvaturea. Liposome flotation assay demonstrating that purified SPHK1 does not bind neutral liposomes (control, ctrl)[PE:PC:chol = 45:50:5], but does bind negatively charged liposomes that contain PS (PS) [PE:PC:PS:chol = 45:10:45:5] or a mixture of acidic phospholipids (mix)[PE:PC:PS:PA:PI(4)P:PI(4,5)P2:chol = 25:10:30:10:10:10:5]. Arrowhead indicates SPHK1-FLAG and arrow indicates fluorescent lipids. b. Curvature-dependent sorting of SPHK1-GFP on an artificial lipid bilayer. SPHK1-GFP is enriched relative to a membrane lipid (red, Texas red-DHPE) on a membrane tether pulled from a low curvature giant unilamellar vesicle (GUV). c. Mean ratio between green (SPHK1-GFP) and red (Texas red-DHPE) fluorescence intensities from the cross-sectional area of the tether, normalized to the two fluorescence intensities on the GUVs, for varying membrane tension (and thus membrane curvature). n = 6 pulled tubules. Each tubule represents one independent experiment, and data from 6 measurements were pooled. Error bars: standard error of the mean. d. Crystal structure of human SPHK1 (PDB ID 3VZB). Ribbon representation (left), surface hydrophobicity (middle) and electrostatic surface (right) are shown. In the ribbon representation, the N-terminal lobe is shown in blue and the C-terminal lobe in magenta. The bound substrate and ADP (superposed from PDB ID 3VZD) are also shown. e. Zoomed-in view of the framed region in d, highlighting the three mutated, surface-exposed and hydrophobic residues, L194, F197 and L198 (green), which are key components of the hydrophobic patch shown in the middle panel of d. f. Coomassie blue-stained SDS-PAGE showing purified SPHK1-FLAG (WT), SPHK1L194Q-FLAG (L194Q) and SPHK1F197A/L198Q-FLAG (F197A/L198Q). g. CD spectrum of purified SPHK1-FLAG (WT), SPHK1L194Q-FLAG (L194Q) and SPHK1F197A/L198Q-FLAG (F197A/L198Q) showing correct folding of the mutant fusion proteins. h. SPHK1L194Q-FLAG (L194Q) and SPHK1F197A/L198Q-FLAG (F197A/L198Q) do not bind either neutral liposomes (ctrl) or negatively charged liposomes (PS and mix). i. Confocal images of cells co-expressing endophilin 2-Ruby (bottom) and either SPHK1WT-GFP, SPHK1L194Q-GFP or SPHK1F197A/L198Q –GFP as indicated, after MβCD treatment. Scale bar: 1 µm in b and 10µm in h.
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Related In: Results  -  Collection

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Show All Figures
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Figure 5: SPHK1 directly binds to negatively charged membranes with a preference for high positive curvaturea. Liposome flotation assay demonstrating that purified SPHK1 does not bind neutral liposomes (control, ctrl)[PE:PC:chol = 45:50:5], but does bind negatively charged liposomes that contain PS (PS) [PE:PC:PS:chol = 45:10:45:5] or a mixture of acidic phospholipids (mix)[PE:PC:PS:PA:PI(4)P:PI(4,5)P2:chol = 25:10:30:10:10:10:5]. Arrowhead indicates SPHK1-FLAG and arrow indicates fluorescent lipids. b. Curvature-dependent sorting of SPHK1-GFP on an artificial lipid bilayer. SPHK1-GFP is enriched relative to a membrane lipid (red, Texas red-DHPE) on a membrane tether pulled from a low curvature giant unilamellar vesicle (GUV). c. Mean ratio between green (SPHK1-GFP) and red (Texas red-DHPE) fluorescence intensities from the cross-sectional area of the tether, normalized to the two fluorescence intensities on the GUVs, for varying membrane tension (and thus membrane curvature). n = 6 pulled tubules. Each tubule represents one independent experiment, and data from 6 measurements were pooled. Error bars: standard error of the mean. d. Crystal structure of human SPHK1 (PDB ID 3VZB). Ribbon representation (left), surface hydrophobicity (middle) and electrostatic surface (right) are shown. In the ribbon representation, the N-terminal lobe is shown in blue and the C-terminal lobe in magenta. The bound substrate and ADP (superposed from PDB ID 3VZD) are also shown. e. Zoomed-in view of the framed region in d, highlighting the three mutated, surface-exposed and hydrophobic residues, L194, F197 and L198 (green), which are key components of the hydrophobic patch shown in the middle panel of d. f. Coomassie blue-stained SDS-PAGE showing purified SPHK1-FLAG (WT), SPHK1L194Q-FLAG (L194Q) and SPHK1F197A/L198Q-FLAG (F197A/L198Q). g. CD spectrum of purified SPHK1-FLAG (WT), SPHK1L194Q-FLAG (L194Q) and SPHK1F197A/L198Q-FLAG (F197A/L198Q) showing correct folding of the mutant fusion proteins. h. SPHK1L194Q-FLAG (L194Q) and SPHK1F197A/L198Q-FLAG (F197A/L198Q) do not bind either neutral liposomes (ctrl) or negatively charged liposomes (PS and mix). i. Confocal images of cells co-expressing endophilin 2-Ruby (bottom) and either SPHK1WT-GFP, SPHK1L194Q-GFP or SPHK1F197A/L198Q –GFP as indicated, after MβCD treatment. Scale bar: 1 µm in b and 10µm in h.
Mentions: C-terminally tagged human SPHK1 (either SPHK1-FLAG or SPHK1-GFP-FLAG) was purified to near homogeneity from extracts of transfected Expi293T cells by anti-FLAG affinity chromatography followed by size-exclusion chromatography (Fig. 5f and Supplementary Fig. 5a). The purified protein was enzymatically active as shown by an in vitro kinase assay involving sphingosine and 32P-ATP as substrates, which revealed an activity of approximately 106 pmol/mg/min (Supplementary Fig. 5b), consistent with previous reports45. SPHK1-FLAG bound to liposomes containing negatively charged lipids (PS or an acidic lipids mixture), but not to liposomes containing neutral lipids only (Fig. 5a). Moreover, when SPHK1-GFP-FLAG was tested for binding to giant unilamellar vesicles (GUVs labeled by a lipid dye) from which narrow tubules had been pulled, it became selectively enriched relative to lipids on the narrow tubules (Fig. 5b), although it did not change the membrane curvature (Supplementary Fig. 5c), suggesting that SPHK1 senses, but does not generate high bilayer curvature (Fig. 5c) under the experimental conditions tested.

Bottom Line: Membrane recruitment of SPHK1 involves a direct, curvature-sensitive interaction with the lipid bilayer mediated by a hydrophobic patch on the enzyme's surface.The knockdown of SPHKs results in endocytic recycling defects, and a mutation that disrupts the hydrophobic patch of Caenorhabditis elegans SPHK fails to rescue the neurotransmission defects in loss-of-function mutants of this enzyme.Our studies support a role for sphingosine phosphorylation in endocytic membrane trafficking beyond the established function of sphingosine-1-phosphate in intercellular signalling.

View Article: PubMed Central - PubMed

Affiliation: 1] Howard Hughes Medical Institute, Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, Connecticut 06510, USA [2] Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA [3].

ABSTRACT
Genetic studies have suggested a functional link between cholesterol/sphingolipid metabolism and endocytic membrane traffic. Here we show that perturbing the cholesterol/sphingomyelin balance in the plasma membrane results in the massive formation of clusters of narrow endocytic tubular invaginations positive for N-BAR proteins. These tubules are intensely positive for sphingosine kinase 1 (SPHK1). SPHK1 is also targeted to physiologically occurring early endocytic intermediates, and is highly enriched in nerve terminals, which are cellular compartments specialized for exo/endocytosis. Membrane recruitment of SPHK1 involves a direct, curvature-sensitive interaction with the lipid bilayer mediated by a hydrophobic patch on the enzyme's surface. The knockdown of SPHKs results in endocytic recycling defects, and a mutation that disrupts the hydrophobic patch of Caenorhabditis elegans SPHK fails to rescue the neurotransmission defects in loss-of-function mutants of this enzyme. Our studies support a role for sphingosine phosphorylation in endocytic membrane trafficking beyond the established function of sphingosine-1-phosphate in intercellular signalling.

Show MeSH
Related in: MedlinePlus