An amphipathic helix in the CRAL-TRIO domain of MOSPD2 mediates its localization at ER–LD contacts. (A) Schematic representation of MOSPD2 showing the position of the amphipathic helix (red) and its sequence. The arrowhead shows the position of residue W201. (B) Helical wheel representation of the WT (left) and W201E mutant (right) AH (aa 189-203) generated with HeliQuest (http://heliquest.ipmc.cnrs.fr/; left). The W201E mutation alters the amphipathic character of the helix by reducing its hydrophobic moment (µH) from 0.436 to 0.254. (C) WebLogo generated from an alignment of MOSPD2 AH sequence from 44 species. The AH is highlighted in light orange and 10 flanking residues from either side are shown. (D) Ribbon diagram of the structure model of the CRAL-TRIO domain of human MOSPD2 (Uniprot Q8NHP6; residues 1-241) obtained with AlphaFold (Jumper et al., 2021). The domain is in light grey except for the amphipathic helix depicted in stick model with residues colored as in B. (E) Far-UV circular dichroism spectrum of the MOSPD2 CRAL-TRIO domain and its W201E variant (6.7 μM) in 20 mM Tris, pH 7.4, 120 mM NaF buffer. MRE, mean residue ellipticity. The percentage of α-helix, β-sheet and turn, deriving from the analysis of the spectrum (WT) are given as well as the values deriving from the structure model (AlphaFold) using the Define Secondary Structure of Protein algorithm. (F) Left: Schematic representation of GFP-MOSPD2 constructs either WT (GFP-MOSPD2), bearing a mutation in the AH (GFP-MOSPD2 W201E), or containing a deletion of the CRAL-TRIO domain together with an insertion of the AH (AH-MOSPD2-ΔCRAL-TRIO). Right: Localization of these constructs in HeLa cells treated with OA; LDs were stained with Nile Red (magenta). Images were acquired on a confocal microscope (Leica SP5, 63× NA 1.4). Scale bars: 10 µm (insets, 2 µm). (G) Quantification of cells showing ring- or comma-shaped staining for these constructs. Mean ± SD; n = 3 independent experiments (MOSPD2 WT:117 cells; MOSPD2 W201E: 156 cells; AH-MOSPD2-ΔCRAL-TRIO: 113 cells). (H) Left: Schematic representation of WT GFP-VAP-A and GFP-AH-VAP-A chimera in which the AH of MOSPD2 was fused at the N-terminus of VAP-A. Right: localization of the different constructs. LDs were stained with Nile Red in HeLa cells treated with OA. Confocal microscope (Leica SP5, 63× NA 1.4) images. Scale bars: 10 µm (insets, 2 µm). (I) Quantification of cells showing ring- or comma-shaped staining for GFP-VAP-A and GFP-AH-VAP-A chimera. Mean ± SD; n = 3 independent experiments (VAP-A WT: 109 cells; AH-VAP-A: 102 cells). In F and H, composite subpanels on the bottom are higher magnification images of the area outlined. The overlay panel shows merged channels.
Figure 7.

An amphipathic helix in the CRAL-TRIO domain of MOSPD2 mediates its localization at ER–LD contacts. (A) Schematic representation of MOSPD2 showing the position of the amphipathic helix (red) and its sequence. The arrowhead shows the position of residue W201. (B) Helical wheel representation of the WT (left) and W201E mutant (right) AH (aa 189-203) generated with HeliQuest (http://heliquest.ipmc.cnrs.fr/; left). The W201E mutation alters the amphipathic character of the helix by reducing its hydrophobic moment (µH) from 0.436 to 0.254. (C) WebLogo generated from an alignment of MOSPD2 AH sequence from 44 species. The AH is highlighted in light orange and 10 flanking residues from either side are shown. (D) Ribbon diagram of the structure model of the CRAL-TRIO domain of human MOSPD2 (Uniprot Q8NHP6; residues 1-241) obtained with AlphaFold (Jumper et al., 2021). The domain is in light grey except for the amphipathic helix depicted in stick model with residues colored as in B. (E) Far-UV circular dichroism spectrum of the MOSPD2 CRAL-TRIO domain and its W201E variant (6.7 μM) in 20 mM Tris, pH 7.4, 120 mM NaF buffer. MRE, mean residue ellipticity. The percentage of α-helix, β-sheet and turn, deriving from the analysis of the spectrum (WT) are given as well as the values deriving from the structure model (AlphaFold) using the Define Secondary Structure of Protein algorithm. (F) Left: Schematic representation of GFP-MOSPD2 constructs either WT (GFP-MOSPD2), bearing a mutation in the AH (GFP-MOSPD2 W201E), or containing a deletion of the CRAL-TRIO domain together with an insertion of the AH (AH-MOSPD2-ΔCRAL-TRIO). Right: Localization of these constructs in HeLa cells treated with OA; LDs were stained with Nile Red (magenta). Images were acquired on a confocal microscope (Leica SP5, 63× NA 1.4). Scale bars: 10 µm (insets, 2 µm). (G) Quantification of cells showing ring- or comma-shaped staining for these constructs. Mean ± SD; n = 3 independent experiments (MOSPD2 WT:117 cells; MOSPD2 W201E: 156 cells; AH-MOSPD2-ΔCRAL-TRIO: 113 cells). (H) Left: Schematic representation of WT GFP-VAP-A and GFP-AH-VAP-A chimera in which the AH of MOSPD2 was fused at the N-terminus of VAP-A. Right: localization of the different constructs. LDs were stained with Nile Red in HeLa cells treated with OA. Confocal microscope (Leica SP5, 63× NA 1.4) images. Scale bars: 10 µm (insets, 2 µm). (I) Quantification of cells showing ring- or comma-shaped staining for GFP-VAP-A and GFP-AH-VAP-A chimera. Mean ± SD; n = 3 independent experiments (VAP-A WT: 109 cells; AH-VAP-A: 102 cells). In F and H, composite subpanels on the bottom are higher magnification images of the area outlined. The overlay panel shows merged channels.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal