FFAT motif phosphorylation controls formation and lipid transfer function of inter-organelle contacts

Abstract

Organelles are physically connected in membrane contact sites. The endoplasmic reticulum possesses three major receptors, VAP-A, VAP-B, and MOSPD2, which interact with proteins at the surface of other organelles to build contacts. VAP-A, VAP-B, and MOSPD2 contain an MSP domain, which binds a motif named FFAT (two phenylalanines in an acidic tract). In this study, we identified a non-conventional FFAT motif where a conserved acidic residue is replaced by a serine/threonine. We show that phosphorylation of this serine/threonine is critical for non-conventional FFAT motifs (named Phospho-FFAT) to be recognized by the MSP domain. Moreover, structural analyses of the MSP domain alone or in complex with conventional and Phospho-FFAT peptides revealed new mechanisms of interaction. Based on these new insights, we produced a novel prediction algorithm, which expands the repertoire of candidate proteins with a Phospho-FFAT that are able to create membrane contact sites. Using a prototypical tethering complex made by STARD3 and VAP, we showed that phosphorylation is instrumental for the formation of ER-endosome contacts, and their sterol transfer function. This study reveals that phosphorylation acts as a general switch for inter-organelle contacts.

Keywords: cholesterol; inter-organelle contact; lipid transfer protein; regulation; small linear motif.

Figure 1. Identification of novel category of FFAT motifs regulated by phosphorylation

1. Schematic representation of STARD3, FIP200, MIGA2, PTPIP51, KCNB1 (Kv2.1), and KCNB2 (Kv2.2) proteins and sequence of their Phospho‐FFAT motif (7 core residues and 4 upstream and downstream residues). Note that the 4^th position of the FFAT (boxed) of these proteins is occupied by a serine or a threonine residue while conventional FFATs have an aspartic acid. Blue rectangles represent transmembrane helices. TPR: tetratrico peptide repeat; LDT: lipid droplet targeting domain; BTB: potassium channel tetramerization‐type BTB domain.

2. Sequence of the peptides used for the pull‐down assays. The peptides are composed of an amino‐terminal biotin, a linker sequence and the FFAT sequence of STARD3 (residues 200–216), FIP200 (residues 725–741), MIGA2 (residues 286–302), PTPIP51 (residues 151–167), Kv2.1 (residues 584–600), Kv2.2 (residues 599–615), and ORP1 (residues 469–485) either without or with a phosphorylated residue (serine or threonine) at position 4 of the core FFAT motif. The negative control peptide is composed of a random sequence.

3. Western blot analysis of proteins pulled down using the peptides described in (B). The input fraction corresponds to HeLa cell total protein extract. Bound proteins were analyzed using anti‐VAP‐A, anti‐VAP‐B and anti‐MOSPD2 antibodies. Actin was used as a loading control.

4. Western blot analysis of proteins pulled down using peptides comprising WT and mutant (D₄₇₈S and D₄₇₈pS) ORP1 FFAT sequence peptides described in (B). Bound proteins were analyzed using anti‐VAP‐A, anti‐VAP‐B, and anti‐MOSPD2 antibodies. Actin was used as a loading control.

5. Venn diagram of VAPs and/or MOSPD2 partners with FFAT scores between 0 and 3. Proteins with a Phospho‐FFAT and a conventional FFAT are shown in green and red, respectively. A total of 42 proteins possesses both a Phospho and a conventional FFAT. Consensus sequences of the 110 Phospho‐FFAT (left) and 94 conventional FFAT motifs (right) identified are shown as sequence logos.

6. List of selected VAP‐A, VAP‐B, or MOSPD2 partners having a potential Phospho‐FFAT motif. For a full list, see Table EV2.

Data information: Acidic (D and E) or phosphorylated residues (pS and pT), alcoholic (S and T), and aromatic (F and Y) residues are in red, green, and blue, respectively; the other residues are in black. Source data are available online for this figure.

Figure 2. STARD3 has a non‐conventional FFAT motif which needs to be phosphorylated to form a complex with VAP proteins

1. A

   Schematic representation of STARD3. Transmembrane helices in the MENTAL domain are in dark blue. The Phospho‐FFAT motif of STARD3 and the conventional FFAT motif sequences are aligned and highlighted in pink. Upper numbers correspond to the position of residues in STARD3. Lower numbers correspond to the position of residues in the FFAT sequence as described in (Loewen et al, 2003). Acidic (D and E), alcoholic (S and T), and aromatic (F and Y) residues are in red, green, and blue, respectively; the other residues are in black. Phosphorylated serines identified by LC/MS/MS are boxed, and the phosphosite probability determined with PhosphoRS is indicated.

2. B

   Coomassie Blue staining of the recombinant wild‐type MSP domains of VAP‐A and VAP‐B, and of the KD/MD mutant of VAP‐A, after SDS–PAGE.

3. C

   Recombinant MSP domains pulled down with peptides corresponding to the Phospho‐FFAT motif of STARD3 (unphosphorylated; monophosphorylated on S₂₀₉ or S₂₁₃; bi‐phosphorylated on S₂₀₉ and S₂₁₃), and with the control peptides were revealed by Coomassie Blue staining. The recombinant proteins subjected to the assay are showed in the input fraction.

4. D, E

   Structure of the MSP domain of VAP‐A in complex with a Phospho‐FFAT motif. Ribbon diagram (D) and surface representation (E) of the MSP domain of VAP‐A in complex with the Phospho‐FFAT of STARD3 depicted in stick model. The protein surface is colored according to the YRB scheme, showing hydrophobic, negatively and positively charged atoms in yellow, red, and blue, respectively; the other atoms are in white (Hagemans et al, 2015).

5. F, G

   Close‐up view of the hydrophobic pocket region in the unbound (F) and the Phospho‐FFAT‐bound (G) VAP‐A; residues of VAP‐A constituting the pocket are indicated.

6. H

   Close‐up view of the structure near the Phospho‐FFAT motif highlighting critical residues (in stick model) of human VAP‐A present in the binding interface.

7. I

   Close‐up view of the structure near the conventional FFAT of ORP1 as described in (PDBID: 1Z9O; Kaiser et al, 2005), highlighting critical residues of rat VAP‐A involved in the interaction. The nomenclature for rat VAP‐A residues is based on the UniProt sequence (Q9Z270) and not on the PDB file 1Z9O.

8. J

   Superposition of the two structures shown in (H) and (I) showing the distinct conformations of the C‐terminal parts of each peptide.

Data information: Phosphorous, nitrogen, oxygen, and sulfur atoms are colored in orange, blue, red, and yellow, respectively. Carbon atoms are shown in green and gray/pink in the MSP domain and the FFAT motif, respectively. The numbers of the peptide residues are noted in subscript. Source data are available online for this figure.

Figure EV1. MS/MS spectrum of peptides phosphorylated on S₂₀₉, S₂₁₃, S₂₁₇, and S₂₂₁

1. A–D

   MS/MS spectrum of peptides phosphorylated on S₂₀₉ (A), S₂₁₃ (B), S₂₁₇ (C), and S₂₂₁ (D). The molecular weight of peptides is indicated on the right. Spectra are an assembly of ions produced by collision‐induced dissociation of the precursor peptides. Fragmentation occurs preferentially at peptide bonds to generate b and y ions, which extend from the amino and carboxy terminus, respectively. The precursor peptide sequence and the different b and y ions identified in the spectrum are shown above. b and y ion peaks are shown in red and blue, respectively. b and y ions with the highest intensity are labeled on the spectrum. Neutral mass losses of H₂O, NH₃, and H₃PO₄ (P) are indicated. Analysis of y and b ion fragmentation patterns showed that S₂₀₉ (A), S₂₁₃ (B), S₂₁₇ (C), and S₂₂₁ (D) were phosphorylated in vivo.

Figure EV2. Interaction of VAP‐A, VAP‐B, and MOSPD2 with the Phospho‐FFAT of STARD3

1. Interaction of VAP‐A K50L, VAP‐B K43L, and MOSPD2 K363L mutants with conventional and Phospho‐FFAT motif. Wild‐type and mutant MSP domains were pulled down with phosphorylated STARD3 (pS₂₀₉ + pS₂₁₃), ORP1 FFAT peptides, and with the negative control peptide. The input fraction corresponds to the recombinant proteins used in the assay. Input and bound proteins were revealed by Coomassie Blue staining. Note that lysine K50 in VAP‐A and K43 in VAP‐B are required for the interaction with a Phospho‐FFAT, and not with a conventional FFAT.

2. Interaction of VAP‐A, VAP‐B, MOSPD2 with phosphomimetic STARD3 Phospho‐FFAT motif. Wild‐type and mutant MSP domains were pulled down with unphosphorylated, phosphorylated (pS₂₀₉), phosphomimetic (S₂₀₉D and S₂₀₉D/P₂₁₀A), and with the negative control peptide. The input fraction corresponds to the recombinant proteins used in the assay. Input and bound proteins were revealed by Coomassie Blue staining. Acidic (D) or phosphorylated residues (pS), and alcoholic (S) residues are in red and green, respectively; the other residues are in black.

3. STARD3 and VAP‐A/VAP‐B or MOSPD2 complex formation requires a unique phosphorylation of the Phospho‐FFAT motif. Western blot analysis of proteins pulled down using the peptides described in Fig EV3A. The input fraction corresponds to the HeLa cell total protein extract. The streptavidin beads were first coupled to the indicated biotinylated peptides, or left without peptide (ø). The soluble fraction after the incubation of the protein extract with the beads (Unbound; left), and proteins attached to the beads (Bound; right) were analyzed by Western blot using anti‐VAP‐A, anti‐VAP‐B, and anti‐MOSPD2 antibodies. Actin was used as a loading control.

4. Immunoprecipitation (GFP‐Trap) experiments between GFP‐tagged VAP‐A and Flag‐tagged STARD3 (WT and P₂₁₀A mutant). Approximatively 15 µg of total protein extracts was analyzed by Western blot using anti‐STARD3, anti‐GFP, and anti‐Actin antibodies. Immunoprecipitated material was analyzed using anti‐STARD3 and anti‐GFP antibodies.

Source data are available online for this figure.

Figure 3. The MSP domain binds the phosphorylated Phospho‐FFAT with an affinity in the micromolar range

1. A–D

   Surface Plasmon resonance analysis of the MSP domain of VAP‐A binding onto immobilized unphosphorylated (A), monophosphorylated pS₂₀₉ (B) and pS₂₁₃ (C), and bi‐phosphorylated pS₂₀₉ + pS₂₁₃ (D) STARD3 FFAT peptides. Representative sensorgrams resulting from the interaction between the MSP domain of VAP‐A injected at different concentrations and the different FFAT peptides are shown in (A), (B left), (C), and (D left). Binding curves display the SPR signal (RU) as a function of time. Some samples were measured twice (concentrations printed in bold). Signal obtained for the negative control peptide immobilized on another flow cell is systematically subtracted, as well as the bulk effect recorded with buffer only. Steady‐state analysis of the interaction between pS₂₀₉ (B right) and pS₂₀₉ + pS₂₁₃ (D right) STARD3 FFAT peptide, and the MSP domain of VAP‐A. Equilibrium responses (Req) extracted from the left panel in association phase were plotted as a function of the dimeric MSP domain of VAP‐A concentration, and fitted with a Langmuir binding model.

2. E

   Dissociation constants between peptides corresponding to different kind of FFAT motifs and the MSP domains of VAP‐A and VAP‐B. The kinetic experiments were performed at 25°C in Tris–HCl 50 mM, pH 7.5, NaCl 75 mM buffer supplemented with 0.005% (v/v) surfactant polysorbate 20 (P20, GE Healthcare). Mean of n independent experiments: n = 2 for pS₂₀₉ and pS₂₀₉ + pS₂₁₃; 3 for pS₂₁₃; and 4 for the non‐phosphorylated FFAT. Uncertainties are obtained from the standard deviation considering a t‐distribution coefficient for a risk factor of 32%.

Figure EV3. The MSP domain of VAP‐A/VAP‐B/MOSPD2 binds the phosphorylated Phospho‐FFAT with an affinity in the micromolar range

1. A

   Sequence of the peptides used for SPR and the pull‐down assays. The peptides are composed of an amino‐terminal biotin, a linker sequence (GAMR) and the FFAT sequence of STARD3 (residues 200–216) either without phosphorylated serine or with combinations of phosphorylated serines (positions 203, 209, and 213 in STARD3 protein). The core FFAT sequence (residues 1–7) is highlighted in red. ORP1 FFAT peptide corresponding to residues 469–485 (Accession Number Q9BXW6‐1), and a random sequence (control peptide) are used as positive and negative control, respectively. Acidic (D and E) or phosphorylated serine (pS), alcoholic (S and T), and aromatic (F, W, and Y) residues are in red, green and blue, respectively; the other residues are in black.

2. B–D

   SPR analysis of the MSP domain of VAP‐A binding onto immobilized ORP1 FFAT peptide (B), pS₂₀₃ (C) or pS₂₀₃ + pS₂₀₉ + pS₂₁₃ (D) STARD3 FFAT peptides. Representative sensorgrams resulting from the interaction between the MSP domain of VAP‐A injected at different concentrations and the different STARD3 FFAT peptides are shown in (B left), (C), and (D left). Binding curves display the SPR signal (RU) as a function of time. Concentrations printed in bold indicate samples measured twice. Steady‐state analysis of the interaction between ORP1 FFAT (B right) and pS₂₀₃ + pS₂₀₉ + pS₂₁₃ (D right) STARD3 FFAT peptide and the MSP domain of VAP‐A. Equilibrium responses (R _eq) extracted from the left panel were plotted as a function of the dimeric MSP domain of VAP‐A concentration, and fitted with a Langmuir binding model.

3. E

   Interaction dissociation constants between the different FFAT peptides and the MSP domains of VAP‐A, VAP‐B, and MOSPD2. MSP concentrations are expressed as dimer for VAP‐A and VAP‐B, and monomer for MOSPD2. The different ionic strength correspond to the following buffers (pH 7.5): low: Tris–HCl 20 mM, NaCl 75 mM; medium: Tris–HCl 50 mM, NaCl 75 mM; high: Tris–HCl 50 mM, NaCl 300 mM. Buffers were supplemented with P20 (0.005% v/v). ND: not determined. Mean values of n independent experiments: n = 2 for pS₂₀₉, pS₂₀₉ + pS₂₁₃, and S₂₀₃ + pS₂₀₉ + pS₂₁₃ (high); 4 for pS₂₀₃ + pS₂₀₉ + pS₂₁₃ (low), ORP1 (low and high), and pS₂₀₃ + pS₂₀₉ + pS₂₁₃ (medium) with MOSPD2; 6 for pS₂₀₃ + pS₂₀₉ + pS₂₁₃ (medium) with VAP‐A and VAP‐B; 12, 11, and 7 for ORP1 (medium) with VAP‐A, VAP‐B, and MOSPD2, respectively. Uncertainties are obtained from the standard deviation considering a t‐distribution coefficient for a risk factor of 32%.

Figure 4. In vivo, a unique phosphorylation of the Phospho‐FFAT motif allows STARD3 and VAP‐A/VAP‐B complex formation and the establishment of ER‐endosome contacts

1. A, B

   Binding assays. Immunoprecipitation (GFP‐Trap) experiments between GFP‐tagged VAP‐A (WT and KD/MD mutant; A), VAP‐B (B) and Flag‐tagged STARD3 (WT and S₂₀₉A, S₂₀₉D, and S₂₀₉D/P₂₁₀A mutants). Please note that the mutations modify the FFAT and Phospho‐FFAT scores of this region of STARD3: compared to WT STARD3 (Phospho‐FFAT: 2.5; conventional FFAT: 6.5), the STARD3 S₂₀₉A, mutant has both low Phospho‐FFAT and FFAT scores (6.5), the S₂₀₉D mutant has a high conventional FFAT score (2.5) but a low Phospho‐FFAT score (6.5), and the double mutant has a conventional FFAT score of 1.5 and a Phospho‐FFAT score of 5.5. Approximatively 5 µg of total protein extracts was analyzed by Western blot using anti‐Flag, anti‐GFP, and anti‐Actin antibodies. Immunoprecipitated material was analyzed using anti‐Flag and anti‐GFP antibodies.

2. C–G

   Recruitment assays. GFP‐VAP‐A (C, D, E, G; green) and GFP‐VAP‐A KD/MD‐expressing cells (F; green) were left untransfected (C) or transfected with Flag‐STARD3 (D, F), Flag‐STARD3 S₂₀₉A (E), and Flag‐STARD3 S₂₀₉D/P₂₁₀A (G), and labeled using anti‐Flag (magenta) antibodies. The subpanels on the right are higher magnification (3.5×) images of the area outlined in white. The Overlay panel shows merged green and magenta images. The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co‐localize are shown in white. Scale bars: 10 µm. Inset scale bars: 2 µm.

3. H

   Pearson’s correlation coefficients between VAP‐A (WT or KD/MD mutant) and STARD3 (WT or S₂₀₉A, S₂₀₉D, S₂₀₉D/P₂₁₀A, FA/YA mutants) staining are shown. Each dot represents a single cell (number of cells: VAP‐A–STARD3: 22; VAP‐A–STARD3 S₂₀₉A: 25; VAP‐A–STARD3 S₂₀₉D: 27; VAP‐A–STARD3 S₂₀₉D/P₂₁₀A: 26; VAP‐A–STARD3 FA/YA: 20; VAP‐A KD/MD–STARD3: 20, from at least three independent experiments). Means and error bars (SD) are shown. Kruskal–Wallis with Dunn’s multiple comparison test (**P < 0.01; ***P < 0.001).

Source data are available online for this figure.

Figure EV4. Effect of non‐phosphorylatable and phosphomimetic mutations of S₂₀₉ on the formation of ER‐endosome contacts involving VAP‐B in vivo

1. A–D

   GFP‐VAP‐B (A–D; green)‐expressing cells were left untransfected (A) or transfected with Flag‐STARD3 (B), Flag‐STARD3 S₂₀₉A (C), and Flag‐STARD3 S₂₀₉D/P₂₁₀A (D), and labeled using anti‐Flag (magenta) antibodies. The subpanels on the right are higher magnification (3.5×) images of the area outlined in white. The Overlay panel shows merged green and magenta images. The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co‐localize are shown in white. Scale bars: 10 µm. Inset scale bars: 2 µm.

2. E

   Pearson’s correlation coefficients between VAP‐B (WT and KD/MD) and STARD3 (WT, S₂₀₉A, S₂₀₉D, S₂₀₉D/P₂₁₀A or FA/YA mutants) staining are shown. Each dot represents a single cell (number of cells: VAP‐B–STARD3: 20; VAP‐B–STARD3 S₂₀₉A: 18; VAP‐B–STARD3 S₂₀₉D: 27; VAP‐B–STARD3 S₂₀₉D/P₂₁₀A: 26; VAP‐B–STARD3 FA/YA: 21; VAP‐B KD/MD–STARD3: 21, from at least three independent experiments). Means and error bars (SD) are shown. Kruskal–Wallis with Dunn’s multiple comparison test (**P < 0.01; ***P < 0.001).

3. F

   Top: Western blot analysis of protein extracts from HeLa cells transfected with control siRNAs (siCtrl) and siRNAs targeting STARD3 (siSTARD3) using anti‐STARD3 and anti‐Actin antibodies. Bottom: After siRNA transfection, the cells were transfected with GFP‐VAP‐A and STARD3 (WT, STARD3 S₂₀₉A, and STARD3 S₂₀₉D/P₂₁₀A). STARD3 expression vectors contained a cDNA with silent mutations rendering it insensitive to siRNAs. The cells were labeled using anti‐STARD3 antibodies. Pearson’s correlation coefficients between STARD3 (WT, STARD3 S₂₀₉A, and STARD3 S₂₀₉D/P₂₁₀A) and VAP‐A staining are shown. Note that the colocalization between VAP‐A and STARD3 is similar in the presence and absence of endogenous STARD3. Means and error bars (SD) are shown. Kruskal–Wallis with Dunn’s multiple comparison test (ns: P ≥ 0.05).

Source data are available online for this figure.

Figure 5. Phosphorylation of STARD3 in its Phospho‐FFAT motif governs membrane attachment and cholesterol transfer in in vitro reconstituted assays

1. Description of the experimental strategy. L_A liposomes (endosome‐like) are decorated with cSTD3 or pS₂₀₉ cSTD3 owing to covalent links with MPB‐PE (1.2‐dioleoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐[4‐(p‐maleimidophenyl) butyramide]), and mixed with L_B liposomes (ER‐like) covered by VAP_HIS6 attached to DOGS‐NTA‐Ni²⁺ (1.2‐dioleoyl‐sn‐glycero‐3‐[(N‐(5‐amino‐1‐carboxypentyl) iminodiacetic acid) succinyl], nickel salt). For dehydroergosterol (DHE) transport experiment, L_B liposomes also contain DHE and a dansyl‐phosphatidylethanolamine (DNS‐PE). The transport of DHE from L_B to L_A liposomes is followed by measuring the change in FRET from DHE to DNS‐PE.

2. SDS–PAGE gel and Western blot analysis of purified cSTD3 and pS₂₀₉ cSTD3 proteins. Top: The gel was stained with SYPRO Orange to visualize proteins and molecular weight markers. Bottom: Two similar gels were blotted onto nitrocellulose and analyzed for the presence of total and STARD3‐pS₂₀₉ using specific antibodies.

3. Aggregation assays. L_A liposomes (50 µM total lipids) were incubated for 5 min with cSTD3 or pS₂₀₉ cSTD3 (380 nM). Then, L_B liposomes (50 µM total lipids) and VAP‐A_HIS6 or VAP‐A KD/MD_HIS6 (700 nM) were successively added. Left panels: mean radius (black dots) and polydispersity (shaded area) over time. Right panels: size distribution before (gray bars) and after the reaction (black bars).

4. DHE transport assay. DOPC (1.2‐dioleoyl‐sn‐glycero‐3‐phosphocholine) liposomes (62.5 µM total lipids, L_A) containing 3 mol% MPB‐PE were mixed with cSTD3 or pS₂₀₉ cSTD3 (475 nM). After 5 min, liposomes (DOPC/DOGS‐NTA‐Ni²⁺/DNS‐PE/DHE liposomes 77.5/10/2.5/10 mol/mol, 62.5 µM total lipids, L_B), covered or not with 1 µM of VAP_HIS6 or VAP‐A KD/MD_HIS6, were added. FRET between DHE and DNS‐PE in the L_B liposomes diminishes as DHE is transported toward L_A liposomes. The signal was converted into amount of DHE present in L_B liposomes (in mol%).

5. Initial DHE transport rate measured with cSTD3 or pS₂₀₉ cSTD3 in the presence or absence of VAP‐A _HIS6 or VAP‐A KD/MD_HIS6. Data are represented as mean ± SEM (n = 3 for cSTD3‐VAP and n = 4 for all other data). Mann–Whitney test (*P < 0.05).

Source data are available online for this figure.

Figure EV5. The attachment of liposomes by pS₂₀₉ cSTD3 does not induce membrane fusion

1. Description of the experimental strategy. L_A liposomes (endosome‐like) are decorated with pS₂₀₉ cSTD3 owing to covalent links with MPB‐PE (1.2‐dioleoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐[4‐(p‐maleimidophenyl) butyramide]), and mixed with L_B liposomes (ER‐like) covered by VAP_HIS6 attached to DOGS‐NTA‐Ni²⁺ (1.2‐dioleoyl‐sn‐glycero‐3‐[(N‐(5‐amino‐1‐carboxypentyl) iminodiacetic acid) succinyl]; nickel salt). L_A liposomes contain the FRET pair NBD‐PE (1,2‐dioleoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐(7‐nitro‐2‐1,3‐benzoxadiazol‐4‐yl)) and Rhod‐PE (1,2‐dioleoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐(lissamine rhodamine B sulfonyl)). The fusion of L_A and L_B liposomes is followed by measuring an increase in NBD‐PE fluorescence, which is initially quenched due to FRET with Rhod‐PE. The percentage of fusion is equal to 100 × ((F − F ₀)/(F _max  − F ₀)) where F ₀ is the signal measured before the addition of L_B liposomes decorated with VAP‐A_His6, and F _max is the signal measured after adding Triton X‐100 (1% v/v final concentration).

2. Aggregation assays. L_A liposomes (50 µM total lipids) were incubated for 5 min with pS₂₀₉ cSTD3 (380 nM). Then, L_B liposomes (50 µM total lipids) and VAP‐A_HIS6 or VAP‐A (KD/MD)_HIS6 (700 nM) were successively added. Left panels: mean radius (black dots) and polydispersity (shaded area) over time. Right panels: size distribution before (gray bars) and after the reaction (black bars).

3. Fusion assay. DOPC (1.2‐dioleoyl‐sn‐glycero‐3‐phosphocholine) liposomes (62.5 µM total lipids, L_A) containing 3 mol% MPB‐PE, 1% NBD‐PE, and 1% Rhod‐PE were mixed with pS₂₀₉ cSTD3 (475 nM). After 5 min, liposomes (DOPC/DOGS‐NTA‐Ni²⁺ liposomes 90/10 mol/mol, 62.5 µM total lipids, L_B), covered or not with 1 µM of VAP_HIS6, were added. At the end of the experiment, Triton X‐100 was added (1% v/v final concentration) to disrupt liposomes and eliminate energy transfer, allowing the determination of the maximal fluorescence of NBD‐PE that would be measured for a complete membrane fusion.

Figure 6. STARD3‐mediated cholesterol transport in endosomes depends on S₂₀₉

1. A–E

   HeLa/Ctrl (A), HeLa/STARD3 (B), HeLa/STARD3 S₂₀₉A (C), HeLa/STARD3 S₂₀₉D/P₂₁₀A (D), and HeLa/STARD3 Conv‐FFAT (E) cells were labeled with anti‐STARD3 antibodies (magenta), anti‐Lamp1 antibodies (green) and with the fluorescent cholesterol probe filipin (Cyan Hot). Nuclei are shown in gray (TO‐PRO‐3). Higher magnifications images (2×) of the area outlined in white are shown on the right. The filipin, STARD3, and Lamp1 merged image is labeled Overlay. Scale bars: 10 µm. Inset scale bars: 5 µm.

2. F

   Pearson’s correlation coefficients between STARD3 and Lamp1 staining in HeLa/STARD3 cells. Each dot represents a single cell (34 cells from 4 independent experiments). Mean and error bars (SD) are shown. Note that STARD3 and Lamp1 signals are highly correlated.

3. G

   Relative fluorescence intensity of intracellular filipin signals in endosomes of HeLa/Ctrl, HeLa/STARD3, HeLa/STARD3 S₂₀₉A, HeLa/STARD3 S₂₀₉D/P₂₁₀A, and HeLa/STARD3 Conv‐FFAT cells. Data are displayed as a Superplot (Lord et al, 2020) showing the relative filipin fluorescence intensity in endosomes of individual cells (small circles) from 6 independent experiments (mean of each experiment shown as a large circle). Independent experiments are color‐coded. Means and error bars (SD) are shown as black bars. Kruskal–Wallis with Dunn’s multiple comparison test (*P < 0.05; **P < 0.01; n = 6 independent experiments).

Figure 7. MOSPD2 interacts with the Phospho‐FFAT motif in a phosphorylation‐dependent manner

1. A

   Coomassie Blue staining of the recombinant MSP domain of MOSPD2 after SDS–PAGE.

2. B

   Recombinant MSP domains pulled down with STARD3 FFAT peptides (unphosphorylated, monophosphorylated on S₂₀₉ or S₂₁₃, bi‐phosphorylated on S₂₀₉ and S₂₁₃, or tri‐phosphorylated on S₂₀₃, S₂₀₉ and S₂₁₃ and control peptides were revealed by Coomassie Blue staining. The recombinant proteins subjected to the assay are shown in the input fraction.

3. C, D

   SPR analysis of the MSP domain of MOSPD2 binding onto immobilized unphosphorylated (C) and pS₂₀₉ (D) STARD3 FFAT. Representative sensorgrams resulting from the interaction between the MSP domain of MOSPD2 injected at different concentrations and the different FFAT peptides are shown in (C) and (D left). Binding curves display the SPR signal (RU) as a function of time. Concentrations printed in bold indicate samples measured twice. Signal obtained for the negative control peptide immobilized on another flow cell is systematically subtracted, as well as the bulk effect recorded with buffer only. (D right) Steady‐state analysis of the interaction between pS₂₀₉ STARD3 FFAT peptide and the MSP domain of MOSPD2. Equilibrium responses (Req) extracted from the left panel were plotted as a function of the monomeric concentration of the MSP domain of MOSPD2, and fitted with a 1:1 binding model. The experiments were performed at 25°C in Tris–HCl 50 mM, pH 7.5, NaCl 75 mM buffer supplemented with 0.005% (v/v) surfactant polysorbate 20 (P20, GE Healthcare). Mean of 2 independent experiments. Uncertainties are obtained from the standard deviation considering a t‐distribution coefficient for a risk factor of 32%.

4. E

   Interaction dissociation constants between the different STARD3 FFAT peptides and the MSP domains of MOSPD2.

5. F

   Immunoprecipitation (GFP‐Trap) experiments between GFP‐tagged MOSPD2 and Flag‐tagged STARD3 (WT and S₂₀₉A, S₂₀₉D, and S₂₀₉D/P₂₁₀A mutants). Approximatively 5 µg of total protein extracts was analyzed by Western blot using anti‐Flag, anti‐GFP and anti‐Actin antibodies. Immunoprecipitated material was analyzed using anti‐Flag and anti‐GFP antibodies.

6. G–I

   GFP‐MOSPD2‐expressing cells (green) were left untransfected (G) or transfected with Flag‐STARD3 (H) and Flag‐STARD3 S₂₀₉A (I), and labeled using anti‐Flag (magenta) antibodies. The subpanels on the right are higher magnification (3.5×) images of the area outlined in white. The Overlay panel shows merged green and magenta images. The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co‐localize are shown in white. Scale bars: 10 µm.

7. J

   Pearson’s correlation coefficients between MOSPD2 (WT or RD/LD) and STARD3 (WT, S₂₀₉A, or FA/YA) staining are shown. Each dot represents a single cell (number of cells: MOSPD2–STARD3: 22; MOSPD2– STARD3 S₂₀₉A: 21; MOSPD2–STARD3 FA/YA: 20; MOSPD2 RD/LD–STARD3: 17, from at least three independent experiments). Means and error bars (SD) are shown. Kruskal–Wallis with Dunn’s multiple comparison test (***P < 0.001).

Source data are available online for this figure.

Figure 8. Structure of the MSP domain of MOSPD2 in its unbound form, and in complex with a conventional FFAT and a Phospho‐FFAT

1. A–C

   Ribbon diagram of the MSP domain of MOSPD2 in its unbound form (A), in complex with the conventional FFAT of ORP1 (B), and in complex with the Phospho‐FFAT of STARD3 (C).

2. D

   Surface representation of the MSP domain of MOSPD2 in complex with the conventional FFAT of ORP1.

3. E

   Close‐up view of the structure near the conventional FFAT motif highlighting critical residues (in stick model) of human MOSPD2 present in the binding interface.

4. F

   Surface representation of the MSP domain of MOSPD2 in complex with the phosphorylated FFAT of STARD3.

5. G

   Close‐up view of the structure near the Phospho‐FFAT motif highlighting critical residues (in stick model) of human MOSPD2 present in the binding interface.

6. H, I

   Close‐up view of the hydrophobic pockets interacting with the 2^nd residue (F₂₀₇) (H) and the 9^th residue (F₂₁₄) (I) of the Phospho‐FFAT of STARD3; residues of MOSPD2 constituting the pockets are indicated.

7. J

   Superposition of the structures shown in (E) and (G) showing the similar trajectories of the peptides.

Data information: Phosphorous, nitrogen, and oxygen atoms are colored in orange, blue, and red, respectively. Carbon atoms are shown in slate blue, gray, and rose in the MSP domain, the classic FFAT motif, and the Phospho‐FFAT motif, respectively. (D, F, H, I): The protein surface is colored according to the YRB scheme, showing hydrophobic, negatively and positively charged atoms in yellow, red, and blue, respectively; the other atoms are in white (Hagemans et al, 2015). (E, G): Red spheres are water molecules.

Figure 9. Phosphorylation acts as a switch for inter‐organelle contact formation

Schematic representation of the two types of FFATs: (left) conventional FFATs (illustrated here with STARD11/CERT) which allow the formation of a stable complex between VAPs/MOSPD2 and thus the formation of MCSs; (right) a novel type of FFATs that we named Phospho‐FFATs (illustrated here with STARD3), which strictly depend on phosphorylation to be active. Thus, phosphorylation acts as a switch mechanism to turn on the interaction between VAPs/MOSPD2 and their partners possessing a Phospho‐FFAT, and thus membrane contact site formation.