Interaction between the PH and START domains of ceramide transfer protein competes with phosphatidylinositol 4-phosphate binding by the PH domain

Structure of the CERT PH and START domain complex

Phosphorylation of the Ser/Thr residues of CERT SR leads to down-regulation of PtdIns(4)P-binding by the PH domain and ceramide transfer by the START domain. This regulation is suggested to involve a direct interaction between the PH and START domains of CERT (12). To understand the structural basis of CERT functional regulation, we solved the crystal structure of a PH–START complex to 2.4 Å, limiting resolution (Table 1). Electron density allowed for building of 97 residues of the 103-residue PH domain and 235 of the 239-residue START domain. The structure contains two potential interfaces between the PH and START domains. The lower energy assembly (EBI PISA server), which also makes the most biological sense, is shown in Fig. 1B (15). The overall structure of the PH domain in the complex is nearly identical to that of the free CERT PH structure (PDB code 4HHV) (7, 8). The root mean square deviation (r.m.s.d.) of superposition across PH Cα atoms is <1 Å with the greatest differences found in the β1/β2 loop. The START domain structure in the complex is also similar to that of the free CERT START domain. The r.m.s.d. of superposition of START domain in the complex with the START domain-alone structure (PDB code 2E3M) across Cα atoms is <1 Å (16–18) (supplemental Fig. S1).

In the complex structure, the β6′/β7′ and β8′/β9′ loops of the START domain clamp onto the β1/β2 loop of the PH domain and buries an interface of 614 Ų (Fig. 1, B and C). Notably, the β6′/β7′ loop of the START domain occupies the cavity of the PH domain that is used for PtdIns(4)P-binding. Moreover, the β1/β2 loop of the PH domain, which makes significant energetic contributions to CERT PH domain association with membranes, is completely sandwiched by the β6′/β7′ and β8′/β9′ loops of the START domain (7, 8). The PtdIns(4)P-binding site and the β1/β2 loop of the PH domain are collectively referred as the “basic groove” due to a significant number of basic residues in that area (7). The electrostatic surface potential maps of the PH and START domains in the complex show that the negatively charged β6′/β7′ loop of the START domain fits snuggly into the basic groove of the PH domain (Fig. 1D). Comparison of the CERT PH–START complex structure with a HADDOCK-generated structure model of the PH/diC6–PtdIns(4)P complex shows a clash between START domain and diC6–PtdIns(4)P-binding to PH domain (7, 8) (Fig. 1E). These observations suggest that PH domain-binding to START domain is incompatible with its association with PtdIns(4)P-containing membranes.

We also used nuclear magnetic resonance (NMR) chemical shift perturbation to examine the binding interface between the PH and START domains. Adding unlabeled START domain to the ¹⁵N-labeled PH domain leads to reduction or complete loss of the peak intensities of some residues, suggesting intermediate exchange in the PH domain in the presence of the START domain (supplemental Fig. S2A). The residues that vanished or showed 90% or more reduction of peak intensity are mapped onto the crystal structure of the PH domain (PDB code 4HHV) (supplemental Fig. S2B). As a comparison, shown in supplemental Fig. S2C are residues that are affected by the addition of diC6–PtdIns(4)P to ¹⁵N labeled PH domain (8). It can be observed that perturbations of the PH domain by START domain and diC6–PtdIns(4)P occur in similar regions, indicating overlapping binding interfaces. The findings from these NMR studies are consistent with the PH–START complex crystal structure (Fig. 1B).

Details of the CERT PH–START complex interface

EBI PISA and CCP4 program CONTACT were used to analyze the PH–START interface (15, 20) (Fig. 2A). The PH and START domain interface consists of 20 residues from the PH domain and 17 residues from the START domain. Specifically, Lys³², Trp³³, Tyr³⁶, Trp⁴⁰, Arg⁴³, Tyr⁵⁴, Arg⁶⁶, and Tyr⁹⁶ of the PH domain form multiple contacts with the START domain. These PH domain interface residues are also involved in binding to PtdIns(4)P-containing membranes either through direct interactions with PtdIns(4)P or through interactions with the membrane interfacial region (7, 8), i.e. the PH domain uses the same set of residues for START domain and PtdIns(4)P-containing membrane interaction. Three residues in the β6′/β7′ loop of the START domain, Glu⁴⁹⁴, Asn⁴⁹⁵, and Glu⁴⁹⁸, form extensive hydrogen bonds with PH domain residues Lys³², Tyr³⁶, Arg⁴³, Tyr⁵⁴, and Arg⁶⁶ (Fig. 2, B and C). Phe⁵⁹⁸, the last residue of the START domain, interacts with Tyr³⁶ through π-π stacking (Fig. 2C). Val⁵³³ and Pro⁵³⁵ in the β8′/β9′ loop make hydrophobic contacts with PH domain residues Trp³³, Trp⁴⁰, and Tyr⁹⁶ (Fig. 2D). In addition, the backbone CO of Val⁵³³ forms a hydrogen bond with Trp³³ indole ring NH group (Fig. 2D). The β8′/β9′ loop of the START domain, although not in direct competition with the PH domain PtdIns(4)P-binding site, shields the β1/β2 loop and would, therefore, prevent PH/membrane association. The PH–START complex structure clearly suggests that PtdIns(4)P and START domain binding by the PH domain are incompatible interactions.

CERT PH domain interacts with START domain with micromolar affinity

Isothermal titration calorimetry (ITC) was used to determine the affinity between the PH and START domains. ITC measurements were performed by injecting PH protein into START protein. Global fitting of three separate measurements using a single-site model yields a ΔH of −12.1 (−13.1, −11.3) kcal/mol and a K_D of 9.2 (8.1, 10.5) μm (Fig. 3).

To evaluate the contributions of interface residues in the interaction, site-directed mutants were generated in both the PH and START domains. The abilities of these mutants to compete for binding against the wild-type PH–START interaction were assessed in an AlphaScreen competition assay (21) (Fig. 4A). IC₅₀ values of 2.0 and 2.2 μm were obtained for the wild-type (WT) PH and START domains, respectively (Fig. 4, B–D). A PH domain R43A mutant showed a >8-fold increase in IC₅₀ (16.8 μm) as compared with PH WT (Fig. 4, B and D). However, the IC₅₀ for the Y54A mutant (1.5 μm) is similar to that of the PH WT, and the IC₅₀ values for the R43A/Y54A and the R43A mutants are also close to each other (Fig. 4, B and D). One-way analysis of variance statistics indicate that the IC₅₀ differences measured for the R43A and R43A/Y54A mutants versus PH WT domain were statistically significant, whereas Y54A was not (Fig. 4E). These results indicate that Arg⁴³ significantly contributes to the PH–START interaction, whereas the contribution from Tyr⁵⁴ is negligible. It is likely that despite the short distance, Tyr⁵⁴ is not positioned at an optimal orientation for hydrogen-bond formation with Glu⁴⁹⁴. It is also conceivable that other nearby basic residues, such as Lys⁵⁶, may engage in interactions with Glu⁴⁹⁴ in the Y54A mutant, thus making this residue less critical for START domain binding. We also attempted to measure the IC₅₀ of a W33A/R43A/Y54A PH domain mutant, but the experiments were unsuccessful. Using NMR ¹H,¹⁵N 2D heteronuclear single-quantum correlation (HSQC) experiments, we found that the W33A/R43A/Y54A mutant maintains the overall tertiary fold of the PH domain (supplemental Fig. S3). We speculate the difficulties with these experiments were likely caused by protein aggregation at high concentrations in the presence of the AlphaScreen beads. As an alternative to the AlphaScreen competition assay, we used 2D HSQC experiments to evaluate the effect of the W33A/R43A/Y54A mutations on START domain binding. The presence of a 1× unlabeled PH WT domain led to extensive broadening of the ¹⁵N-labeled START domain peaks, likely from intermediate binding exchange (supplemental Fig. S4A). In contrast, the addition of 1× PH W33A/R43A/Y54A mutant lead to little change in the START domain spectrum, suggesting a much-reduced affinity between the PH W33A/R43A/Y54A mutant and the START domain (supplemental Fig. S4B).

To validate the role of the β6′/β7′ and β8′/β9′ loops of the START domain in the PH–START interaction, START domain mutants E494R/N495K and E494R/N495K/P535R/E537K and a mutant in which a (GGS)₂ linker replaces the β6′/β7′ loop (Δ493–498 (GGS)₂) were tested in the AlphaScreen competition assay (Fig. 4A). The E494R/N495K and the Δ493–498 (GGS)₂ mutants both displayed significant decreases in affinity compared with START WT domain with an IC₅₀ of 34.7 and 59.5 μm, respectively, demonstrating the importance of the β6′/β7′ loop in mediating the PH–START interaction (Fig. 4, C–E). The E494R/N495K/P535R/E537K mutant showed the most loss in affinity with an IC₅₀ of 86.1 μm, confirming that both loops of the START domain contribute to PH domain binding (Fig. 4, C–E). Altogether, these competition assays establish that the START domain uses the β6′/β7′ and β8′/β9′ loops to engage with the PtdIns(4)P-binding site of the PH domain.

CERT PH–START interaction is incompatible with PH domain binding to PtdIns(4)P

The crystal structure of the PH–START complex predicts that the START domain competes with PtdIns(4)P-containing membranes for binding to the PH domain (7, 8). To test this hypothesis, an AlphaScreen competition assay in which the ability of START domain to compete against PtdIns(4)P-embedded liposomes for PH domain association was evaluated (Fig. 5A). An IC₅₀ value of 1.5 μm was obtained for the PH WT protein, which is comparable with the reported affinity of the PH domain for PtdIns(4)P-embedded liposomes (7, 8). The START domain yielded an IC₅₀ value of 4.4 μm, indicating that it is able to compete with the PH/liposome interaction (Fig. 5B). The START domain has been shown to interact with membranes weakly (16, 18). To evaluate how START domain association with membrane affects the competition, the assays were repeated in the presence of a START domain inhibitor (1R,3S)-N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl) dodecanamide (HPA-12), which has been shown to abolish START domain membrane-binding (22–24). The presence of HPA-12 slightly increased the IC₅₀ of the START domain, but the difference was not significant (supplemental Fig. S5A and B), indicating START/PH interaction is likely responsible for the competition.

Disrupting the PH–START interaction restores CERT SR phosphorylation mimic PtdIns(4)P-binding and ceramide transfer activity

Our investigations on isolated PH and START domains indicate that PH–START-binding competes with PH domain-binding to PtdIns(4)P-containing membranes. To further investigate the role of this interaction in the regulation of full-length CERT, we made use of a SR phosphorylation mimic of CERT, termed CERT10E, where the Ser/Thr residues of SR are mutated to Glu residues (12). We generated a CERT10E E494R/N495K/P535R/E537K mutant and investigated the effects of disrupting the PH–START interaction on CERT10E PtdIns(4)P binding and ceramide transfer activity.

Fluorescence resonance energy transfer (FRET) between protein Trp residues and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(5-dimethylamino-1-naphthalenesulfonyl (dansyl-PE) lipids embedded in liposomes was used to assess the binding of proteins to the liposomes (25). In the absence of PtdIns(4)P, neither CERT10E nor the CERT10E E494R/N495K/P535R/E537K mutant exhibited any detectable FRET, indicating the absence of association with the liposomes (Fig. 6A). In the presence of 4 mol% of PtdIns(4)P in the liposomes, low FRET was detected between CERT10E and liposomes, suggesting weak association. This is consistent with previous studies demonstrating that phosphorylated CERT has low affinity for PtdIns(4)P-embedded membranes (12–14). The CERT10E E494R/N495K/P535R/E537K mutant showed a clear increase in FRET signal as compared with CERT10E, indicating increased affinity for PtdIns(4)P-containing liposomes in the absence of the PH–START interaction. The data shown in Fig. 6A were performed with 40 μm total liposome concentration. The binding experiments were also carried out with 60 and 80 μm liposomes, and similar increases in PtdIns(4)P-binding of CERT10E E494R/N495K/P535R/E537K compared with CERT10E were observed (supplemental Fig. S6, A and B). This result was also consistent with prior work showing that the affinity of CERT10E for PtdIns(4)P increased after cleavage of either the PH or START domain (12).

To investigate if the PH–START complex interface revealed in this study is involved in the phosphorylation inhibition of CERT ceramide transfer activity, the activities of CERT10E and the CERT10E E494R/N495K/P535R/E537K mutant were compared. The transfer activity assay utilized two types of liposomes: donor liposomes that contained anthrylvinyl-ceramide (AV-Cer), a fluorescent ceramide analogue, and its quencher, 3,3′-dihexadecyloxacarbocyanine (DiO-C₁₆), and acceptor liposomes that contain 4 mol% of PtdIns(4)P. Transfer of AV-Cer into acceptor liposomes that do not contain the quencher allowed for detection of AV-Cer emission (26, 27). CERT10E, consistent with previous results, exhibits very low ceramide transfer activity (12) (Fig. 6B and supplemental Fig. S6C). In comparison, the CERT10E E494R/N495K/P535R/E537K showed much higher ceramide transfer activity than CERT10 despite the fact that the E494R/N495K/P535R/E537K mutations decreased the intrinsic ceramide transfer activity of the isolated START domain (Fig. 6C and supplemental Fig. S6D). These results indicate that the PH–START interface identified in our structural investigation is involved in SR phosphorylation regulation of CERT ceramide transfer activity. These results are also consistent with prior studies showing that removal of the PH domain leads to an increase in the ceramide transfer activity of CERT10E (12). Ideally, mutations on the PH domain that disrupt PH–START-binding but not the intrinsic ceramide transfer activity of the START domain should have been used here. However, such mutations on the PH domain would also impact PtdIns(4)P-binding by the PH domain so as a compromise the E494R/N495K/P535R/E537K mutant that was used in these experiments.

Although the PH–START complex crystal structure provides a clear explanation for the inhibition of PtdIns(4)P-binding by the START domain, it is not immediately clear how this interaction affects the ceramide transfer activity of the START domain. To address this question, the ceramide transfer activity of the isolated START domain in the presence of PH WT or PH W33A/R43A/Y54A mutant was compared. Due to the moderate affinity between the isolated PH and START domains and the relatively low concentration of the START domain used in the transfer assay, an excess amount of PH domain was used so that ∼90% of the START domain would exist in the PH domain-bound form. The activity of the START domain in the presence of PH WT was decreased, although not significantly, compared with the activity in the presence of PH W33A/R43A/Y54A (supplemental Fig. S7, A, D, and E). However, the excess of both PH WT and mutant proteins caused unexpected changes to the activity of the START domain (supplemental Fig. S7B). Because non-vesicular lipid transfer mediated by lipid transfer proteins is a multistep process that requires the transfer protein to adopt different conformations during lipid extraction, lipid transport, and lipid release, the high concentrations of protein in solution may have led to modulations of the conformational equilibrium of the START domain and its affinity for membranes, which could result in changes in the transfer activity. To determine if these changes to activity were the result of nonspecific effects from the high concentrations of PH proteins used in this assay, the ceramide transfer activity of START domain was measured in the presence of excess B1 immunoglobulin-binding domain of streptococcal protein G (GB1), which does not interact with START domain and is not related to the PH domain (28–30) (supplemental Fig. S7, B and C). Indeed, the presence of GB1 altered the rate for transfer in a manner nearly identical to PH W33A/R43A/Y54A protein, which also does not bind the START domain, indicating a nonspecific effect on START activity (supplemental Fig. S7, D and E). Altogether, these results show that the START domain is not inhibited by binding to the isolated PH domain alone. Therefore, although the PH–START interaction does play a role in the phosphorylation inhibition of CERT ceramide transfer activity, the interaction is not directly responsible for inhibiting the START domain, and additional factors are involved in the SR phosphorylation inhibition of CERT function.

Disrupting the PH–START interaction changes the cellular distribution of CERT

The FRET-based liposome-binding experiments show that disrupting the PH–START interaction increased PtdIns(4)P binding of the CERT SR phosphorylation mimic, CERT10E (Fig. 6A). Based on this result, one would predict that disrupting the PH–START interaction would increase the Golgi localization of CERT. To test this hypothesis, immunofluorescence microscopy was used to assess the role of the PH–START interaction in the cellular localization of CERT. HEK293 cells were transiently transfected with HA-tagged CERT or CERT E494R/N495K/P535R/E537K mutant. Using GM130 as a Golgi marker, the cells were visualized by indirect immunofluorescence with anti-HA and anti-GM130 antibodies (Fig. 7). HA-CERT exhibited even distribution in the perinuclear region, likely due to association with vesicle-associated membrane protein (VAMP)-associated protein A (VAP-A) in the ER membrane (5) (Fig. 7). In contrast, HA-CERT E494R/N495K/P535R/E537K showed clear preferential co-localization with GM130, indicating release of inhibition of CERT binding to the Golgi membrane. Interestingly, the cellular distribution of HA-CERT E494R/N495K/P535R/E537K is very similar to that of the CERT S132A mutant, where SR cannot be phosphorylated, suggesting HA-CERT E494R/N495K/P535R/E537K resembles the non-phosphorylated CERT (31). These similarities further illustrate that the PH–START interaction is responsible for SR phosphorylation inhibition of CERT association with the Golgi membrane. Our study provides a structural explanation for the previous observations that the inhibition of SR phosphorylated CERT-binding to the Golgi membrane requires the START domain (12).

Materials

Lipids were obtained from Avanti Polar Lipids unless otherwise specified. AlphaScreen technology materials and equipment were purchased from PerkinElmer Life Sciences. The primary rat monoclonal anti-HA antibody was purchased from Sigma. The primary mouse polyclonal antibody to GM130 and the secondary antibodies, anti-rat IgG labeled with Alexa Fluor® 488 and anti-mouse IgG labeled with Alexa Fluor® 594, were purchased from Abcam.

Protein expression and purification

The human CERT PH domain-containing residues 20–122 and START domain-containing residues 360–598 were cloned into the prokaryotic overexpression vector pHis₆-GB1 as previously described (8). DNA molecules encoding the CERT PH domain with an N-terminal c-Myc epitope tag and the START domain with a Cys residue added to the N terminus were purchased from GenScript in the pHis₆-GB1 vector. The site-specific biotinylated START domain was produced using the EZ-Link Maleimide-PEG₂-Biotin reagent (Thermo Scientific) according to the manufacturer's instructions. Mass spectrometry confirmed single or double biotinylation of the START domain. The full-length, phosphorylation mimic CERT (CERT10E) was created by site-directed mutagenesis of wild-type human CERT (CERT) to mutate Ser¹³², Ser¹³⁵, Ser¹³⁸, Ser¹⁴¹, Ser¹⁴⁴, Thr¹⁴⁶, Ser¹⁴⁷, Thr¹⁴⁸, Ser¹⁴⁹, and Ser¹⁵⁰ to glutamate (12). CERT10E was cloned into the pHis₆-GB1 vector with six extra amino acids (GEFKGL) added at the N terminus after cloning. All additional mutations were created by site-directed mutagenesis, and individual clones were confirmed by DNA sequencing. Proteins were expressed and purified as previously described (8). The PH and START domain mutants were uniformly labeled with ¹⁵N, and 2D ¹H,¹⁵N NMR HSQC spectra show the mutants maintain the overall fold of the wild-type protein (data not shown).

Crystallization, X-ray diffraction data collection, structure solution, and refinement

Crystallization experiments were carried out by vapor diffusion of hanging drops. CERT PH–START co-crystals appeared in 5–7 days at 20 °C using 0.1 m MES bis-tris (pH 6.0), 6% (w/v) polyethylene glycol 10,000, and 10 μm calcium chloride. Individual drops consisted of 1 μl of PH–START complex sample (8 mg/ml) and 1 μl of precipitant solution. Crystals were cryoprotected by a brief incubation in precipitant solution containing 24% (w/v) polyethylene glycol 400. Crystal samples were preserved by flash-cooling in liquid nitrogen.

X-ray diffraction data were collected at −173 °C using beamline 22-BM of the Advanced Photon Source (Argonne National Laboratory). Individual reflections were indexed, integrated, and scaled using HKL2000 (32). Initial-phase information was obtained by maximum-likelihood molecular replacement using the program Phaser using available crystal structures of the PH (PDB code 4HHV) and START (PDB code 2E3M) domains (33, 34). An initial model was built in PHENIX.AUTOBUILD. The final model was obtained after manual building in COOT followed by additional rounds of refinement using the refine program of Phenix (20, 35–37). The model was analyzed and validated using MOLPROBITY before PDB deposition with the accession code 5JJD (38). Structure representations were generated using PyMOL (39). Additional structural information and refinement statistics are presented in Table 1.

Isothermal titration calorimetry

CERT PH and START domains were exchanged into buffer containing 25 mm Hepes (pH 7.5), 100 mm sodium chloride, and 2 mm β-mercaptoethanol. All ITC experiments were carried out on a MicroCal VP-ITC instrument (Northampton, MA). The first set of ITC data were collected by injecting 290 μl of 300 μm PH domain into 1.4 ml of 30 μm START domain with 10-μl injections. Later experiments were performed with 3-μl and 7-μl injections of 400 μm PH domain into 40 μm START domain. Protein solutions were held at 30 °C with a 290-rpm stirring speed and a 120-s delay between injections.

Each data set was first analyzed with the NITPIC program which determined the injection peak baselines, integrated the areas of injection heats, prepared an isotherm, and estimated the initial parameter values (40, 41). Global fitting of all data sets was carried out using SEDPHAT 10.58d (42, 43). ITC data plots were then prepared with GUSSI 1.0.8d (44).

AlphaScreen competition assay

Binding between CERT PH and START domains or PtdIns(4)P-embedded liposomes was evaluated using a luminescent microbead AlphaScreen technology (21). In this assay c-Myc PH domain was mixed with equimolar biotinylated START domain or biotinylated-PE- and PtdIns(4)P-embedded liposomes and increasing concentrations of untagged competitor proteins. Acceptor beads coated with anti-c-Myc antibody and streptavidin-coated donor beads were added to adsorb the c-Myc PH domain and biotinylated START domain or biotinylated-PE and PtdIns(4)P-embedded liposomes, respectively. Upon illumination at 680 nm, a photosensitizer in the donor beads converts ambient oxygen to singlet oxygen. If the acceptor beads are within close proximity to the donor beads, energy is transferred from the singlet oxygen to thioxene derivatives on the acceptor bead, resulting in emission at 520–620 nm.

Using the AlphaScreen c-Myc detection kit from PerkinElmer Life Sciences, an equilibrium competition-binding assay was carried out in 96-well format ½ area opaque plates with the following procedures. Competition assays with CERT PH and START domains were carried out in a final reaction volume of 25 μl. Each component was added to the following final concentrations in buffer containing 25 mm Hepes (pH 7.5), 100 mm sodium chloride, and 0.1% (w/v) BSA: 200 nm c-Myc PH domain, 200 nm biotinylated START domain, 20 μg/ml anti-c-Myc AlphaScreen acceptor beads, and 20 μg/ml AlphaScreen Streptavidin donor beads. A dilution series of the untagged competitor protein was prepared with final concentrations ranging from 20 nm to 200 μm. The reaction began by mixing c-Myc PH domain, biotinylated START domain, and varying concentrations of the unlabeled competitor. After incubation for 1 h at room temperature, acceptor beads were added and incubated for 1 h. Donor beads were then added, and after a 1-h incubation the AlphaScreen signal was read on an EnSpire multimode plate reader. Competition assays with c-Myc PH domain and biotinylated-PE and PtdIns(4)P-embedded liposomes were carried out with the same procedure but with a final concentration of 200 nm liposomes in place of biotinylated START domain. Liposomes containing 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS), porcine brain PtdIns(4)P, and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (biotinyl-cap PE) were prepared at a molar ratio of 84.6/9.4/4/2 by the extrusion method using 0.1-μm pore size membranes (Avanti Polar Lipids).

The AlphaScreen signal was normalized to wells without competitor. A dose-response curve was generated by plotting normalized signal (%) versus log[competitor (M)]. IC₅₀ values were calculated by nonlinear curve-fitting to a one-site competition model using GraphPad Prism7 software (GraphPad; La Jolla, CA). IC₅₀ values are reported as averages ± S.D. from two or three experiments.

Fluorescence resonance energy transfer measurement of protein-liposome interaction

FRET between dansyl-PE embedded in liposomes and Trp residues of CERT10E or CERT10E E494R/N495K/P535R/E537K protein was used to monitor the protein/liposome interaction. Liposomes that contain either DOPC/DOPS/dansyl-PE/PtdIns(4)P at a molar ratio of 77.4/8.6/10/4 or DOPC/DOPS/dansyl-PE at a molar ratio of 81/9/10 were prepared by the extrusion method using 0.1-μm pore size membranes. Liposomes were suspended in a total volume of 1.2 ml of buffer containing 25 mm Tris (pH 7.5), 100 mm NaCl, 1 mm EDTA, and 1 mm tris(2-carboxyethyl)phosphine. The buffer also contained 8.25 μm HPA-12 to abolish START domain interaction with liposome (22–24). CERT10E or CERT10E E494R/N495K/P535R/E537K protein was added to the vesicle suspension under constant stirring at 25 °C. Trp residues were excited at 290 nm and dansyl-PE emission was monitored at 400–550 nm. Emission intensity at 530 nm was chosen for FRET analyses to ensure the absence of intensity contribution from the protein. The normalized FRET intensity (ΔF) at a given protein concentration was calculated with the following equation: ΔF = (F_i − F₀)/F₀, where F₀ is the intensity in the absence of protein and F_i is the intensity after protein addition. The titration series were performed with liposome concentrations of 40, 60, and 80 μm. For each vesicle concentration, the normalized FRET values were plotted against protein concentration to compare the binding of CERT10E and CERT10E E494R/N495K/P535R/E537K to liposomes.

FRET measurement of ceramide transfer activity

The ceramide transfer activities of CERT10E and START proteins were measured using donor liposomes containing ceramide and acceptor liposomes that do not (26, 27). The donor liposomes were prepared with DOPC, DOPS, DiO-C₁₆ (Genolite Biotek), N-octanoyl-d-erythro-sphingosine (C8-Cer), and a fluorescent ceramide analogue called AV-Cer (Cayman Chemical) at a molar ratio of DOPC/DOPS/DiO-C₁₆/C8-Cer/AV-Cer (79.2/8.8/3/8/1) (27). The acceptor liposomes were composed of DOPC/DOPS/PtdIns(4)P at a molar ratio of 86.4/9.6/4.0. Liposomes were prepared by the extrusion method using 0.1-μm pore-size membranes. AV-Cer emission was quenched by DiO-C₁₆ in the donor liposomes but became detectable upon transfer to the acceptor liposomes.

The ceramide transfer assays were performed in buffer containing 25 mm Tris (pH 7.5), 100 mm sodium chloride, 1 mm EDTA, and 1 mm tris(2-carboxyethyl)phosphine with a 1.4-ml total volume at 25 °C with constant stirring. The emission of AV-Cer at 420 nm was monitored after excitation at 370 nm. 6 μm donor, and 60 μm acceptor liposomes were added first. After ∼100 s, ceramide transfer was initiated by adding 80 nm protein. No spontaneous transfer was observed in the absence of protein addition. At the end of the assay, 3.5% (v/v) Triton X-100 was added to disrupt the liposomes and release all AV-Cer molecules to give the maximum fluorescence. Fluorescence intensities were corrected for background by subtracting the intensity before the initiation of ceramide transfer. The percent of AV-Cer transferred at each time point was calculated by dividing the corresponding intensity by the maximum intensity. Ceramide transfer activity was measured in triplicate for each protein.

Immunofluorescence microscopy of CERT cellular localization

An HA-epitope tag was added to the N terminus of CERT and CERT E494R/N495K/P535R/E537K by PCR. HEK293 cells were transfected with either HA-CERT or HA-CERT-E494R/N495K/P535R/E537K in pcDNA6/V5-His expression plasmids by calcium phosphate precipitation. After 24 h, the transfected cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.2% Triton X-100 in PBS for 20 min, and then incubated in BlockAid™ Blocking Solution (Molecular Probes) for 1 h at room temperature. The cells were then incubated with primary antibodies, rat anti-HA, and mouse anti-GM130 (Golgi marker), or control IgG, diluted in PBS at 4 °C overnight. The cells were briefly rinsed with PBS then incubated with secondary antibodies, anti-rat IgG labeled with Alexa Fluor® 488, and anti-mouse IgG labeled with Alexa Fluor® 594 for 1 h at room temperature. After intensive washing with PBS, the slides were mounted by cold Vectashield Hard Set mounting medium and visualized by an Olympus Fluoview 300 confocal microscope. The images were acquired with the PlanApo 100× objective using the Olympus FluoView 5.0 software. Images for Fig. 7 were prepared with ImageJ (45).