Osh6p and ORP8 transfer saturated and unsaturated PS species differently
We first measured in vitro the speed at which Osh6p and the ORD of ORP8 (ORP8 [370-809] [14], hereafter called ORD8) transferred PS subspecies with different acyl chains between two membranes (Fig. 1a). Our series comprised subspecies with saturated acyl chains of increasing length (12:0/12:0, 14:0/14:0, 16:0/16:0, 18:0/18:0), with two C18 acyl chains that are more or less unsaturated (18:0/18:1, 18:1/18:1, 18:2/18:2) and with one saturated C16 acyl chain at the sn-1 position and one C18 acyl chain, with a different unsaturation degree, at the sn-2 position (16:0/18:1, 16:0/18:2). Note that 16:0/18:1-PS is the dominant PS species in S. cerevisiae yeast [22, 24, 28, 37] (under standard growing conditions), whereas in humans, 18:0/18:1-PS and 16:0/18:1-PS are the two most abundant species [25, 38,39,40]. PS transfer was measured between LA liposomes, made of DOPC and containing 5% PS (mol/mol) and 2% Rhod-PE, and LB liposomes only made of DOPC, using the fluorescent sensor NBD-C2Lact. In each measurement, NBD-C2Lact was initially bound to LA liposomes and its fluorescence was quenched due to energy transfer to Rhod-PE; when LTP transferred PS to LB liposomes, NBD-C2Lact translocated onto these liposomes and the fluorescence increased (Fig. 1b). By normalizing the NBD signal, we established transfer kinetics (Additional file 1: Fig. S1a) and initial transfer rates (Fig. 1c). Osh6p transferred saturated PS rapidly, with rates between 7.6 ± 1 (mean ± s.e.m., 14:0/14:0-PS) and 35.2 ± 2 PS min−1 (18:0/18:0-PS). The transfer of unsaturated PS species was much slower (from 0.8 ± 0.1 to 3.1 ± 0.8 PS min−1 per Osh6p). A different picture was obtained when we measured PS transfer in a situation of PS/PI(4)P exchange using LB liposomes containing 5% 16:0/16:0-PI(4)P. The transfer rates measured with 14:0/14:0-PS and 16:0/16:0-PS were similar to those measured in non-exchange contexts, and significantly lower with 12:0/12:0-PS and 18:0/18:0-PS. In contrast, the transfer rate of unsaturated PS, with the exception of 18:2/18:2-PS, strongly increased (by a factor from 3.2 to 19.5).
Overall, ORD8 transported PS more slowly than Osh6p in both exchange and non-exchange situations (Fig. 1c, Additional file 1: Fig. S1b). Nevertheless, the activity of ORD8 changed depending on the PS subspecies in a manner similar to that of Osh6p, as highlighted by the correlation of transfer rates measured for the two LTPs with each PS ligand (R2 ~ 0.75, Additional file 1: Fig. S1c). Like Osh6p, ORD8 transferred saturated PS species more rapidly than unsaturated ones when PI(4)P was absent. In a situation of PS/PI(4)P exchange, the transfer of unsaturated PS species (except for 18:2/18:2-PS) was much more rapid (up to 29-fold) whereas the transfer of saturated PS was slightly enhanced or inhibited. Collectively, these data did not point to a monotonic relationship between PS transfer rates and the length of PS acyl chains or the degree of unsaturation of these chains. However, they indicated that PS species were transported and exchanged with PI(4)P quite differently depending on whether or not they had at least one double bond.
Coupling between the transfer rate of PS species and PI(4)P under exchange conditions
Next, we determined whether the rate of 16:0/16:0-PI(4)P transfer was different depending on the nature of the PS species under the exchange conditions. Using a fluorescent PI(4)P sensor (NBD-PHFAPP) and a FRET-based strategy akin to that used to measure PS transfer, we measured the speed at which PI(4)P, at 5% in LB liposomes, was transported by Osh6p and ORD8 to LA liposomes devoid of PS or containing a given PS species (at 5%) (Fig. 1b, Additional file 1: Fig. S2). With PS-free LA liposomes, the initial PI(4)P transfer rate was 8.4 ± 1.3 PI(4)P min−1 for Osh6p and 4.2 ± 0.5 PI(4)P min−1 for ORD8 (Fig. 1d). In a situation of lipid exchange, these transfer rates increased to a different degree when LA liposomes contained a PS species other than 18:0/18:0-PS and, in experiments with ORD8, 16:0/16:0-PS (Fig. 1d). For each PS species, we calculated an acceleration factor corresponding to the ratio (expressed as a log value) of the PI(4)P transfer rate, measured in the presence of this PS species, to the PI(4)P transfer rate measured in the absence of counterligand. Also, acceleration factors based on PS transfer rates reported in Fig. 1c were determined. Then for each LTP, these two factors were plotted against each other, allowing saturated and unsaturated PS species to cluster in two groups (Fig. 1e). With Osh6p, the group including saturated PS species was characterized by null or negative acceleration factors for PS (down to − 0.49) associated with low or moderate acceleration factors for 16:0/16:0-PI(4)P (from 0.20 to 0.58). In contrast, the group corresponding to unsaturated PS species was characterized by higher acceleration factors for both PS (from 0.30 to 1.30) and PI(4)P (from 0.49 to 0.79). With ORD8, the acceleration factors for saturated PS were negative, null, or moderate (from − 0.15 to 0.47) and associated with null or moderate acceleration factors for PI(4)P (from 0 to 0.48). For unsaturated PS, the acceleration factors were higher, ranging from 0.44 to 1.47 for PS and from 0.40 to 0.55 for PI(4)P. The observation of high acceleration factors for both unsaturated PS and 16:0/16:0-PI(4)P, and much lower or even negative values for saturated PS species, suggests that the LTPs exchange unsaturated PS for PI(4)P much more efficiently than saturated PS.
Exchange activity with prominent cellular PS and PI(4)P species
We next measured how Osh6p and ORD8 exchanged PS and PI(4)P species that are dominant in the yeast and/or human repertoire. With Osh6p, we tested 16:0/18:1-PS and 18:0/18:1-PS with 16:0/18:1-PI(4)P, one of the two most abundant yeast PI(4)P species [22, 23]. As a comparison, we tested a non-yeast species, 18:0/20:4-PI(4)P, which is the main constituent of purified brain PI(4)P. With ORD8, we tested the two same PS species with 18:1/18:1-PI(4)P that resembles unsaturated PI(4)P species (36:1 and 36:2) found in transformed cells [41, 42] and 18:0/20:4-PI(4)P, which is prominent in primary cells and tissues [42]. As a comparison, we used 16:0/16:0-PI(4)P as in our previous assays.
Osh6p slowly transferred the two PS species from LA to LB liposomes in the absence of PI(4)P but ten times faster when LB liposomes contained 16:0/18:1-PI(4)P or 16:0/16:0-PI(4)P. Smaller accelerations of PS transfer were seen with 18:0/20:4-PI(4)P as counterligand. Conversely, in the absence of PS, Osh6p hardly transported any 16:0/18:1-PI(4)P and 18:0/20:4-PI(4)P (< 0.4 lipids min−1) compared to saturated PI(4)P (5.4 lipids min−1) (Fig. 2a, Additional file 1: Fig. S3a, b). When LA liposomes contained PS, the transfer rate of all PI(4)P species increased but with rates that were high for 16:0/16:0-PI(4)P (25.8–39.6 lipids min−1), intermediate for 16:0/18:1-PI(4)P (7–13.3 lipids min−1) and low for 18:0/20:4-PI(4)P (1.63–2.4 lipids min−1). Interestingly, the 16:0/18-1-PS and 16:0/18:1-PI(4)P transfer rates were both similar and high in a situation of lipid exchange, suggesting that Osh6p can execute an efficient one-for-one exchange of these major yeast PS and PI(4)P species.
In the absence of PI(4)P, ORD8 slowly transferred 16:0/18:1-PS and 18:0/18:1-PS between membranes and much faster in a situation of exchange, by a factor of 4.8–6.8 and 11.5–16.6, respectively, depending on the PI(4)P species used as counterligand (Fig. 2b, Additional file 1: Fig. S3c, d). Like Osh6p, ORD8 barely transferred unsaturated PI(4)P under non-exchange conditions (< 0.26 lipids min−1), compared to 16:0/16:0-PI(4)P. Under exchange conditions, ORD8 transferred these PI(4)P species more rapidly but far less than PS in the opposite direction. This suggests that ORP8 cannot efficiently exchange unsaturated PS for PI(4)P. We conclude that the acyl chain composition of PI(4)P, and primarily its unsaturation degree, impacts how Osh6p and ORP8 transfer and use this PIP in exchange for PS.
Osh6p and ORP8 activities drastically change if the sn-1 or sn-2 chain of PS is monounsaturated
Striking differences were seen between 18:0/18:0-PS and unsaturated forms of this lipid in our transfer assays. In particular, data obtained with 18:0/18:0-PS and 18:0/18:1 PS suggested that only one double bond in PS was sufficient to drastically change LTP activity. Whether this depends on the location of this double bond in the sn-2 chain was unclear. Therefore, we compared how Osh6p transferred 18:0/18:1 PS and 18:1/18:0-PS, in which the saturated and monounsaturated acyl chains are permuted, between membranes (Fig. 3a). In mere transfer assays, 18:0/18:1-PS and 18:1/18:0-PS were transported at rates that were slightly different (4.7 vs 2.1 PS min−1) but ten-fold more slowly than 18:0/18:0-PS (Fig. 3b). In the presence of 16:0/18:1-PI(4)P, the transfer of the two unsaturated PS species was faster whereas the transfer of 18:0/18:0-PS was inhibited. The opposite transfer of 16:0/18:1-PI(4)P was inhibited if 18:0/18:0-PS was tested as counterligand but enhanced using the two unsaturated PS forms (Fig. 3b). Similar results were obtained with ORD8 except that the transfer of 18:0/18:0-PS was slightly more rapid (by 2.2-fold) in exchange conditions (here 18:1/18:1-PI(4)P was used as counterligand (Fig. 3c3c)). However, the rate of acceleration was low compared to that measured with 18:0/18:1-PS and 18:1/18:0-PS (by 24.0 and 8.4-fold, respectively). Jointly, these results indicate that only one double bond, in one or the other acyl chain of PS, is enough to dramatically change how the two LTPs transport and exchange this lipid for PI(4)P.
Osh6p and ORD8 have a higher affinity for unsaturated than saturated PS and PI(4)P species
Our results suggest that Osh6p and ORD8 transport and exchange PS and PI(4)P at a different speed depending on the unsaturation degree of these lipids. To further analyze why, we devised assays to determine the relative affinity of these LTPs for each PS and PI(4)P species. We established that the intrinsic fluorescence of both proteins (from tryptophan, with a maximum intensity at λ = 335 and 340 nm, respectively) was quenched by ~ 25% when mixed with liposomes doped with 2% NBD-PS, a PS species whose C12:0 acyl chain at the sn-2 position bears an NBD moiety (Additional file 1: Fig. S4). Concomitantly, a higher NBD fluorescence was measured at λ = 540 nm. Adding each LTP to liposomes doped with 2% NBD-PC provoked a slighter decrease of tryptophan fluorescence, yet similar to the changes recorded with pure PC liposomes, and no change in NBD fluorescence. Likely, FRET exclusively occurs between these proteins and NBD-PS because this lipid is specifically trapped in their binding pocket and close to a number of tryptophan residues. Interestingly, we found subsequently that Osh6p and ORP8 fluorescence, pre-mixed with NBD-PS-containing liposomes, increased when adding incremental amounts of liposomes containing unlabelled PS. This allowed us to measure how each PS species competes with NBD-PS for occupation of the Osh6p and ORD8 pocket and thus determine the relative affinity of each ORD for different lipid ligands (Fig. 4a, b, Additional file 1: Fig. S5a and Additional file 2: Table S1 and Table S5). Remarkably, Osh6p had a very low affinity for 18:0/18:0-PS and 12:0/12:0-PS. It showed a higher affinity for 14:0/14:0-PS and 16:0/16:0-PS. Highest affinities were found with unsaturated PS and more particularly 16:0/18:1-PS, 18:1/18-1-PS and 18:0/18:1-PS. Interestingly, Osh6p had a higher affinity for 18:0/18:1-PS than its mirror 18:1/18:0-PS counterpart (Fig. 4b and Additional file 2: Table S1). Using this assay, we also found that Osh6p had a high affinity for 16:0/18:1-PI(4)P and 18:0/20:4-PI(4)P and less for 16:0/16:0-PI(4)P (Fig. 4c, Additional file 2: Table S2). With ORD8, competition assays revealed that it had a much lower affinity for saturated PS than for unsaturated PS (Additional file 1: Fig. S5a, Additional file 2: Table S5). ORD8 had a higher affinity for 18:1/18:1-PI(4)P and 18:0/20:4-PI(4)P than for 16:0/16:0-PI(4)P (Additional file 1: Fig. S5b, Additional file 2: Table S6). Collectively, our data indicated that Osh6p and ORD8 had a higher affinity for unsaturated than saturated lipid ligands.
Alternatively, Osh6p was incubated with liposomes doped with a given PS or PI(4)P subspecies, isolated, and then subjected to thermal shift assays (TSAs) to evaluate to what extent it formed a stable complex with each ligand ((Fig. 4d,e and Additional file 2: Table S3). Low melting temperatures (Tm) were observed with Osh6p exposed to liposomes containing saturated PS species (from 45 to 47.4 °C), near the Tm value of Osh6p incubated with DOPC liposomes devoid of ligand (44.9 ± 0.7 °C). In contrast, significantly higher values were obtained with unsaturated PS (from 47.8 to 50.1 °C). The highest Tm values were found with Osh6p loaded with 16:0/18:1 and 18:0/20:4-PI(4)P species (56.1 and 62.1 °C, respectively), and a slightly lower Tm was found with 16:0/16:0-PI(4)P (52 °C). These results suggest that Osh6p is more prone to capture and hold unsaturated PS and PI(4)P than saturated PS, corroborating the results from competition assays.
The affinity of Osh6p and ORP8 for PS and PI(4)P species dictates how they transfer and exchange them
Next, we analyzed how the affinity of Osh6p and ORD8 for lipid ligands was related to their capacity to transfer them. To do so, we plotted the PS and PI(4)P transfer rates measured in non-exchange contexts (reported in Fig. 1c, d, Fig. 2, and Fig. 3) as a function of 1/[L]50, with [L]50 being the concentration of each species necessary to displace 50% NBD-PS from each LTP in the competition assays. Remarkably, this revealed an inverse relationship between the transfer rates and 1/[L]50 values (Fig. 4f and Additional file 1: Fig. S5c) for each LTP. For Osh6p, plotting the transfer rates as a function of Tm values uncovered a comparable relationship (Fig. 4 g). This suggested that the less affinity these LTPs have for a ligand, the faster they transfer it between membranes. We also plotted the acceleration factors established under exchange conditions with different PS species and 16:0/16:0-PI(4)P (showed in Fig. 1e) against 1/[L]50 values determined with each PS species and LTP (Fig. 4 h, Additional file 1: Fig. S5d). Interestingly, a positive relationship was found between the 1/[L]50 values and each of these factors. Moreover, we noted that PS and PI(4)P transfer rates measured under exchange conditions were overall higher when the LTPs had a higher affinity for PS than PI(4)P (1/[PS]50 > 1/[16:0/16:0-PI(4)P]50). Collectively, these analyses reveal an inverse correlation between the affinity of an LTP for a ligand and its ability to simply transfer it down its concentration gradient. Moreover, they indicate that in a situation of lipid exchange, the acceleration of the PS and PI(4)P transfer rate is more pronounced with high-affinity PS species.
Simulation of transfer and exchange activity of the ORD as a function of its affinity for PS and PI(4)P
To understand why the affinity of Osh6p and ORD8 for PS and PI(4)P species governed how they transferred and exchanged these lipids, we built a simplified kinetic model (Fig. 5a). It was assumed that the ORD interacts similarly with A and B membranes during a transfer process (kON-Mb= 10µM–1 s−1and kOFF-Mb = 0.1 s−1) with an equal ability to capture and release a given lipid (similar kON-lipid and kOFF-lipid). We simulated initial PS transfer rates for kON-PS values ranging from 10−2 to 104 μM−1 s−1 to evaluate how the affinity of the ORD for PS (proportional to kON-PS) governs how it transfers this lipid. The kON-PI4P values was set to 10 μM−1 s−1 and kOFF-PS and kOFF-PI4P values to 1 s−1; the ORD concentration was 200 nM, with an A membrane including 5 μM accessible PS and a B membrane devoid of ligand (as in our transfer assays). A bell-shaped curve (Fig. 5b, black dots) was obtained with a maximum at kON-PS = 3.7 μM−1 s−1 and minima near zero for very low and high kON-PS values. Remarkably, our simulations indicate that an LTP can transfer a low-affinity ligand more rapidly than a high-affinity one, as seen for instance when comparing rates at kON-PS = 10 and 100 μM−1 s−1, and as observed experimentally with saturated and unsaturated PS.
Next, using the same range of kON-PS values, we simulated PS and PI(4)P transfer rates in a situation of lipid exchange between A and B membranes that initially contained 5 μM PS and PI(4)P, respectively (Fig. 5b, pink dots for PS and orange dots for PI(4)P). Acceleration factors were determined from rates established for each lipid in exchange and non-exchange contexts. The PS transfer rate found to be maximal at kON-PS = 3.7 μM−1 s−1 when PI(4)P was absent, slightly increased in the presence of PI(4)P (acceleration factor = 0.13). If kON-PS > 3.7 μM−1 s−1, the PS transfer rates were lower in a non-exchange situation but considerably higher if PI(4)P was present as counterligand. In contrast, for kON-PS < 3.7 μM−1 s−1, the PS transfer rate decreased toward zero, even if PI(4)P was present, and acceleration factors were almost null. In parallel, the PI(4)P transfer rates were found to be systematically higher in the presence of PS yet to a degree that depended on kON-PS values (Fig. 5b). Finally, we noted that an ORD efficiently exchanges PS and PI(4)P if it has a higher affinity for PS than PI(4)P (kON-PS > kON-PI(4)P, Fig. 5b, gray area). These simulations again corroborated our experimental data. In non-exchange situations, saturated PS species, which are low-affinity ligands compared to 16:0/16:0-PI(4)P, are transferred at the fastest rates; yet these rates barely or marginally increase once PI(4)P is present. In contrast, unsaturated PS species, which are globally better ligands than 16:0/16:0-PI(4)P, are slowly transferred in non-exchange situations, but much faster when PI(4)P is present. In all cases, the PI(4)P transfer rate is unchanged or higher in the presence of PS.
To consolidate this analysis, we plotted the acceleration factors for PS and PI(4)P, simulated for different kON-PS and kON-PI(4)P values (Additional file 1: Fig. S6a), against each other and compared the curves obtained with experimental factors shown in Fig. 1e. By setting kON-PI(4)P at 40 or 75 μM−1 s−1, we obtained curves that follow the distribution of acceleration factors obtained with ORD8 and Osh6p, respectively (Fig. 5c).
Finally, simulations performed with variable kON-PI(4)P values (10, 40, 75, or 100 μM−1 s−1) indicated that, when the ORD had a higher affinity for PI(4)P, the PI(4)P transfer rate decreased in a non-exchange context (dashed lines, Additional file 1: Fig. S6a) but increased to a greater extent in a situation of PS/PI(4)P exchange. Interestingly, a different picture emerged if the affinity of the ORD for PI(4)P was increased by ten, by lowering the kOFF-PI(4)P value from 1 to 0.1 s−1 instead of increasing the kON-PI4P value from 10 to 100 μM−1 s−1 (Additional file 1: Fig. S6b): PI(4)P transfer rates were low in non-exchange conditions and only slightly higher in exchange conditions, for all tested kON-PS values. PS transfer was more rapid in exchange conditions with high kON-PS values. This resembled our data showing that unsaturated PI(4)P species were poorly transferred and exchanged for PS while PS transfer was enhanced by these PI(4)P species (Fig. 2). Overall, our model showed how variations in the capacity of the ORD to extract and deliver PS and PI(4)P could modify how it transfers and exchanges these lipids.
Osh6p and ORD8 cannot use PI(4,5)P2 if PI(4)P is present in membranes
ORP5/8 have been suggested, notably based on in vitro data, to use PI(4,5)P2 instead of PI(4)P as a counterligand to supply the PM with PS [17] but this conclusion is disputed [19]. To address this issue, we measured how ORD8 transferred PI(4)P and PI(4,5)P2 (with 16:0/16:0 acyl chains) from LB liposomes that contained only one kind of PIP or, like the PM, both PIPs, to LA liposomes. NBD-PHFAPP, which can detect PI(4,5)P2 in addition to PI(4)P [17], was used as sensor. ORD8 transported PI(4,5)P2 more swiftly than PI(4)P (Fig. 6a, b), as previously shown, but surprisingly, when both PIPs were in LB liposomes, the transfer kinetics was comparable to that measured with PI(4)P alone.
To understand why, we specifically measured the transfer of PI(4,5)P2 with a sensor based on the PH domain of the phospholipase C-δ1 (PHPLCδ1), which has a high affinity and specificity for the PI(4,5)P2 headgroup [43]. This domain was reengineered to include, near its PI(4,5)P2-binding site [44], a unique solvent-exposed cysteine (C61) to which a NBD group was attached (Additional file 1: Fig. S7a). In flotation assays this NBD-PHPLCδ1 construct associated with liposomes doped with PI(4,5)P2 but not with liposomes only made of DOPC or containing either PI or PI(4)P (Additional file 1: Fig. S7b). Fluorescence assays also indicated that NBD-PHPLCδ1 bound to PI(4,5)P2-containing liposomes, as its NBD signal underwent a blue-shift and a 2.2-fold increase in intensity (Additional file 1: Fig. S7c). A binding curve was established by measuring this change as a function of the incremental addition of these liposomes (Additional file 1: Fig. S7d, Additional file 2: Table S7). In contrast, no signal change occurred with PI(4)P-containing liposomes, indicative of an absence of binding (Additional file 1: Fig. S7c, d, Additional file 2: Table S7). NBD-PHPLCδ1 was thus suitable to detect PI(4,5)P2 but not PI(4)P. It was substituted for NBD-PHFAPP to measure to what extent ORD8 specifically transported PI(4,5)P2 from LB liposomes, containing or not PI(4)P, to LA liposomes. Remarkably, we found that ORD8 efficiently transferred PI(4,5)P2 but only if PI(4)P was absent (Fig. 6a,b). Similar conclusions were reached using each PIP sensor by assaying ORD8 with PI(4)P and PI(4,5)P2 ligands with 18:0/20:4 acyl chains (Fig. 6c, Additional file 1: Fig. S8a), and Osh6p using PIPs with a 16:0/16:0 composition (Additional file 1: Fig. S8b, c). These data indicate that ORP8 and Osh6p preferentially extract PI(4)P from a membrane that contains both PI(4)P and PI(4,5)P2, suggesting that they use PI(4)P rather than PI(4,5)P2 in exchange cycles with PS at the PM.
To address this possibility in vitro, we devised an assay with three liposome populations (Fig. 6d, e) to examine whether ORP8 delivers PS in a PI(4)P-containing membrane or in a PI(4,5)P2-containing membrane. First, LA liposomes doped with 5% PS were mixed with NBD-C2Lact. Then LB liposomes, containing 5% PI(4)P and 2% Rhod-PE, and LC liposomes, only made of PC, were successively added. Injecting ORD8 provoked a quenching of the NBD signal, indicating that the C2Lact domain moved onto the LB liposomes. The signal normalization indicated that ~ 1 μM of PS was transferred to LB liposomes. Equivalent data were obtained with LC liposomes doped with PI(4,5)P2, suggesting that this lipid has no influence on the PI(4)P-driven transfer of PS to LB liposomes mediated by ORD8. We performed mirror experiments with LB liposomes that contained PI(4,5)P2 and LC liposomes with or without PI(4)P. Remarkably, PS was transferred to LB liposomes but not if LC liposomes contained PI(4)P. We concluded that ORP8 selectively delivers PS in a compartment that harbors PI(4)P if PI(4,5)P2 is present in a second compartment. This suggests that PI(4)P, and not PI(4,5)P2, is used by ORP5/8 to transfer PS intracellularly.
These observations suggest that ORD8 and Osh6p have a lower affinity for PI(4,5)P2 than for PI(4)P. Confirming this, the NBD-PS-based competition assay showed that each protein barely bound to PI(4,5)P2 compared to PI(4)P (with 16:0/16:0 or 18:0/20:4 acyl chains, Fig. 6f and Additional file 2: Table S4). Likewise, TSAs indicated that Osh6p incubated with liposomes containing 16:0/16:0- and 18:0/20:4-PI(4)P or PI(4,5)P2 was loaded with and stabilized by PI(4)P but not PI(4,5)P2 (Additional file 1: Fig. S8d, e and Additional file 2: Table S8). Finally, we evaluated the conformational state of Osh6p in the presence of each PIP. To this end, we used a version of the protein, Osh6p(noC/S190C), which has a unique cysteine at position 190; this residue is solvent-exposed only if the molecular lid that controls the entrance of the binding pocket of the protein is open [45]. This construct was added to liposomes devoid of PIPs or containing 2% PI(4)P or PI(4,5)P2. Then, 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin (CPM), a molecule that becomes fluorescent only when forming a covalent bond with accessible thiol, was added to each sample. After a 30-min incubation, a high fluorescence signal was measured with Osh6p(noC/S190C) mixed with PC liposomes, indicating that the protein remained essentially open over time (Fig. 6g). In contrast, almost no fluorescence was recorded when the protein was incubated with PI(4)P-containing liposomes, indicating that it remained mostly closed, as previously shown [45]. Remarkably, a high signal was obtained with liposomes doped with 2% PI(4,5)P2, indicating that Osh6p remained open as observed with pure PC liposomes. Altogether, these data suggest that Osh6p/7p and ORP5/8 have a low affinity for PI(4,5)P2 compared to PI(4)P, likely as they cannot form stable and closed complexes with this lipid.
Sterol abundance in membrane does not enhance PS delivery and retention
Like PS, sterol is synthesized in the ER and enriched in the PM, where it constitutes 30–40% of all lipids [46]. PS is thought to associate laterally with sterol, thus retaining sterol in the inner leaflet of the PM and controlling its transbilayer distribution [30, 31]. However, it was not known whether sterol stabilizes PS and thereby aids ORP/Osh proteins to accumulate PS in the PM. To explore this possibility, we measured in vitro the speed at which Osh6p transported PS from LA liposomes to LB liposomes, containing or not 30% cholesterol or ergosterol, and doped or not with 5% PI(4)P. These assays were performed using 16:0/18:1-PS and 16:0/18:1-PI(4)P, as in yeast PM, these predominant PS and PI(4)P species are thought to co-distribute in the presence of sterol [22]. However, we observed that the transfer of PS was not markedly impacted by higher contents of sterol in LB liposomes in non-exchange and exchange conditions (Fig. 7a, Additional file 1: Fig. S9a). We then replaced 16:0/18:1-PS in our assays with 18:0/18:1-PS shown to segregate with cholesterol in vitro [31]. Yet, with LB liposomes containing 0, 30 or even 50% cholesterol, no change was seen in the 18:0/18:1-PS transfer rate in a non-exchange context. If PI(4)P was present in LB liposomes, PS was transferred more rapidly but at a slightly lesser extent when LB liposomes also contained 50% cholesterol (Fig. 7b,c). This suggests that sterols do not favor PS transfer, possibly as they are dispensable for PS retention. To examine more extensively how cholesterol controls the retention of PS in the complex context of the PM, we used a GFP-C2Lact probe to examine whether the steady-state accumulation of PS in the PM of HeLa cells was impacted when cholesterol was depleted from the PM. This depletion was achieved by treating the cells for 24 h with U18666A, a compound that blocks lysosomal-to-PM sterol movement through the inhibition of Niemann-Pick disease, type C1 (NPC1) protein [31, 47, 48]. Such a treatment lowers sterol levels without provoking the remodeling of the PM that occurs following a faster and acute sterol removal [30]. Using the sterol sensor mCherry-D4, we detected sterol in the PM of untreated cells but not in U18666A-treated cells (Fig. 7d). The same results were obtained with a mCherry-D4H construct (carrying the D434S mutation) even if it can detect lower sterol density [31], confirming that the PM was highly deprived in sterol (Additional file 1: Fig. S9b). However, no change was seen in the distribution of PS, which remained in the PM. This was ascertained by measuring the relative distribution of GFP-C2Lact between the PM and the cytosol, in treated and untreated cells, using Lyn11-FRB-mCherry as a stable PM marker and internal reference (Fig. 7e, Additional file 1: Fig. S9c). We conclude that a high, normal level of sterol in the PM is not critical for PS retention and for PS/PI(4)P exchange. However, because we primarily impacted a NPC1-regulated pool of sterol, and as D4 and D4H probes do not detect sterol below a certain threshold [49], we cannot rule out that a sterol pool, inaccessible to our depletion procedure, contributes to stabilizing PS in the PM.