Fig 1.
Kinetics of brominated PC exchange between mixed DDM/lipid micelles and SERCA1a solubilized at the same detergent/lipid ratio.
(A) Trace a1, to 2 mL of SR vesicles suspended at 0.04 mg protein/mL (together with 0.02 mg endogenous lipid/mL) in buffer A at 20°C, 0.6 mM EGTA (EG) followed by 0.6 mM CaCl2 (Ca) were first added as controls, to illustrate the classical Ca2+-dependent changes in SERCA1a intrinsic fluorescence as well as the usual down-drift in fluorescence intensity accompanying such measurements (see Materials and Methods). SERCA1a was then solubilized by adding mixed DDM/egg PC micelles (D/L, containing the unbrominated lipid at a ratio of 5 g detergent/g lipid, i.e. ~7 mol/mol) at final concentrations of 0.4 and 0.08 mg/mL for DDM and egg PC, respectively. Mixed micelles with brominated lipids (D/BrL) were then added and this was followed by addition of excess DDM (to reach a final total DDM concentration of 4 mg/mL). For Trace a2, additions were performed first with brominated (D/BrL) and then with unbrominated (D/L) lipid. Traces a3 and a4 correspond to experiments similar to those in a1 and a2, respectively, but now measuring light scattering at 290 nm. (B) In Traces b1 and b2, unbrominated lipid was used but DDM was replaced by 5,6-brominated DDM (BrD) at the same molar concentration. Traces here have not been corrected for dilution effects. This dilution effect only becomes somewhat significant when adding excess DDM at 3.2 mg/mL (32 μL of a 200 mg/mL stock solution resulting in a 1.6% dilution). Numbers indicate the concentrations of detergent and lipid added at each step to the cuvette, in mg/mL. The cartoon on top depicts the principle of the experiment. Lipids are represented with a grey headgroup, detergent molecules are represented with a white headgroup and bromine ions are displayed as black dots. Each trace corresponds to one experiment representative of three independent experiments.
Fig 2.
Kinetics of BrPC exchange in the presence of different amounts of the same mixed micelles, and at various temperatures.
For these experiments, buffer B was used. (A) For Traces a1 and a2, the temperature was 30°C. To SR vesicles at 0.04 mg protein/mL and 0.02 mg endogenous lipid/mL, DDM was initially added at a concentration of either 0.2 mg/mL (Trace a1) or 0.6 mg/mL (Trace a2), resulting in membrane solubilization in both cases. Then BrPC in DDM was added, either 0.08 mg/mL in 0.2 mg/mL (Trace a1) or 0.24 mg/mL in 0.6 mg/mL (Trace a2). The total DDM concentrations therefore were 0.4 or 1.2 mg/mL, but the BrPC/DDM ratio remained the same. At the end, ‘excess’ DDM was added, at 1.2 or 3.6 mg/ml. The final total DDM concentrations therefore were 1.6 or 4.8 mg/mL. Traces a3 and a4, same as a1 and a2 but the temperature was 20°C. Traces a5 and a6, same as a1 and a2 but the temperature was 8°C. Traces have not been corrected for dilution effects. In the case of Traces a2, a4, and a6, this dilution effect only becomes somewhat significant when adding excess DDM at 3.6 mg/mL (36 μL of a 200 mg/mL stock solution resulting in a 1.8% dilution). The cartoon on top depicts the principle of the experiment. Each trace corresponds to one experiment representative of three to four independent experiments. Numbers in panel A indicate the concentrations of detergent and lipid added to the cuvette at each step, in mg/mL. (B) Half-times for BrPC exchange calculated from traces displayed in (A). Note that when experiments were performed at 8°C and in the presence of mixed D/BrL micelles at only a 1X concentration (Trace a5), because of the much slower rate of phospholipid exchange at this temperature, we could not reliably extract rate constants for the exchange process. Data are presented as the mean ± S.D. (error bars) of three to four independent experiments.
Fig 3.
BrPC transfer is greatly accelerated during asymmetrical transfer, as well as in the additional presence of C12E8.
(A) Temperature was 20°C and buffer A was used, as for Fig 1. Each trace corresponds to one experiment representative of three independent experiments. Trace a1 illustrates an experiment similar to that previously shown as Trace a2 of Fig 1 (identical assay conditions but independent experiments). Trace a2 shows a related experiment, where brominated lipid was first added at a higher bromolipid to detergent ratio, as indicated, while subsequently only DDM was added, in two steps, to monitor the kinetics of fluorescence recovery under ‘asymmetrical’ conditions. The histograms on the right represent half-times for BrPC transfer from solubilized SERCA1a to either mixed egg PC/DDM micelles (open bars, as in Trace a1) or pure DDM micelles (grey bars, as in Trace a2). Data are presented as the mean ± S.D. (error bars) of three independent experiments. (B) Temperature was here 8°C and buffer B was used. Each trace corresponds to one experiment representative of three independent experiments. Trace b1 illustrates an experiment similar to that previously shown as Trace a6 of Fig 2 (identical assay conditions but independent experiment). Trace b2 shows a related experiment, where membranes were initially solubilized with C12E8 at 0.6 mg/mL instead of DDM. Then BrPC in DDM was added (at 0.24 mg/mL and 0.6 mg/mL, respectively), as for the left trace. Phospholipid exchange therefore now took place in the presence of 0.24 mg/mL BrPC, 0.6 mg/mL DDM and 0.6 mg/mL C12E8 instead of 0.24 mg/mL BrPC and 1.2 mg/mL DDM. Since detergent itself is thought to exchange rapidly, DDM and C12E8 presumably soon exchanged with each other, hence the cartoon on the right representing a mixed ternary micelle and a protein belt with both detergents. In this cartoon, the polar headgroup of C12E8 is represented as triangles. At the end, excess DDM was added at 3.6 mg/ml, so that total DDM and C12E8 concentrations were 4.2 and 0.6 mg/mL, respectively. Numbers indicate the concentrations of detergent and lipid added to the cuvette at each step, in mg/mL. The histograms on the right represent half-times for BrPC transfer to solubilized SERCA1a calculated from traces of the same panel. Data are presented as the mean ± S.D. (error bars) of three independent experiments.
Fig 4.
Slow phospholipid transfer can also be detected by functional measurements.
(A) ATPase activity measurements with SERCA1a. SERCA1a at 2 mg protein/mL was solubilized with mixed micelles at 10 mg/mL DDM and 2 mg/mL BrPC. This solubilized enzyme was diluted 500-fold into either (Trace a1) buffer A to which a total of 1 mg/mL DDM and 0.2 mg/mL BrPC had been added (and excess DDM at 9 mg/mL was added at the end), or (Trace a2) buffer A to which 1 mg/mL DDM only had been added. In the latter case, mixed micelles were added after some time, resulting in final concentrations of detergent and lipid twice higher than those in Trace a1 (2 mg/mL DDM and 0.4 mg/mL BrPC), but at the same detergent/lipid ratio. ATPase activity was measured at 20°C. The cartoon on top depicts the principle of the experiment. Lipids and detergent molecules are represented with grey and white headgroups, respectively. (B) ATPase activity measurements with purified and chymotrypsin-treated Drs2p/Cdc50p complex. To be able to reveal the ATPase activity of the complex using the enzyme-coupled assay, we first subjected the purified Drs2p/Cdc50p to limited proteolysis with chymotrypsin in the presence of DDM, PS and PI4P (see Materials and Methods). This treatment relieves auto-inhibition of Drs2p/Cdc50p, increasing significantly its ATP hydrolysis rate (manuscript in preparation). The protein concentration of the purified and proteolyzed sample was 0.12 mg/mL. After dilution, its ATPase activity was assayed at 30°C in buffer B, supplemented with 20% glycerol as well as with 0.8 mg/mL DDM and 0.06 mg/mL POPS. For Trace b1, this medium was also supplemented with 0.025 mg/mL PI4P and with 0.25 mg/mL DDM, and the chymotrypsin-treated Drs2p/Cdc50p sample was then added, with 180-fold dilution. For Trace b2, the Drs2p/Cdc50p sample was directly added, and 0.025 mg/mL PI4P together with 0.25 mg/mL DDM was only added after a few tens of minutes. For Trace b3, after adding the Drs2p/Cdc50p sample, 0.025 mg/mL egg PC (PC) was first added together with 0.25 mg/mL DDM, and 0.025 mg/mL PI4P together with 0.25 mg/mL DDM was subsequently added. The cartoon on the left depicts the principle of the experiment illustrated by Trace b1 while the cartoon on the right depicts the principle of the experiments illustrated by Traces b2 and b3. PI4P is represented with a black headgroup while POPS is represented with a grey headgroup. Numbers at the edge of dotted lines represent the specific activity (in μmol.min-1.mg-1) calculated from the slopes of the corresponding traces. Each trace corresponds to one experiment representative of two to three independent experiments.