Determination of protein-mediated intervesicular phospholipid transfer
by time-resolved measurements
Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama
Minoru Nakano
1. Introduction
Bilayer structure of biomembranes consists of several thousand of lipids, and the composition differs at
each organelle. Since most lipids are synthesized in the endoplasmic reticulum (ER), the subsequent distribution
to each organelle determines the composition and function of the biomembranes (J Lipid Res 1981, 22, 391), andlipid transfer proteins are members that take the role. Among yeast proteins, Sec14 is known to transfer
phosphatidylcholine (PC) and phosphatidylinositol (PI) (Nature 1990, 347, 56). However, the mechanism bywhich this protein transfers lipids between membranes has not been discussed in detail based on quantitative
analysis.
We have utilized time-resolved small-angle neutron scattering (TR-SANS) for determination of the
spontaneous phospholipid transfer between vesicles (Phys. Rev. Lett. 2007, 98, 238101). This technique takes
advantage of the large difference in the scattering length density between hydrogenated and deuterated
phospholipids. Exchange of these lipids between vesicles brings about a decrease in the scattering intensity. By
this method, we have reported that 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), represents extremely slow
intervesicular exchange dynamics (t1/2 ~ 90 h), and that methyl-β-cyclodextrin (MβCD) can mediate andaccelerate the lipid exchange (J. Phys. Chem. B 2009, 113, 6745). Based on these findings, we have got an ideathat TR-SANS can be used to determine phospholipid transfer activity of proteins using the proteins instead ofMβCD. Hence, in this study we carried out the TR-SANS experiments for quantitative analysis of Sec14-mediatedtransfer of phospholipids.
2. ExperimentLarge

unilamellarvesicles(diameter~120nm)

consistingof90%POPC

and

10%

1-palmitoyl-2-oleoylphosphatidylserine (POPS) or 90% d31-POPC and 10% POPS (denoted as H-LUV and
D-LUV, respectively) were prepared. TR-SANS experiments were performed at J-PARC BL15 TAIKAN.
Measurements were started immediately after mixing several concentrations of Sec14 to mixtures of D-LUV (15mM) and H-LUV (15 mM) in Tris buffer containing 30% D2O at 37 °C. The D2O volume fraction of 30% wassettled on so that the solvent has the intermediate value of the scattering length density between that of D-LUVand H-LUV. Scattering data collected for every one minute were processed including conversion to I-Q data,subtraction for the solvent scattering, and integration of I(Q) between 0.007 < Q < 0.011 Å-1 to obtain the total
scattering intensity. The normalized contrast, Δρ(t)/Δρ(0), is calculated by Δρ(t)/Δρ(0) = {I(t)/I(0)}0.5, where I(0)and I(t) are the total scattering intensity at time 0 and t, respectively, after the protein’s mixing. Because POPC
does not flip spontaneously (J. Phys. Chem. B 2009, 113, 6745), reduction in the scattering intensity is solely
ascribed to the protein-mediated intervesicular exchange of POPC. Because only POPC (but not POPS) in D-LUV
is deuterated, transfer of POPS, even if it occurs, does not change the scattering intensity and, thus, only the POPC
transfer is detected in this study.
3. Results
The normalized contrasts, calculated from the SANS intensities, were plotted against time after the1

addition of Sec14 into the mixture of D- and H-LUVs (Figure 1). Decay in the normalized contrast was observed
depending on the Sec14 concentration, suggesting the protein-mediated POPC transfer. The decay curves could
not be fitted by a single-exponential function, although the reason is unknown. Thus, we used adouble-exponential function with a constant term of 0.6, which is the value obtained for the mixture of D- andH-LUVs incubated with MβCD for 6 h. Initial rates of the transfer were calculated by the differential of the fitting
curve at t = 0, and were plotted against the protein concentration in Figure 2. The transfer rate of POPC in thepresence of 10% POPS was significantly faster than that in the absence of POPS, which was previously measured
at SANS-U using 100% POPC vesicles. Since lipid transfer is caused by the proteins that are on the membranes,
the initial rate (y-axis of Figure 2) should be proportional to the amount of Sec14 binding to the membranes. Thus,
the dissociation constant (KD) could be figured out to be 262 µg/mL (7.5 µM) and 116 µg/mL (3.3 µM) for POPC
membranes with and without 10% POPS, respectively. In addition, from the initial slope of the curves in Figure 2
the maximum transfer rate per protein could be calculated, which was 0.88 or 3.79 PC･protein–1･s–1 in theabsence or presence of POPS, respectively.

1Sec14(µg/mL)
10254050
100150300

Contrast

0.90.8
0.7
0.6
0.5〜〜

101001000

Time (min)Figure 1. Temporal changes in the normalized
contrasts of POPC/POPS (9:1) vesicles withdifferent concentrations of Sec14.

Figure 2. Initial POPC exchange rates for POPC
and POPC/POPS (9:1) vesicles with different
concentrations of Sec14. Open circles are the datapreviously obtained at SANS-U.

4. Conclusion
PC transfer activity of Sec14 could be detected by TR-SANS. The PC transfer rate per protein wasincreased 4.3-fold (from 0.88 to 3.79 PC･protein–1･s–1) by the incorporation of 10% of POPS into POPC vesicles.
Because the binding affinity (1/KD) was not increased by the incorporation of POPS, this acidic lipid would raise
the PC transfer activity not by increasing membrane-binding rate but by enhancing lipid-exchanging process of
Sec14 on membranes.

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