Transmembrane protein (perfringolysin o) association with ordered membrane domains (rafts) depends upon the raft-associating properties of protein-bound sterol

Abstract

Because transmembrane (TM) protein localization, or nonlocalization, in ordered membrane domains (rafts) is a key to understanding membrane domain function, it is important to define the origin of protein-raft interaction. One hypothesis is that a tight noncovalent attachment of TM proteins to lipids that have a strong affinity for ordered domains can be sufficient to induce raft-protein interaction. The sterol-binding protein perfringolysin O (PFO) was used to test this hypothesis. PFO binds both to sterols that tend to localize in ordered domains (e.g., cholesterol), and to those that do not (e.g., coprostanol), but it does not bind to epicholesterol, a raft-promoting 3α-OH sterol. Using a fluorescence resonance energy transfer assay in model membrane vesicles containing coexisting ordered and disordered lipid domains, both TM and non-TM forms of PFO were found to concentrate in ordered domains in vesicles containing high and low-Tm lipids plus cholesterol or 1:1 (mol/mol) cholesterol/epicholesterol, whereas they concentrate in disordered domains in vesicles containing high-Tm and low-Tm lipids plus 1:1 (mol/mol) coprostanol/epicholesterol. Combined with previous studies this behavior indicates that TM protein association with ordered domains is dependent upon both the association of the protein-bound sterol with ordered domains and hydrophobic match between TM segments and rafts.

Figure 1

PFO interaction with vesicles composed of DSPC/DMoPC/sterol. Trp fluorescence (A) of 55 nM PFO and acrylodan fluorescence (B) of 25 nM acrylodan-labeled PFO interacting with MLVs (500 μM total lipid) composed of 1:1 DSPC/DMoPC with different amounts of cholesterol (●), coprostanol (), epicholesterol (), 1:1 cholesterol/epicholesterol (▾), or 1:1 coprostanol/epicholesterol (♢) were measured in PBS pH 5.1. Average (mean) values and SD values from triplicates are shown. Error bars are not shown where they are too small to illustrate.

Figure 2

Detection of domain formation in vesicles composed of DSPC/DMoPC/sterol by FRET. MLVs (500 μM total lipid) composed of DMoPC with 45 mol % cholesterol (), DMoPC with 45 mol % 1:1 coprostanol/epicholesterol (), 1:1 DSPC/DMoPC with 45 mol % cholesterol (▪), 1:1 DSPC/DMoPC with 45 mol % 1:1 cholesterol/epicholesterol (), or 1:1 DSPC/DMoPC with 45 mol % 1:1 coprostanol/epicholesterol (♦) were prepared in PBS pH 5.1. F samples contained both FRET donor (0.05 mol % pyrene-DPPE) and FRET acceptor (2 mol % Rho-DOPE). Fo samples only contained FRET donor (0.05 mol % pyrene-DPPE). The ratio of donor fluorescence in the presence of acceptor to that in its absence (F/Fo) is graphed. Average (mean) values and SD values from triplicates are shown. Abbreviations: chol = cholesterol; epichol = epicholesterol; and cop = coprostanol.

Figure 3

Fluorescence micrographs of GUVs show formation of coexisting Lo and Ld domains in the presence of epicholesterol and epicholesterol plus coprostanol. (A) GUVs composed of high-T_m lipid, low-T_m lipid, and epicholesterol: (a) 1:1 DSPC/DMoPC with 22.5 mol % epicholesterol; (b) 1:1 SM/DMoPC with 22.5 mol % epicholesterol; (c) 1:1 DSPC/DPhPC with 20 mol % epicholesterol; and (d) 1:1 SM/DPhPC with 20 mol % epicholesterol. The scale bar is 5 μm. Dashed white line is added to help visualize vesicle perimeter. (B) Two representative micrographs of GUVs composed of 1:1 DSPC/DPhPC with 10 mol % coprostanol and 20 mol % epicholesterol. The scale bar for the micrograph on the left is 10 μm and on the right is 5 μm. Abbreviation: epichol = epicholesterol. Vesicles in (A) and (B) were labeled with 0.02 mol % Ld-marker Rho-DOPE. To see this figure in color, go online.

Figure 4

FRET-detected raft affinity of PFO in DSPC/DMoPC/sterol vesicles forming coexisting Lo and Ld domains. Raft affinity of LW peptide, CT-B, and PFO in MLVs (500 μM total lipid) composed of 1:1 DSPC/DMoPC with 45 mol % cholesterol (black bars), 45 mol % 1:1 cholesterol/epicholesterol (gray bars), or 45 mol % 1:1 coprostanol/epicholesterol (striped bars) was measured in PBS pH 5.1. 2 mol % NBD-DPhPE was used as the FRET acceptor. The C_LoLd/C_Ld ratio represents the average local acceptor concentration of acceptor around the donor (protein) in vesicles containing Lo and Ld domains (C_LoLd) relative to that in a homogenous bilayer (DMoPC with 45 mol % sterol) lacking domains (C_Ld). C_LoLd/C_Ld is high for a protein in Ld domains and low for a protein in Lo domains. The actual FRET (F/Fo) data are shown in Fig. S3. For vesicles containing a mixture of coprostanol and epicholesterol, in which PFO binding is not complete, corrected C_LoLd/C_Ld values are shown (see Fig. S4 and Materials and Methods). Uncorrected F/Fo and C_LoLd/C_Ld data are shown in Fig. S5. Average (mean) values and SD values from triplicates are shown.

Figure 5

FRET-detected raft affinity of PFO in vesicles containing various lipid compositions forming coexisting Lo and Ld domains. (A) Raft affinity of LW peptide, CT-B, and PFO in MLVs (500 μM total lipid) composed of 1:1 SM/DMoPC with 45 mol % cholesterol (black bars) or 45 mol % 1:1 coprostanol/epicholesterol (striped bars). (B) Raft affinity of LW peptide, CT-B, and PFO in MLVs (500 μM total lipid) composed of 1:1 DSPC/DPhPC with 40 mol % cholesterol (black bars) or 40 mol % 1:1 coprostanol/epicholesterol (striped bars). (C) Raft affinity of LW peptide, CT-B, and PFO in MLVs (500 μM total lipid) composed of 1:1 SM/DPhPC with 40 mol % cholesterol (black bars) or 40 mol % 1:1 coprostanol/epicholesterol (striped bars). Samples were prepared in PBS pH 5.1. 2 mol % NBD-DPhPE was used as the FRET acceptor. The C_LoLd/C_Ld ratio represents the average local acceptor concentration of acceptor around the donor (protein) in vesicles containing Lo and Ld domains (C_LoLd) relative to that in a homogenous bilayer (DMoPC with 40 mol % or 45 mol % sterol) lacking domains (C_Ld). (We could not use DPhPC/sterol for the experiments in (B) and (C) because we could not make MLVs of DPhPC with a high sterol concentration.) The actual FRET (F/Fo) data are shown in Fig. S3. For vesicles containing a mixture of coprostanol and epicholesterol, in which PFO binding is not complete, corrected C_LoLd/C_Ld values are shown (see Fig. S4 and Materials and Methods). Uncorrected F/Fo and C_LoLd/C_Ld data are shown in Fig. S5. Average (mean) values and SD values from triplicates are shown.