Our examination of the cholesterol dependence of PFO binding to and interaction with membranes has provided insights into the mechanisms by which PFO accomplishes the initial cholesterol recognition step during pore formation. By applying a number of different experimental approaches, we were able to assess several different possibilities for the nature of the PFO–cholesterol interaction and ultimately identify the most important factor in determining whether PFO binds to the membrane surface. This factor, the presence of free cholesterol in the membrane, is critical for triggering the PFO–membrane interaction, while the packing of the acyl chains of the phospholipids and the nature of the phospholipids in the bilayer are only important to the extent that they influence cholesterol chemical activity or the exposure of cholesterol to the surface (55
This conclusion is based primarily on the epicholesterol data that revealed that the efficacy of PFO binding to the membrane surface was dictated by both the presence of cholesterol and the total sterol content in the bilayer (). PFO binds directly to pure cholesterol (26
), but PFO–membrane recognition and binding requires PFO to access cholesterol that is membrane-embedded, yet surface-exposed. Cholesterol interacts more favorally with saturated acyl chain lipids than with unsaturated acyl chains lipids (ref 41
and references cited therein), and recent studies with monolayers and bilayers have demonstrated the existence of cholesterol-enriched regions in membranes whose formation is heavily dependent on the degree of phospholipid acyl chain unsaturation, head-group structure, and acyl chain length (56
). Since epicholesterol has similar membrane packing characteristics as cholesterol (60
), but is not functional in PFO pore formation [, lane A; (26
)], the ability to partially replace cholesterol with epicholesterol without blocking PFO binding and oligomerization shows that some sterols in the membrane associate with phospholipids while others are free to interact directly with PFO. Hence, PFO binding was observed to membranes containing as little as 19 mol% cholesterol because the added epicholesterol (, lane C) associates and interacts with phospholipids in the membrane allowing some of the cholesterol molecules to be free to interact with PFO. Epicholesterol apparently intercalates in the bilayer and competes with cholesterol for association with phospholipids, as reported for other membrane intercalating agents (61
). These data therefore reveal that there are at least two distinctive states of cholesterol in a typical membrane bilayer: one in which cholesterol is readily accessible for binding to proteins such as PFO (free cholesterol), and one in which the sterol is associated with surrounding membrane components that reduce its exposure to the surface (e.g., phospholipid head-groups may obscure access to sterols associated with phospholipid acyl chains).
Consistent with our view, recent work done on two other cytolysins that bind cholesterol (streptolysin O and Vibrio cholerae
toxin) indicated that reducing the size of the phospholipid headgroup caused an increase in cholesterol exposure and consequently an increase in the binding of the cytolysins (62
). We have confirmed these observations by replacing some of the choline-containing POPC with ethanolamine-bearing POPE, a phospholipid with the same acyl chains but a smaller headgroup. The addition of POPE reduced the amount of cholesterol required for PFO binding (data not shown). In contrast, the presence of 10 mol% POPS in a POPC/POPS/cholesterol mixture did not affect the binding profile of PFO when cholesterol is varied from 34 to 55 mol%, either in the presence or absence of 2 mM Ca2+
(data not shown).
Similarly, PFO binding requires less cholesterol when the liposomal phospholipids contain unsaturated rather than saturated acyl chains [; (29
)], presumably because acyl chain kinks introduced by the double bonds occupied more space within the bilayer and therefore pushed adjacent sterols out from under the headgroup where they were less accessible to PFO. We also observed that less cholesterol was required for PFO binding when the phospholipid acyl chain length increased up to 18 carbons (). It have been shown that sterol/phospholipid interactions are affected by the hydrophobic mismatch between these two lipids because the hydrophobic “length” of cholesterol is equivalent to the length of a PC with 17 carbon saturated acyl chains (41
). Phospholipid acyl chains with more than 17 carbons must bend to accommodate the shorter cholesterol molecule, and as a consequence the cholesterol molecules are pushed out from under the headgroup and become more exposed to PFO. Thus, the steric demands and interactions of lipid molecules in the bilayer will influence whether or not cholesterol is accessible to PFO. These findings agreed with those reported previously (34
), though no clear conclusions evolved from their more limited investigation.
We also examined other membrane properties that did not correlate with the cholesterol dependence of PFO binding to the membrane. The relative acyl chain packing was one such property, as PFO was just as capable of binding to membranes displaying a low amount of lipid order (i.e., DOPC/cholesterol) as to membranes having a more restricted lipid motion (i.e., DPPC/cholesterol) (). In addition, we also investigated whether PFO had a preference for binding to detergent-resistant or detergent-soluble membranes. Using cold TX-100 as an assay for membrane resistant to solubilization, we found the PFO bound both to DOPC/cholesterol liposomes that were completely soluble in TX-100 and to DPPC/cholesterol liposomes that were insoluble in TX-100 (). These data indicate that the detergent solubility properties of a liposome comprised of cholesterol and a single phospholipid does not correlate with the binding of PFO to the membrane.
However, when we examined more complex lipid mixtures containing DOPC, SM, and a constant amount of cholesterol, we discovered that the ratio of DOPC/SM in the membranes affected PFO binding and oligomerization (). Increasing the SM content of the membranes enhanced their resistant to cold TX-100 solubilization and reduced the ability of PFO to recognize and/or bind to cholesterol. PFO binding to membranes therefore appeared to be negatively influenced by the presence of SM. More importantly, this result suggests that PFO does not recognize or bind to cholesterol that is sequestered in SM-rich microdomains, structures that are commonly associated with the presence of “rafts” in cell membranes (63
Our data therefore conflict with an earlier report that PFO interacts preferentially with cholesterol located in lipid “rafts” in natural membranes (64
). Their conclusions were based on the colocalization of a protease-treated PFO fragment with lipid raft-associated proteins during TX-100 detergent-insolubility assays. In our experience, such an experimental approach is susceptible to multiple interpretations and this may explain the difference between their conclusions and ours. Using the detergent and pelleting conditions reported in their work to examine PFO localization in “rafts”, we found that the majority of the PFO was located in the pellet after treatment with TX-100 when either DOPC- or DPPC-liposomes containing 50 mol% cholesterol were used (data not shown). Since DOPC/cholesterol membranes were soluble in TX-100 (), the PFO in the pellet was not bound to any detergent-resistant membranes. This result demonstrated that the membrane-inserted PFO oligomer was resistant to dissociation by either SDS or TX-100 since oligomeric PFO was found in the pellet. The resistance of the PFO oligomer to TX-100 solubilization may therefore complicate the interpretation of the sedimentation data because PFO oligomers may sediment in the presence of TX-100 even if they were formed on nonraft membrane surfaces. Since our data were obtained with full-length PFO and uniform chemically defined liposomes, we believe that PFO does not necessarily bind to lipid “rafts” in membranes. But we also recognize that the conflicting conclusions may reflect limitations in the TX-100 assay as applied to natural and/or synthetic membranes (47
It is clear from the data presented in this work that the total amount of cholesterol required to trigger PFO binding to a particular membrane is influenced by the lipid composition of the bilayer. Moreover, the total amount of cholesterol required to initiate PFO binding will also be affected by the presence of membrane proteins due to specific lipid binding to, association with, or intercalation into the transmembrane segments of these proteins. We speculate that the latter effect may be the reason that PFO binds to and forms pores in ER membranes (37
) even though their total cholesterol content is very low, because the nonsterol lipid components are more likely to pack within the membrane proteins than are the relatively rigid cholesterol molecules. One effect of this selective partitioning is that the mole fraction of cholesterol in the bulk lipid in the exposed membrane surface area will increase (35
). Whatever the explanation for the sensitivity of ER membranes to PFO, one should be cautious when using PFO or any PFO derivatives to track the total cholesterol content in cellular membranes. PFO binding may depend on the “free cholesterol” or cholesterol chemical activity, rather than on the total amount of cholesterol present in the membrane, and the chemical activity will be influenced by the lipid composition and the presence of other membrane components.
In summary, exposure of free cholesterol at the membrane surface is essential for PFO binding to the bilayer and the initiation of the sequence of events that culminate with the spontaneous formation of a transmembrane pore. Bending of the phospholipids acyl chains by introducing double bonds or reducing the size of the phospholipid head groups decreased the threshold of cholesterol required to trigger binding. In contrast, changes in the relative packing of lipids in the membrane core or the presence or absence of detergent-resistant membranes did not correlate with PFO binding. Finally, addition of molecules that do not interact with PFO, but intercalate into the membrane and displace cholesterol from its association with phospholipids (e.g., epicholesterol), reduced the amount of cholesterol required to trigger PFO binding. Since exposure of free cholesterol at the surface of membranes is necessary for the binding of CDCs and other bacterial and viral proteins (15
), the results reported here may be generally applicable to other protein–membrane interactions involved in human pathogenesis.