Abstract
Recent studies using two cholesterol-binding bacterial toxin proteins, perfringolysin O (PFO) and domain 4 of anthrolysin O (ALOD4), have shown that cholesterol in the plasma membranes (PMs) of animal cells resides in three distinct pools. The first pool comprises mobile cholesterol, accessible to both PFO and ALOD4, that is rapidly transported to the endoplasmic reticulum (ER) to signal cholesterol excess and maintain cholesterol homeostasis. The second is a sphingomyelin (SM)-sequestered pool inaccessible to PFO and ALOD4 but that becomes accessible by treatment with SM-degrading sphingomyelinase (SMase). The third is an essential pool also inaccessible to PFO and ALOD4 that cannot be liberated by SMase treatment. The accessible cholesterol pool can be trapped on PMs of live cells by nonlytic ALOD4, blocking its transport to the ER. However, studies of the two other pools have been hampered by a lack of available tools. Here, we used ostreolysin A (OlyA), which specifically binds SM/cholesterol complexes in membranes, to study the SM-sequestered cholesterol pool. Binding of nonlytic OlyA to SM/cholesterol complexes in PMs of live cells depleted the accessible PM cholesterol pool detectable by ALOD4. Consequently, transport of accessible cholesterol from PM to ER ceased, thereby activating SREBP transcription factors and increasing cholesterol synthesis. Thus, OlyA and ALOD4 both control movement of PM cholesterol, but through different lipid-binding mechanisms. We also found that PM-bound OlyA was rapidly internalized into cells, whereas PM-bound ALOD4 remained on the cell surface. Our findings establish OlyA and ALOD4 as complementary tools to investigate cellular cholesterol transport.
Highlights
Recent studies using two cholesterol-binding bacterial toxin proteins, perfringolysin O (PFO) and domain 4 of anthrolysin O (ALOD4), have shown that cholesterol in the plasma membranes (PMs) of animal cells resides in three distinct pools
Pleurotolysin B (PlyB), a co-factor that is recruited by membrane-bound ostreolysin A (OlyA) to form oligomeric pores in cells [20, 22], did not cause cell lysis when added alone (Fig. 1C, lane 5); addition of both PlyB and OlyA resulted in complete lysis (Fig. 1C, lane 6)
Trated in Fig. 8, cholesterol in the PMs of LDL-treated cells is divided into three distinct pools: (i) an accessible pool that is mobile and transports to the endoplasmic reticulum (ER) to signal cholesterol sufficiency; (ii) a SM-sequestered pool that does not transport to the ER but can be liberated for transport by SMase; and (iii) an essential pool bound to other PM factors that does not transport to the ER and cannot be released by SMase
Summary
We overexpressed and purified recombinant His6-tagged versions of both ALOD4 and OlyA, as described under “Experimental procedures.” The homogeneity of the purified proteins was confirmed by Coomassie staining (Fig. 1A). Addition of ALOD4 and OlyA at concentrations of 30 M did not result in any cell lysis (Fig. 1C, lanes 2 and 3). We assessed the lipid specificities of nonlytic ALOD4 and OlyA for binding to CHO-K1 cells at 37 °C For these experiments, cells were set up in lipoprotein-rich FCS. Mass spectrometry analysis showed that SMase treatment reduced overall cellular SM content by 76% (Fig. 2B). We examined their binding dependence on cholesterol by treating cells with hydroxypropyl--cyclodextrin (HPCD), a reagent that extracts cholesterol from membranes (Fig. 2C). When treated with increasing concentrations of HPCD, binding of ALOD4 declined sharply and was completely eliminated after treatment with 1% HPCD (Fig. 2C, lanes 1– 6).
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
