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Detection of cholesterol-rich microdomains in the inner leaflet of the plasma membrane
BBRC Biochemical and Biophysical Research Communications 351 (2006) 713–718 www.elsevier.com/locate/ybbrc
Detection of cholesterol-rich microdomains in the inner leaflet of the plasma membrane Masami Hayashi, Yukiko Shimada, Mitsushi Inomata, Yoshiko Ohno-Iwashita
*
Cellular Signaling Group, Research Team for Functional Genomics, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan Received 13 October 2006 Available online 26 October 2006
Abstract The C-terminal domain (D4) of perfringolysin O binds selectively to cholesterol in cholesterol-rich microdomains. To address the issue of whether cholesterol-rich microdomains exist in the inner leaflet of the plasma membrane, we expressed D4 as a fusion protein with EGFP in MEF cells. More than half of the EGFP-D4 expressed in stable cell clones was bound to membranes in raft fractions. Depletion of membrane cholesterol with b-cyclodextrin reduced the amount of EGFP-D4 localized in raft fractions, confirming EGFP-D4 binding to cholesterol-rich microdomains. Subfractionation of the raft fractions showed most of the EGFP-D4 bound to the plasma membrane rather than to intracellular membranes. Taken together, these results strongly suggest the existence of cholesterol-rich microdomains in the inner leaflet of the plasma membrane. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Lipid raft; Cholesterol; Plasma membrane; Inner leaflet; GFP-fusion protein; Perfringolysin O; Mouse fibroblast
Accumulating evidence suggests that functional lipid microdomains, termed lipid rafts, exist in the plasma membrane [1–3]. Specific proteins, including caveolins, flotillins, and Src-family kinases, assemble in these microdomains where they play a role in signal transduction and many other cellular events [2–4]. Cholesterol has been suggested to play an essential role in the structural maintenance and function of lipid rafts, since changes in cholesterol content affect the localization of proteins associated with lipid rafts, and thus affect their function [2–4]. Attractive forces between cholesterol and sphingolipids induce the formation of liquid-ordered (lo) domains, segregated from bulk unsaturated glycerophospholipids [1]. These segregated lo domains are thought to be the physical basis for lipid rafts [1,2]. It is generally assumed that lipid rafts exist in both the outer and inner leaflets of the plasma membrane. It is commonly accepted that outer leaflet rafts
*
Corresponding author. Fax: +81 3 3579 4776. E-mail address: [email protected] (Y. Ohno-Iwashita).
0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.10.088
are enriched in cholesterol and sphingolipids [1–3,5]; however, such microdomains cannot be mirrored in the inner leaflet because sphingolipids are mainly concentrated in the outer leaflet. The coupling of raft domains at the outer and inner leaflets is thought to be important for signal transduction via lipid rafts [2,4,6,7]. Nevertheless, little is known about lipid rafts in the inner leaflet due to the lack of a suitable detection method. We have been attempting to detect cholesterol-rich microdomains in the inner leaflet by expressing Domain 4 (D4) of h-toxin, a protein targeting these microdomains, inside cells. h-Toxin (perfringolysin O) is a cholesterolbinding, pore-forming cytolysin produced by Clostridium perfringens [8–10]. h-Toxin and its non-cytolytic derivatives bind to membranes with high cholesterol contents, but scarcely bind to liposomes containing less than 20 mol% cholesterol [8,11] nor to cell membranes when cell cholesterol is depleted by more than 30% [8,12,13], indicating their selective binding to cholesterol-enriched membranes. D4, the C-terminal domain (110 a.a.), is the smallest functional unit that has the same cholesterol-binding activity as
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the full-size toxin [12]. We prepared recombinant D4 and EGFP-tagged D4 (EGFP-D4) by overexpression in Escherichia coli, and demonstrated that cell-bound D4 was predominantly recovered in detergent-insoluble low-density membrane fractions (raft fractions) where cholesterol and other raft marker molecules are enriched [8,12]. In addition to D4, we previously established another non-cytolytic derivative of h-toxin, named BCh, as a probe to detect cholesterol in cholesterol-rich microdomains on the cell surface [8,13–16]. Recently it was revealed that BCh is useful for isolating plasma membrane-derived, cholesterol-rich membrane vesicles from raft fractions [16]. In this study, we expressed EGFP-D4 inside MEF cells, and demonstrate that the expressed EGFP-D4 is bound to the inner side of the plasma membrane. It has been reported that upon interaction with membranes, rather than spanning the membrane bilayer, only the tip of D4 is shallowly embedded in the non-polar interior of the bilayer [10], indicating that D4 primarily recognizes cholesterol not in both leaflets of the bilayer, but in just one leaflet facing the toxin. Thus it is expected that D4, if expressed inside cells, could capture cholesterol in the inner leaflet of the plasma membrane. Our finding strongly suggests the existence of cholesterol-enriched microdomains in the inner leaflet of the plasma membrane. Materials and methods Materials. A rabbit antibody specific to the C-terminus of D4, which corresponds to the C-terminus of EGFP-D4, was produced as described [12]. Anti-GFP (clone JL-8) was purchased from BD Bioscience (San Jose, CA, USA). Anti-caveolin-1 was from Transduction Laboratories (Lexington, KY, USA). Anti-flotillin-1 was from IBL (Takasaki, Japan). Phenylmethylsulfonyl fluoride (PMSF), aprotinin, and pepstatin A were from Sigma–Aldrich (St. Louis, MO, USA). Leupeptin was from Peptide Institute (Osaka, Japan). Dynabeads M-280 conjugated with streptavidin were purchased from Dynal (Oslo, Norway). BCh was prepared by biotinylation of protease-nicked h-toxin as described previously [14]. Plasmid construction. DNA constructs were prepared to express a fusion protein of EGFP and D4. Since the C-terminus of D4 is essential for maintaining the protein structure and thus for binding to cholesterol [12], EGFP was fused not to the C-terminus but to the N-terminus of D4 so that the fusion protein retained cholesterol-binding activity. A DNA fragment containing the EGFP-encoding region was amplified from pEGFP-N3 (Clontech) by the polymerase chain reaction with forward primer A (5 0 -CGTTCTAGAGCCACCATGGTGAGCAAGGGCGAG3 0 ) and reverse primer B (5 0 -ATCTACGTCGGCTAGCCTTGTACAG CTCGTCCATGCCGAG-3 0 ). The fragment was then ligated into the NheI site of a mammalian expression vector pTRE2hyg (Clontech), and served as an EGFP expression plasmid, pTRE2hyg-EGFP. A DNA fragment containing the D4-encoding region was prepared by digesting a plasmid encoding His-tag-D4 [12] with NheI and SalI. Then the fragment was inserted into the plasmid pTRE2hyg-EGFP using NheI/SalI sites on the 3 0 side of the sequence encoding EGFP, and this served as an EGFPD4 expression plasmid, pTRE2hyg-EGFP-D4. Constructs were confirmed by sequencing before use. Isolation of inducible MEF cell clones. A Swiss mouse embryonic fibroblast (3T3)-derived cell line (MEF Tet-Off cell line) that expresses the tetracycline-controlled transactivator was purchased from Clontech. MEF Tet-Off cells were cultured in DMEM supplemented with 10% FBS and antibiotics (50 lg/ml streptomycin, 50 U/ml penicillin, and 100 lg/ml G418). The pTRE2hyg-EGFP-D4 construct was transfected into MEF
Tet-Off cells, using Lipofectamine (Invitrogen). For transient expression, cells were used for experiments 24–48 h after transfection. To generate stable transfectants, the transfected cells were selected in the presence of the antibiotics, 350 lg/ml hygromycin, and 1 lg/ml doxycycline. Single colonies were isolated and cells were grown and tested for the expression of EGFP-D4 upon induction. The pTRE2hyg-EGFP construct was transfected into MEF Tet-Off cells and single colonies were isolated as described above to serve as control cell clones expressing EGFP. Fluorescence microscopy. Transfected MEF Tet-Off cells were cultured in poly-d-lysine-coated glass-bottom dishes (Mat Tek Corp., Ashland, MA, USA) for 24 h after induction, washed with phosphate-buffered saline (PBS), and fixed with 4% formaldehyde (Polysciences, Inc.) for 10 min. The EGFP fluorescence of the fixed cells was monitored with an inverted fluorescence microscope (IX-70, Olympus, Japan) equipped with a digital camera (Penguin 600CL, Pixera Corp, Los Gatos, CA, USA). Preparation of detergent-insoluble, low-density membrane fractions (raft fractions). MEF Tet-Off cells were washed with PBS, incubated with 1% Triton X-100 in TN buffer (25 mM Tris–HCl, pH 6.8, 150 mM NaCl) containing 1 mM PMSF, 1 mM leupeptin, 20 lM pepstatin A, and 20 lg/ml aprotinin for 15 min on ice, and homogenized. The homogenate was fractionated by sucrose-density gradient centrifugation as described [16]. After centrifugation, a total of 11 fractions were collected from the tops of the tubes. The pellet was suspended in TN buffer, sonicated, and designated as P. In some experiments MEF Tet-Off cells were incubated with 10 lg/ml BCh in PBS containing 1 mg/ml BSA for 30 min on ice, and washed twice with PBS prior to the incubation with Triton X-100. Isolation of a BCh-bound membrane subpopulation from raft fractions. The BCh-bound membrane subpopulation was prepared from raft fractions as described previously [16]. Briefly, BCh was first bound to the plasma membrane surface of EGFP-D4-expressing MEF cells, and then raft fractions were prepared as described above. The raft fractions were pooled and mixed with avidin-conjugated magnet beads on ice. Membrane vesicles bound to BCh were retained with avidin-magnet beads via biotinavidin interaction and separated from BCh-unbound vesicles that were recovered in the bead-unbound fraction. Detection of proteins and lipids. Proteins were analyzed using a bicinchoninic acid (BCA) protein assay kit (Pierce). To analyze the distributions of proteins in the gradient, equal volumes of each fraction of the gradient were loaded onto a gel and SDS–PAGE was performed. The molecules in the gels were transferred to PVDF membranes and visualized using ECL plus (Amersham Bioscience). EGFP-D4 was detected with anti-GFP and/or antibody against the h-toxin C-terminal peptide [12]. BCh was detected with anti-h-toxin [14]. Lipids were extracted by the method of Bligh and Dyer [17] with slight modification. Cholesterol was quantified using an assay kit for free cholesterol, Determiner L FC (Kyowa Medex, Japan).
Results Isolation of cell clones stably expressing EGFP-D4 MEF Tet-Off cells were transfected with a plasmid encoding an EGFP-D4 fusion protein, and several cell clones were isolated that expressed the full-size EGFP-D4 with no obvious degradation products upon induction by removal of doxycycline. The expressed protein was detected as a band with the expected molecular size by immunoblotting with anti-GFP and anti-D4-C-terminus (Fig. 1). The growth rates of the cell clones were almost the same under conditions both with and without EGFP-D4 induction (data not shown), indicating that the expressed EGFPD4 does not affect cell growth, at least not at the expression level. However, since the expression levels of EGFP-D4 in these stable cell clones are lower than those in transient
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compels them to localize in non-raft fractions. Rather the result suggests that EGFP-D4 molecules in non-raft fractions might have lost their capacity to bind to raft membranes. To determine whether the high cholesterol content of lipid rafts is required for the binding of EGFP-D4, we depleted cholesterol from EGFP-D4-expressing cells and analyzed the distribution profile of EGFP-D4 (Fig. 3B). When cells were incubated with 20 mM 2-hydroxypropylb-cyclodextrin for 15 min at 37 °C, cholesterol was depleted by ca. 47% from the cells. The treatment caused a significant reduction in the amount of EGFP-D4 recovered in raft fractions (Fig. 3B), indicating its cholesterol-dependent binding to lipid rafts.
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Fig. 1. Expression of EGFP-D4 in MEF cells. Expression patterns of EGFP-D4 and EGFP in stable transfectants were analyzed by Western blotting with anti-GFP and anti-D4-C-terminus and compared with the patterns of cells expressing EGFP-D4 and EGFP transiently. Arrowheads on the right side show the expected migration position of full-size EGFPD4.
transfectants (Fig. 1), higher level of constitutive expression of EGFP-D4 may be toxic for the cells. Upon induction, the expressed EGFP-D4 exhibited a different pattern in fluorescence microscopy from EGFP expressed in a control cell clone (Fig. 2). EGFP-D4 shows an expression pattern suggesting localization at the plasma membrane and intracellular vesicles and in cytoplasm. On the other hand, EGFP exhibited localized only in the cytoplasm. Cholesterol-dependent localization of EGFP-D4 in lipid rafts The clones were characterized further to examine whether the fusion protein is localized in lipid rafts. After induction, the cells were grown for 24 h, and then cell lysates were prepared in the presence of Triton X-100 and fractionated by sucrose-density gradient centrifugation (Fig. 3A). More than half of the EGFP-D4 was recovered in raft fractions (fr.#3–5) where raft marker proteins, flotillin-1 and caveolin-1, were localized (Fig. 3A). In contrast EGFP expressed in a control cell clone was never recovered in raft fractions (Fig. 3A), indicating that the D4 portion of EGFP-D4 determines its localization in lipid rafts. The rest of EGFP-D4 was recovered in non-raft fractions (fr.#10–11). EGFP-D4 molecules in non-raft fractions never redistributed to raft fractions even after incubation with newly added raft membranes from EGFP-D4-non-expressing MEF cells (data not shown). Therefore, it is unlikely that a shortage of raft membranes
EGFP-D4 binds to cholesterol-rich microdomains in the inner leaflet of the plasma membrane It is expected that EGFP-D4 molecules recovered in raft fractions are bound to cholesterol-rich microdomains in the inner leaflet of the plasma membrane and/or in intracellular organelle/vesicular membranes. We examined the proportion of EGFP-D4 bound to the plasma membrane of that recovered in raft fractions. We previously isolated plasma membrane-derived, cholesterol-rich membranes from raft fractions prepared from BCh-pre-bound Jurkat cells [16]. This procedure was applied to subfractionate the raft fractions of EGFP-D4-expressing cells. Prior to the subfractionation, we examined whether BCh binding to the cell surface affects the binding of expressed EGFPD4 to raft membranes inside cells. As a result, the amount of EGFP-D4 recovered in raft fractions of BCh-bound cells was almost the same as that of BCh-unbound cells (Fig. 4A). Since it was confirmed that BCh binding to the cell surface does not affect EGFP-D4 recovery in raft fractions, we then carried out subfractionation experiments. Raft fractions were prepared from BCh-bound EGFPD4-expressing MEF cells, pooled and subfractionated into avidin-magnet-bead-bound (BCh-bound) and -unbound (BCh-unbound) membranes as described in Materials and methods. When the bead-bound and -unbound fractions were analyzed, most of the EGFP-D4 was recovered in bead-bound fraction, namely in the BCh-bound membrane fraction (Fig. 4B). This is in contrast to the recovery of flotillin and caveolin, which were detected in both BCh-bound and -unbound membrane fractions (Fig. 4B and data not shown). When raft fractions were prepared in the absence of BCh (Fig. 4B, BCh ()), no EGFP-D4 was retained with the magnet beads, indicating that retention by the beads is not due to non-specific adsorption. Since BCh was bound to the cell surface prior to cell fractionation, it is expected that the BCh-bound membranes were derived from the plasma membrane. Therefore, these results indicate that most of the EGFP-D4 recovered in raft fractions is bound to the inner leaflet of the plasma membrane, and not to intracellular membranes. Thus our data clearly
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EGFP-D4
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Fig. 2. Flourescence microscopy of EGFP-D4- and EGFP-expressing cells. MEF Tet-Off cells were grown under inducible (Dox()) and non-inducible (Dox(+)) conditions, fixed, and subjected to fluorescence measurement as described in Materials and methods. (Left) Phase contrast; (Right) fluorescence.
demonstrate the existence of cholesterol-rich microdomains in the inner leaflet of the plasma membrane. Discussion In this study, we constructed an intracellular expression system for EGFP-D4, a probe that recognizes cholesterol in cholesterol-rich microdomains. By using this system, we demonstrate the existence of detergent-resistant, cholesterol-enriched microdomains in the inner leaflet of the plasma membrane, as has been shown for the outer leaflet. Although sphingolipids are mainly concentrated in the outer leaflet, cholesterol is present in both leaflets [18]. Furthermore, it has been reported that high concentrations of
cholesterol result in lo domains not only with sphingolipids but also with other saturated phospholipids segregated from the bulk unsaturated glycerophospholipids [7,18]. Therefore, cholesterol probably forms such domains by interacting with saturated phospholipids other than sphingolipids in the inner leaflet. Considering previous observations that showed the requirement of cholesterol for assembly/coupling of outer and inner rafts and for downstream signaling [2,4,6,7], it is conceivable that these cholesterol-rich microdomains couple between the two leaflets upon cell stimulation and serve as platforms for signaling. It is interesting to note that BCh and EGFP-D4 bind to the same subpopulation of raft membranes from the outside and inside, respectively (Fig. 4B). This indicates
M. Hayashi et al. / Biochemical and Biophysical Research Communications 351 (2006) 713–718
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Fig. 3. Cholesterol-dependent localization of EGFP-D4 in raft fractions. (A) EGFP-D4-expressing cells were treated with 1% Triton X-100, homogenized, and subjected to sucrose-density gradient centrifugation. The distributions of EGFP-D4, flotillin-1, and caveolin-1 were detected with specific antibodies. In parallel experiments, EGFP-expressing cells were also analyzed as a control. (B) Cholesterol depletion reduces EGFP-D4 binding to lipid rafts. EGFPD4-expressing cells were incubated with 0 or 20 mM of 2-hydroxypropyl-b-cyclodextrin (bCD) for 15 min at 37 °C. After washing with PBS, the cell homogenates were fractionated as described in Materials and methods. The distributions of cholesterol (upper) and EGFP-D4 (lower two) were analyzed.
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flotillin-1 Fig. 4. (A) BCh-bound to the outer leaflet of the plasma membrane does not interfere with the binding of expressed EGFP-D4 to lipid rafts. EGFP-D4expressing cells were preincubated in the presence (BCh (+)) or absence (BCh ()) of BCh as described in Materials and methods. After washing, cells were treated with Triton X-100, homogenized, and fractionated by sucrose-density gradient centrifugation. The distributions of EGFP-D4 were detected with anti-GFP. (B) EGFP-D4 was recovered in a BCh-bound subpopulation of lipid rafts. Total raft fractions (T) were prepared from BCh-bound (BCh (+)) and -unbound (BCh ()) EGFP-D4-expressing cells as described in (A), and mixed with avidin-conjugated magnet beads. Bead-bound (B) and -unbound (UB) fractions were collected, and the proteins in each fraction were detected with specific antibodies. Due to the tight binding of biotin with avidin, a majority of the bead-bound BCh was not released from avidin-magnet beads even after boiling in SDS-containing buffer at 100 °C (shown as an asterisk in a gel used for Western blotting with anti-h-toxin) [16,22].
that membranes in the raft subpopulation are rich in cholesterol in both the outer and the inner leaflets. The result also indicates that BCh binding to the outer surface of the plasma membrane does not interfere with the binding of EGFP-D4 to the inner leaflet (Fig. 4A). This observation is consistent with the shallow insertion model of h-toxin [10], where D4 captures cholesterol in only the bilayer leaflet exposed to D4; that is, EGFP-D4 expressed inside cells binds to cholesterol in the inner leaflet, while externally added BCh binds to cholesterol in the outer leaflet.
Most of the EGFP-D4 molecules recovered in raft fractions are bound to the inner leaflet of the plasma membrane (Fig. 4), whereas the localization is unclear for EGFP-D4 species recovered in non-raft fractions. Experiments suggest that these species might have lost their capacity to bind to raft membranes. A specific antibody against the D4 C-terminus shows that the C-terminal region of D4 essential for binding remains intact, suggesting that proteolysis is not a cause for the inactivation. Inactivation might be caused by misfolding of the polypeptides during biosynthesis or by a post-translational modification,
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such as the oxidation of the thiol group in the unique Cys of D4 [19]. Such inactivated species are likely to accumulate in the cytoplasm, consistent with our fluorescence microscopy observation (Fig. 2). We showed that intracellular vesicular structures were also stained with EGFP-D4 in some cells (Fig. 2). EGFPD4 bound to such structures is another candidate for the EGFP-D4 recovered in non-raft fractions. If intracellular rafts are more sensitive to detergent solubilization than plasma membrane rafts, as has been shown for caveolincontaining monolayer membranes at the surface of lipid droplets [20], it is possible that EGFP-D4 species bound to intracellular vesicular structures would be solubilized by Triton X-100 and recovered in non-raft fractions. It has been proposed that intracellular rafts are involved in protein sorting at various sites in the cell [21]. It is possible that the intracellular cholesterol-rich vesicular structures detected by EGFP-D4 are those that participate in intracellular trafficking. Attempts to identify these structures are under way in our laboratory. At present it is not known how rafts in the outer and inner leaflets couple with each other. From recent physico-chemical studies it is argued that the coupling may be transient and occur after receptor activation and oligomerization [7,18]. To approach the issue, methods for detecting dynamic features of lipid rafts are required. In this report, we describe an expression system involving EGFP-D4 inside cells that offers a potential tool for visualizing inner leaflet cholesterol-rich microdomains in live cells. Along with this expression system, we have recently prepared fluorescent dye-conjugated h-toxin derivatives, such as Alexa dye-labeled BCh and D4, as tools for monitoring cell-surface cholesterol-rich microdomains in live cells (Ref. [12] and Y. Shimada and Y. Ohno-Iwashita, unpublished results). A combination of these tools might help to increase understanding of the dynamic behavior and function of lipid rafts in future studies. Acknowledgments We thank Dr. Koichi Suzuki, former Director of the Tokyo Metropolitan Institute of Gerontology, for support and encouragement. We thank Dr. M.M. Dooley-Ohto for reading the manuscript. This work was supported in part by Grants-in-Aid for Science Research from the Japan Society for the Promotion of Science (to M.H. and to Y.O.-I.). References [1] D.A. Brown, E. London, Structure of detergent-resistant membrane domains: does phase separation occur in biological membranes? Biochem. Biophys. Res. Commun. 240 (1997) 1–7. [2] K. Simons, D. Toomre, Lipid rafts and signal transduction, Mol. Cell. Biol. 1 (2000) 31–39.
[3] L.J. Pike, Lipid rafts: bringing order to chaos, J. Lipid Res. 44 (2003) 655–667. [4] T. Harder, P. Scheiffele, P. Verkade, K. Simons, Lipid domain structure of the plasma membrane revealed by patching of membrane components, J. Cell Biol. 141 (1998) 929–942. [5] D.A. Brown, J.K. Rose, Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface, Cell 68 (1992) 533–544. [6] G. Gri, B. Molon, S. Manes, T. Pozzan, A. Viola, The inner side of T cell lipid rafts, Immunol. Lett. 94 (2004) 247–252. [7] W.K. Subczynski, A. Kusumi, Dynamics of raft molecules in the cell and artificial membranes: approaches by pulse EPR spin labeling and single molecule optical microscopy, Biochim. Biophys. Acta 1610 (2003) 231–243. [8] Y. Ohno-Iwashita, Y. Shimada, A.A. Waheed, M. Hayashi, M. Inomata, M. Nakamura, M. Maruya, S. Iwashita, Perfringolysin O, a cholesterol-binding cytolysin, as a probe for lipid rafts, Anaerobe 10 (2004) 125–134. [9] J. Rossjohn, S.C. Feil, W.J. McKinstry, R.K. Tweten, M.W. Parker, Structure of a cholesterol-binding, thiol-activated cytolysin and a model of its membrane form, Cell 89 (1997) 685–692. [10] R. Ramachandran, A.P. Heuck, R.K. Tweten, A.E. Johnson, Structural insights into the membrane-anchoring mechanism of a cholesterol-dependent cytolysin, Nat. Struct. Biol. 9 (2002) 823–827. [11] Y. Ohno-Iwashita, M. Iwamoto, S. Ando, S. Iwashita, Effect of lipidic factors on membrane cholesterol topology—mode of binding of theta-toxin to cholesterol in liposomes, Biochim. Biophys. Acta 1109 (1992) 81–90. [12] Y. Shimada, M. Maruya, S. Iwashita, Y. Ohno-Iwashita, The Cterminal domain of perfringolysin O is an essential cholesterolbinding unit targeting to cholesterol-rich microdomains, Eur. J. Biochem. 269 (2002) 6195–6203. [13] A.A. Waheed, Y. Shimada, H.F.G. Heijinen, M. Nakamura, M. Inomata, M. Hayashi, S. Iwashita, J.W. Slot, Y. Ohno-Iwashita, Selective binding of perfringolysin O derivative to cholesterol-rich membrane microdomains (rafts), Proc. Natl. Acad. Sci. USA 98 (2001) 4926–4931. [14] M. Iwamoto, I. Morita, M. Fukuda, S. Murota, S. Ando, Y. OhnoIwashita, A biotinylated perfringolysin O derivative: a new probe for detection of cell surface cholesterol, Biochim. Biophys. Acta 1327 (1997) 222–230. [15] M. Inomata, Y. Shimada, M. Hayashi, H. Kondo, Y. Ohno-Iwashita, Detachment-associated changes in lipid rafts of senescent human fibroblasts, Biochem. Biophys. Res. Commun. 343 (2006) 489–495. [16] Y. Shimada, M. Inomata, H. Suzuki, M. Hayashi, A.A. Waheed, Y. Ohno-Iwashita, Separation of a cholesterol-enriched microdomain involved in T-cell signal transduction, FEBS J. 272 (2005) 5454–5463. [17] E.G. Bligh, W.J. Dyer, A rapid method of total lipid extraction and purification, Can. J. Biochem. Physiol. 37 (1959) 911–917. [18] P.F. Devaux, R. Morris, Transmembrane asymmetry and lateral domains in biological membranes, Traffic 5 (2004) 241–246. [19] M. Iwamoto, Y. Ohno-Iwashita, S. Ando, Role of the essential thiol group in the thiol-activated cytolysin from Clostridium perfringens, Eur. J. Biochem. 167 (1987) 425–430. [20] K. Tauchi-Sato, S. Ozeki, T. Houjou, R. Taguchi, T. Fujimoto, The surface of lipid droplets is a phospholipid monolayer with a unique fatty acid composition, J. Biol. Chem. 277 (2002) 44507–44512. [21] J.B. Helms, C. Zurzolo, Lipids as targeting signals: lipid rafts and intracellular trafficking, Traffic 5 (2004) 247–254. [22] S. Wada, M. Morishima-Kawashima, Y. Qi, H. Misono, Y. Shimada, Y. Ohno-Iwashita, Y. Ihara, Gamma-secretase activity is present in rafts but is not cholesterol-dependent, Biochemistry 42 (2003) 13977–13986.
Detection of cholesterol-rich microdomains in the inner leaflet of the plasma membrane Masami Hayashi, Yukiko Shimada, Mitsushi Inomata, Yoshiko Ohno-Iwashita
*
Cellular Signaling Group, Research Team for Functional Genomics, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan Received 13 October 2006 Available online 26 October 2006
Abstract The C-terminal domain (D4) of perfringolysin O binds selectively to cholesterol in cholesterol-rich microdomains. To address the issue of whether cholesterol-rich microdomains exist in the inner leaflet of the plasma membrane, we expressed D4 as a fusion protein with EGFP in MEF cells. More than half of the EGFP-D4 expressed in stable cell clones was bound to membranes in raft fractions. Depletion of membrane cholesterol with b-cyclodextrin reduced the amount of EGFP-D4 localized in raft fractions, confirming EGFP-D4 binding to cholesterol-rich microdomains. Subfractionation of the raft fractions showed most of the EGFP-D4 bound to the plasma membrane rather than to intracellular membranes. Taken together, these results strongly suggest the existence of cholesterol-rich microdomains in the inner leaflet of the plasma membrane. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Lipid raft; Cholesterol; Plasma membrane; Inner leaflet; GFP-fusion protein; Perfringolysin O; Mouse fibroblast
Accumulating evidence suggests that functional lipid microdomains, termed lipid rafts, exist in the plasma membrane [1–3]. Specific proteins, including caveolins, flotillins, and Src-family kinases, assemble in these microdomains where they play a role in signal transduction and many other cellular events [2–4]. Cholesterol has been suggested to play an essential role in the structural maintenance and function of lipid rafts, since changes in cholesterol content affect the localization of proteins associated with lipid rafts, and thus affect their function [2–4]. Attractive forces between cholesterol and sphingolipids induce the formation of liquid-ordered (lo) domains, segregated from bulk unsaturated glycerophospholipids [1]. These segregated lo domains are thought to be the physical basis for lipid rafts [1,2]. It is generally assumed that lipid rafts exist in both the outer and inner leaflets of the plasma membrane. It is commonly accepted that outer leaflet rafts
*
Corresponding author. Fax: +81 3 3579 4776. E-mail address: [email protected] (Y. Ohno-Iwashita).
0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.10.088
are enriched in cholesterol and sphingolipids [1–3,5]; however, such microdomains cannot be mirrored in the inner leaflet because sphingolipids are mainly concentrated in the outer leaflet. The coupling of raft domains at the outer and inner leaflets is thought to be important for signal transduction via lipid rafts [2,4,6,7]. Nevertheless, little is known about lipid rafts in the inner leaflet due to the lack of a suitable detection method. We have been attempting to detect cholesterol-rich microdomains in the inner leaflet by expressing Domain 4 (D4) of h-toxin, a protein targeting these microdomains, inside cells. h-Toxin (perfringolysin O) is a cholesterolbinding, pore-forming cytolysin produced by Clostridium perfringens [8–10]. h-Toxin and its non-cytolytic derivatives bind to membranes with high cholesterol contents, but scarcely bind to liposomes containing less than 20 mol% cholesterol [8,11] nor to cell membranes when cell cholesterol is depleted by more than 30% [8,12,13], indicating their selective binding to cholesterol-enriched membranes. D4, the C-terminal domain (110 a.a.), is the smallest functional unit that has the same cholesterol-binding activity as
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the full-size toxin [12]. We prepared recombinant D4 and EGFP-tagged D4 (EGFP-D4) by overexpression in Escherichia coli, and demonstrated that cell-bound D4 was predominantly recovered in detergent-insoluble low-density membrane fractions (raft fractions) where cholesterol and other raft marker molecules are enriched [8,12]. In addition to D4, we previously established another non-cytolytic derivative of h-toxin, named BCh, as a probe to detect cholesterol in cholesterol-rich microdomains on the cell surface [8,13–16]. Recently it was revealed that BCh is useful for isolating plasma membrane-derived, cholesterol-rich membrane vesicles from raft fractions [16]. In this study, we expressed EGFP-D4 inside MEF cells, and demonstrate that the expressed EGFP-D4 is bound to the inner side of the plasma membrane. It has been reported that upon interaction with membranes, rather than spanning the membrane bilayer, only the tip of D4 is shallowly embedded in the non-polar interior of the bilayer [10], indicating that D4 primarily recognizes cholesterol not in both leaflets of the bilayer, but in just one leaflet facing the toxin. Thus it is expected that D4, if expressed inside cells, could capture cholesterol in the inner leaflet of the plasma membrane. Our finding strongly suggests the existence of cholesterol-enriched microdomains in the inner leaflet of the plasma membrane. Materials and methods Materials. A rabbit antibody specific to the C-terminus of D4, which corresponds to the C-terminus of EGFP-D4, was produced as described [12]. Anti-GFP (clone JL-8) was purchased from BD Bioscience (San Jose, CA, USA). Anti-caveolin-1 was from Transduction Laboratories (Lexington, KY, USA). Anti-flotillin-1 was from IBL (Takasaki, Japan). Phenylmethylsulfonyl fluoride (PMSF), aprotinin, and pepstatin A were from Sigma–Aldrich (St. Louis, MO, USA). Leupeptin was from Peptide Institute (Osaka, Japan). Dynabeads M-280 conjugated with streptavidin were purchased from Dynal (Oslo, Norway). BCh was prepared by biotinylation of protease-nicked h-toxin as described previously [14]. Plasmid construction. DNA constructs were prepared to express a fusion protein of EGFP and D4. Since the C-terminus of D4 is essential for maintaining the protein structure and thus for binding to cholesterol [12], EGFP was fused not to the C-terminus but to the N-terminus of D4 so that the fusion protein retained cholesterol-binding activity. A DNA fragment containing the EGFP-encoding region was amplified from pEGFP-N3 (Clontech) by the polymerase chain reaction with forward primer A (5 0 -CGTTCTAGAGCCACCATGGTGAGCAAGGGCGAG3 0 ) and reverse primer B (5 0 -ATCTACGTCGGCTAGCCTTGTACAG CTCGTCCATGCCGAG-3 0 ). The fragment was then ligated into the NheI site of a mammalian expression vector pTRE2hyg (Clontech), and served as an EGFP expression plasmid, pTRE2hyg-EGFP. A DNA fragment containing the D4-encoding region was prepared by digesting a plasmid encoding His-tag-D4 [12] with NheI and SalI. Then the fragment was inserted into the plasmid pTRE2hyg-EGFP using NheI/SalI sites on the 3 0 side of the sequence encoding EGFP, and this served as an EGFPD4 expression plasmid, pTRE2hyg-EGFP-D4. Constructs were confirmed by sequencing before use. Isolation of inducible MEF cell clones. A Swiss mouse embryonic fibroblast (3T3)-derived cell line (MEF Tet-Off cell line) that expresses the tetracycline-controlled transactivator was purchased from Clontech. MEF Tet-Off cells were cultured in DMEM supplemented with 10% FBS and antibiotics (50 lg/ml streptomycin, 50 U/ml penicillin, and 100 lg/ml G418). The pTRE2hyg-EGFP-D4 construct was transfected into MEF
Tet-Off cells, using Lipofectamine (Invitrogen). For transient expression, cells were used for experiments 24–48 h after transfection. To generate stable transfectants, the transfected cells were selected in the presence of the antibiotics, 350 lg/ml hygromycin, and 1 lg/ml doxycycline. Single colonies were isolated and cells were grown and tested for the expression of EGFP-D4 upon induction. The pTRE2hyg-EGFP construct was transfected into MEF Tet-Off cells and single colonies were isolated as described above to serve as control cell clones expressing EGFP. Fluorescence microscopy. Transfected MEF Tet-Off cells were cultured in poly-d-lysine-coated glass-bottom dishes (Mat Tek Corp., Ashland, MA, USA) for 24 h after induction, washed with phosphate-buffered saline (PBS), and fixed with 4% formaldehyde (Polysciences, Inc.) for 10 min. The EGFP fluorescence of the fixed cells was monitored with an inverted fluorescence microscope (IX-70, Olympus, Japan) equipped with a digital camera (Penguin 600CL, Pixera Corp, Los Gatos, CA, USA). Preparation of detergent-insoluble, low-density membrane fractions (raft fractions). MEF Tet-Off cells were washed with PBS, incubated with 1% Triton X-100 in TN buffer (25 mM Tris–HCl, pH 6.8, 150 mM NaCl) containing 1 mM PMSF, 1 mM leupeptin, 20 lM pepstatin A, and 20 lg/ml aprotinin for 15 min on ice, and homogenized. The homogenate was fractionated by sucrose-density gradient centrifugation as described [16]. After centrifugation, a total of 11 fractions were collected from the tops of the tubes. The pellet was suspended in TN buffer, sonicated, and designated as P. In some experiments MEF Tet-Off cells were incubated with 10 lg/ml BCh in PBS containing 1 mg/ml BSA for 30 min on ice, and washed twice with PBS prior to the incubation with Triton X-100. Isolation of a BCh-bound membrane subpopulation from raft fractions. The BCh-bound membrane subpopulation was prepared from raft fractions as described previously [16]. Briefly, BCh was first bound to the plasma membrane surface of EGFP-D4-expressing MEF cells, and then raft fractions were prepared as described above. The raft fractions were pooled and mixed with avidin-conjugated magnet beads on ice. Membrane vesicles bound to BCh were retained with avidin-magnet beads via biotinavidin interaction and separated from BCh-unbound vesicles that were recovered in the bead-unbound fraction. Detection of proteins and lipids. Proteins were analyzed using a bicinchoninic acid (BCA) protein assay kit (Pierce). To analyze the distributions of proteins in the gradient, equal volumes of each fraction of the gradient were loaded onto a gel and SDS–PAGE was performed. The molecules in the gels were transferred to PVDF membranes and visualized using ECL plus (Amersham Bioscience). EGFP-D4 was detected with anti-GFP and/or antibody against the h-toxin C-terminal peptide [12]. BCh was detected with anti-h-toxin [14]. Lipids were extracted by the method of Bligh and Dyer [17] with slight modification. Cholesterol was quantified using an assay kit for free cholesterol, Determiner L FC (Kyowa Medex, Japan).
Results Isolation of cell clones stably expressing EGFP-D4 MEF Tet-Off cells were transfected with a plasmid encoding an EGFP-D4 fusion protein, and several cell clones were isolated that expressed the full-size EGFP-D4 with no obvious degradation products upon induction by removal of doxycycline. The expressed protein was detected as a band with the expected molecular size by immunoblotting with anti-GFP and anti-D4-C-terminus (Fig. 1). The growth rates of the cell clones were almost the same under conditions both with and without EGFP-D4 induction (data not shown), indicating that the expressed EGFPD4 does not affect cell growth, at least not at the expression level. However, since the expression levels of EGFP-D4 in these stable cell clones are lower than those in transient
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compels them to localize in non-raft fractions. Rather the result suggests that EGFP-D4 molecules in non-raft fractions might have lost their capacity to bind to raft membranes. To determine whether the high cholesterol content of lipid rafts is required for the binding of EGFP-D4, we depleted cholesterol from EGFP-D4-expressing cells and analyzed the distribution profile of EGFP-D4 (Fig. 3B). When cells were incubated with 20 mM 2-hydroxypropylb-cyclodextrin for 15 min at 37 °C, cholesterol was depleted by ca. 47% from the cells. The treatment caused a significant reduction in the amount of EGFP-D4 recovered in raft fractions (Fig. 3B), indicating its cholesterol-dependent binding to lipid rafts.
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Fig. 1. Expression of EGFP-D4 in MEF cells. Expression patterns of EGFP-D4 and EGFP in stable transfectants were analyzed by Western blotting with anti-GFP and anti-D4-C-terminus and compared with the patterns of cells expressing EGFP-D4 and EGFP transiently. Arrowheads on the right side show the expected migration position of full-size EGFPD4.
transfectants (Fig. 1), higher level of constitutive expression of EGFP-D4 may be toxic for the cells. Upon induction, the expressed EGFP-D4 exhibited a different pattern in fluorescence microscopy from EGFP expressed in a control cell clone (Fig. 2). EGFP-D4 shows an expression pattern suggesting localization at the plasma membrane and intracellular vesicles and in cytoplasm. On the other hand, EGFP exhibited localized only in the cytoplasm. Cholesterol-dependent localization of EGFP-D4 in lipid rafts The clones were characterized further to examine whether the fusion protein is localized in lipid rafts. After induction, the cells were grown for 24 h, and then cell lysates were prepared in the presence of Triton X-100 and fractionated by sucrose-density gradient centrifugation (Fig. 3A). More than half of the EGFP-D4 was recovered in raft fractions (fr.#3–5) where raft marker proteins, flotillin-1 and caveolin-1, were localized (Fig. 3A). In contrast EGFP expressed in a control cell clone was never recovered in raft fractions (Fig. 3A), indicating that the D4 portion of EGFP-D4 determines its localization in lipid rafts. The rest of EGFP-D4 was recovered in non-raft fractions (fr.#10–11). EGFP-D4 molecules in non-raft fractions never redistributed to raft fractions even after incubation with newly added raft membranes from EGFP-D4-non-expressing MEF cells (data not shown). Therefore, it is unlikely that a shortage of raft membranes
EGFP-D4 binds to cholesterol-rich microdomains in the inner leaflet of the plasma membrane It is expected that EGFP-D4 molecules recovered in raft fractions are bound to cholesterol-rich microdomains in the inner leaflet of the plasma membrane and/or in intracellular organelle/vesicular membranes. We examined the proportion of EGFP-D4 bound to the plasma membrane of that recovered in raft fractions. We previously isolated plasma membrane-derived, cholesterol-rich membranes from raft fractions prepared from BCh-pre-bound Jurkat cells [16]. This procedure was applied to subfractionate the raft fractions of EGFP-D4-expressing cells. Prior to the subfractionation, we examined whether BCh binding to the cell surface affects the binding of expressed EGFPD4 to raft membranes inside cells. As a result, the amount of EGFP-D4 recovered in raft fractions of BCh-bound cells was almost the same as that of BCh-unbound cells (Fig. 4A). Since it was confirmed that BCh binding to the cell surface does not affect EGFP-D4 recovery in raft fractions, we then carried out subfractionation experiments. Raft fractions were prepared from BCh-bound EGFPD4-expressing MEF cells, pooled and subfractionated into avidin-magnet-bead-bound (BCh-bound) and -unbound (BCh-unbound) membranes as described in Materials and methods. When the bead-bound and -unbound fractions were analyzed, most of the EGFP-D4 was recovered in bead-bound fraction, namely in the BCh-bound membrane fraction (Fig. 4B). This is in contrast to the recovery of flotillin and caveolin, which were detected in both BCh-bound and -unbound membrane fractions (Fig. 4B and data not shown). When raft fractions were prepared in the absence of BCh (Fig. 4B, BCh ()), no EGFP-D4 was retained with the magnet beads, indicating that retention by the beads is not due to non-specific adsorption. Since BCh was bound to the cell surface prior to cell fractionation, it is expected that the BCh-bound membranes were derived from the plasma membrane. Therefore, these results indicate that most of the EGFP-D4 recovered in raft fractions is bound to the inner leaflet of the plasma membrane, and not to intracellular membranes. Thus our data clearly
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Fig. 2. Flourescence microscopy of EGFP-D4- and EGFP-expressing cells. MEF Tet-Off cells were grown under inducible (Dox()) and non-inducible (Dox(+)) conditions, fixed, and subjected to fluorescence measurement as described in Materials and methods. (Left) Phase contrast; (Right) fluorescence.
demonstrate the existence of cholesterol-rich microdomains in the inner leaflet of the plasma membrane. Discussion In this study, we constructed an intracellular expression system for EGFP-D4, a probe that recognizes cholesterol in cholesterol-rich microdomains. By using this system, we demonstrate the existence of detergent-resistant, cholesterol-enriched microdomains in the inner leaflet of the plasma membrane, as has been shown for the outer leaflet. Although sphingolipids are mainly concentrated in the outer leaflet, cholesterol is present in both leaflets [18]. Furthermore, it has been reported that high concentrations of
cholesterol result in lo domains not only with sphingolipids but also with other saturated phospholipids segregated from the bulk unsaturated glycerophospholipids [7,18]. Therefore, cholesterol probably forms such domains by interacting with saturated phospholipids other than sphingolipids in the inner leaflet. Considering previous observations that showed the requirement of cholesterol for assembly/coupling of outer and inner rafts and for downstream signaling [2,4,6,7], it is conceivable that these cholesterol-rich microdomains couple between the two leaflets upon cell stimulation and serve as platforms for signaling. It is interesting to note that BCh and EGFP-D4 bind to the same subpopulation of raft membranes from the outside and inside, respectively (Fig. 4B). This indicates
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Fig. 3. Cholesterol-dependent localization of EGFP-D4 in raft fractions. (A) EGFP-D4-expressing cells were treated with 1% Triton X-100, homogenized, and subjected to sucrose-density gradient centrifugation. The distributions of EGFP-D4, flotillin-1, and caveolin-1 were detected with specific antibodies. In parallel experiments, EGFP-expressing cells were also analyzed as a control. (B) Cholesterol depletion reduces EGFP-D4 binding to lipid rafts. EGFPD4-expressing cells were incubated with 0 or 20 mM of 2-hydroxypropyl-b-cyclodextrin (bCD) for 15 min at 37 °C. After washing with PBS, the cell homogenates were fractionated as described in Materials and methods. The distributions of cholesterol (upper) and EGFP-D4 (lower two) were analyzed.
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flotillin-1 Fig. 4. (A) BCh-bound to the outer leaflet of the plasma membrane does not interfere with the binding of expressed EGFP-D4 to lipid rafts. EGFP-D4expressing cells were preincubated in the presence (BCh (+)) or absence (BCh ()) of BCh as described in Materials and methods. After washing, cells were treated with Triton X-100, homogenized, and fractionated by sucrose-density gradient centrifugation. The distributions of EGFP-D4 were detected with anti-GFP. (B) EGFP-D4 was recovered in a BCh-bound subpopulation of lipid rafts. Total raft fractions (T) were prepared from BCh-bound (BCh (+)) and -unbound (BCh ()) EGFP-D4-expressing cells as described in (A), and mixed with avidin-conjugated magnet beads. Bead-bound (B) and -unbound (UB) fractions were collected, and the proteins in each fraction were detected with specific antibodies. Due to the tight binding of biotin with avidin, a majority of the bead-bound BCh was not released from avidin-magnet beads even after boiling in SDS-containing buffer at 100 °C (shown as an asterisk in a gel used for Western blotting with anti-h-toxin) [16,22].
that membranes in the raft subpopulation are rich in cholesterol in both the outer and the inner leaflets. The result also indicates that BCh binding to the outer surface of the plasma membrane does not interfere with the binding of EGFP-D4 to the inner leaflet (Fig. 4A). This observation is consistent with the shallow insertion model of h-toxin [10], where D4 captures cholesterol in only the bilayer leaflet exposed to D4; that is, EGFP-D4 expressed inside cells binds to cholesterol in the inner leaflet, while externally added BCh binds to cholesterol in the outer leaflet.
Most of the EGFP-D4 molecules recovered in raft fractions are bound to the inner leaflet of the plasma membrane (Fig. 4), whereas the localization is unclear for EGFP-D4 species recovered in non-raft fractions. Experiments suggest that these species might have lost their capacity to bind to raft membranes. A specific antibody against the D4 C-terminus shows that the C-terminal region of D4 essential for binding remains intact, suggesting that proteolysis is not a cause for the inactivation. Inactivation might be caused by misfolding of the polypeptides during biosynthesis or by a post-translational modification,
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such as the oxidation of the thiol group in the unique Cys of D4 [19]. Such inactivated species are likely to accumulate in the cytoplasm, consistent with our fluorescence microscopy observation (Fig. 2). We showed that intracellular vesicular structures were also stained with EGFP-D4 in some cells (Fig. 2). EGFPD4 bound to such structures is another candidate for the EGFP-D4 recovered in non-raft fractions. If intracellular rafts are more sensitive to detergent solubilization than plasma membrane rafts, as has been shown for caveolincontaining monolayer membranes at the surface of lipid droplets [20], it is possible that EGFP-D4 species bound to intracellular vesicular structures would be solubilized by Triton X-100 and recovered in non-raft fractions. It has been proposed that intracellular rafts are involved in protein sorting at various sites in the cell [21]. It is possible that the intracellular cholesterol-rich vesicular structures detected by EGFP-D4 are those that participate in intracellular trafficking. Attempts to identify these structures are under way in our laboratory. At present it is not known how rafts in the outer and inner leaflets couple with each other. From recent physico-chemical studies it is argued that the coupling may be transient and occur after receptor activation and oligomerization [7,18]. To approach the issue, methods for detecting dynamic features of lipid rafts are required. In this report, we describe an expression system involving EGFP-D4 inside cells that offers a potential tool for visualizing inner leaflet cholesterol-rich microdomains in live cells. Along with this expression system, we have recently prepared fluorescent dye-conjugated h-toxin derivatives, such as Alexa dye-labeled BCh and D4, as tools for monitoring cell-surface cholesterol-rich microdomains in live cells (Ref. [12] and Y. Shimada and Y. Ohno-Iwashita, unpublished results). A combination of these tools might help to increase understanding of the dynamic behavior and function of lipid rafts in future studies. Acknowledgments We thank Dr. Koichi Suzuki, former Director of the Tokyo Metropolitan Institute of Gerontology, for support and encouragement. We thank Dr. M.M. Dooley-Ohto for reading the manuscript. This work was supported in part by Grants-in-Aid for Science Research from the Japan Society for the Promotion of Science (to M.H. and to Y.O.-I.). References [1] D.A. Brown, E. London, Structure of detergent-resistant membrane domains: does phase separation occur in biological membranes? Biochem. Biophys. Res. Commun. 240 (1997) 1–7. [2] K. Simons, D. Toomre, Lipid rafts and signal transduction, Mol. Cell. Biol. 1 (2000) 31–39.
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