(Received for publication, October 19, 1994; and in revised form, December 23, 1994)
From the
SPB-1, a Chinese hamster ovary cell variant defective in serine palmitoyltransferase activity for sphingolipid synthesis, provides a useful system for studying the effects of sphingolipids and/or cholesterol deprivation on cellular functions and membrane properties. To investigate whether there was an interaction among sphingolipids, cholesterol, and glycosylphosphatidylinositol (GPI)-anchored proteins in biological membranes, we introduced human placental alkaline phosphatase (PLAP) in SPB-1 and in wild type cells by stable transfection and examined the effects of sphingolipid and/or cholesterol deprivation on the solubility of PLAP in Triton X-100. Although the PLAP solubility of the membranes isolated from the control cells in Triton X-100 was only 10%, deprivation of sphingolipid and cholesterol further enhanced the solubility, which reached 50% when both sphingolipids and cholesterol were deprived. The enhanced solubility was suppressed to the control level by metabolic complementation with exogenous sphingosine and cholesterol. The sphingolipid and cholesterol content of the isolated membranes changed independently, eliminating the possibility that sphingolipid deprivation induced a reduction in cellular cholesterol and enhanced PLAP solubility and vice versa. It was also unlikely that the enhanced solubility was due to structural changes in PLAP molecules since, regardless of sphingolipid and cholesterol deprivations, almost all PLAP had the GPI-anchor moiety and there were no differences in the apparent molecular weight of the protein in supernatant and precipitate fractions of the detergent-treated membranes. In addition, the expression level of caveolin in the isolated membranes was not significantly affected by sphingolipids and/or cholesterol depletion. These results indicated that both sphingolipids and cholesterol were involved in the PLAP insolubility and suggested that these lipids coordinately played a role in formation of Triton X-100resistant complexes.
Lipids are now recognized not only as constituents of the fluid matrix of biological membranes but also are thought to play a role in the formation of microdomains in membranes(1, 2) . Membrane lipids of mammalian cells consist mainly of three different classes of lipids (glycerolipids, sterols, and sphingolipids), which are categorized according to the structure of their hydrophobic backbones. Although the lipid composition of biological membranes depends on cell type and on the types of intracellular organelles, glycerolipids are the most abundant class of membrane lipids with ratios of phosphatidylcholine and phosphatidylethanolamine to total phospholipids of 30-60% and 10-30%, respectively, in various organelles(3) . Cholesterol, which is the major sterol in most mammalian cells, is preferentially localized to the plasma membrane and amounts to about 10-20% of the total plasma membrane lipid(4, 5) . Sphingolipids are less abundant lipids but are also ubiquitous in mammalian cells(6) . Like cholesterol, complex sphingolipids (sphingomyelin and glycosphingolipids) are preferentially localized to the plasma membrane, and, moreover, both cholesterol and complex sphingolipids are highly enriched in the exoplasmic leaflet of the plasma membrane bilayer(5, 7) . Although it is still unclear whether the similarity in distribution of these lipids has a physiological significance, several investigators have shown that cholesterol has a stronger affinity for sphingomyelin than for glycerophospholipids in model membrane systems (reviewed in (8) ), suggesting cooperative functions of these lipids in biological membranes.
GPI()-anchored proteins, which are widely present in
various types of cells from lower eukaryotes to mammalian cells,
associate with the plasma membrane by integration of their
phosphatidylinositol moiety in the exoplasmic leaflet of the membrane
bilayer(9, 10) . Various GPI-anchored proteins in
mammalian membranes are poorly soluble in Triton X-100 at low
temperatures(11, 12, 13) , and Brown and Rose (14) recently demonstrated that, when Madin-Darby canine kidney
cells expressing PLAP are treated with Triton X-100, most PLAP
molecules are recovered as insoluble membranous materials where
sphingomyelin and glycosphingolipids are also enriched. Their findings
raised the possibility that the insolubility of GPI-anchored proteins
might be conferred by the lipid environment(14) .
Interestingly, lipid composition analysis showed that cholesterol is
also enriched, compared to glycerophospholipids, in the Triton
X-100-insoluble fraction, although sphingolipids are enriched in the
insoluble fraction to a much greater extent than
cholesterol(14) . Participation of membrane cholesterol in the
insoluble complexes was suggested by Cerneus et al.(15) who demonstrated that treatment of cells with
saponin, a detergent which extracts cholesterol, enhances solubility of
a GPI-anchored protein in Triton X-100; however, no analysis of
sphingolipids was presented in their study. While an interaction
between sphingolipids and GPI-anchored proteins was suggested by our
previous findings that sphingolipid deficiency induces hypersensitivity
of a GPI-anchored protein to PI-PLC(16) , in the previous study
we did not show any evidence for participation of sphingolipids in the
Triton X-100 insolubility of GPI-anchored protein. Thus, there is no
direct evidence that sphingolipids participate in the Triton X-100
insolubility of GPI-anchored proteins or that there is an additive or
synergistic effect of sphingolipids and cholesterol on the insolubility
of GPI-anchored proteins. To address these points, we introduced human
placental alkaline phosphatase (PLAP), a typical GPI-anchored protein,
into wild type CHO and mutant CHO cells defective in sphingolipid
biosynthesis and examined the effects of deprivation of sphingolipids
and/or cholesterol on the insolubility of PLAP in Triton X-100.
Alkaline
phosphatase activity was determined as described previously (20) with the following modifications. Briefly, 100 µl of
sample (containing up to 10 µg of protein in 1% Triton X-100/buffer
S) was added to 800 µl of 0.1 M diethanolamine containing
2 mM MgCl and 5 mMp-nitrophenyl
phosphate, and absorbance at 410 nm of the mixture was monitored at
room temperature for 30 s with a Shimadzu UV-160 spectrophotometer
using a time-scanning mode. One arbitrary unit of alkaline phosphatase
activity was defined as the activity producing 0.1 A
per min.
For Western blotting, proteins were separated by SDS-polyacrylamide gel (10% acrylamide) electrophoresis(27) , and the separated proteins were transferred to a polyvinylidene difluoride membrane with a Mini Trans-Blot electrophoretic transfer system (Bio-Rad)(28) . PLAP and caveolin were detected on the membrane by enhanced chemiluminescence using rabbit anti-PLAP antibody and anti-caveolin antibody, respectively, as the primary antibodies and horseradish peroxidase-conjugated goat anti-rabbit IgG as the secondary antibody.
For deprivation of membrane sphingolipids, SPB-1/PLAP cells were cultured in a lipid-deficient medium at 39 °C for 3 days. Membranes prepared from these cells were incubated in 1% Triton X-100 at 4 °C, and the recovery of alkaline phosphatase activity in the supernatant after high speed centrifugation was compared between CHO-K1/PLAP and SPB-1/PLAP cell membranes. Solubility of PLAP of the wild type cell membranes in Triton X-100 was only about 10% (Table 2). Interestingly, SPB-1 cell membranes showed three times higher solubility of PLAP than the wild type cell membranes (Table 2).
For deprivation of membrane cholesterol by inhibiting cholesterol synthesis, cells were cultured in lipid-deficient medium at 39 °C in the presence of compactin, a potent inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase(29, 30) . During the 3-day compactin treatment, 0.1 mM mevalonate was added daily to the culture medium to complement non-sterol isoprenoid products essential for cell growth(31) , so that the cells could sustain viability (estimated by trypan blue extrusion). Compactin treatment of CHO-K1/PLAP cells increased the solubility of PLAP about 2-fold (Table 2). Also, compactin treatment of SPB-1/PLAP cells increased the solubility to 50% (Table 2).
To verify that the
GPI-anchor was an essential part of PLAP for the insolubility in Triton
X-100, we used PLAP-HA, a chimeric protein which has a single
membrane-spanning domain of influenza hemagglutinin at the
carboxyl-terminal region of PLAP in place of GPI-anchor (Table 1). Nearly 100% of the activity of PLAP-HA of
membranes prepared from CHO-K1 and SPB-1 transfectants was solubilized
in Triton X-100 (Table 2), confirming that the GPI-anchor moiety
of PLAP was an essential part of PLAP insolubility.
Figure 1: Restoration of PLAP insolubility in SPB-1/PLAP cell membranes by metabolic complementation. SPB-1/PLAP cells were cultured in Nutridoma-BO medium at 39 °C for 3 days in the presence (+) or the absence(-) of compactin, sphingosine, and cholesterol, and membranes were prepared from these cells as described under ``Experimental Procedures.'' Solubility of PLAP (open bars) and membrane proteins (hatched bars) in Triton X-100 were determined and are represented as the means ± S.D. (n = 3).
Figure 2: PI-PLC sensitivity of PLAP. Membranes prepared from the indicated cells (when indicated, the cells were cultured with compactin) were incubated with (+) or without(-) PI-PLC at 37 °C for 1 h and subsequently subjected to partitioning by Triton X-114 phase separation as described under ``Experimental Procedures.'' Distribution of alkaline phosphatase activity to Triton X-114 and aqueous phases after partitioning are shown as the percentages of the total activity (Triton X-114 plus aqueous fractions). Bars shown are the means ± S.D. (n = 3).
Figure 3: Western blotting of PLAP. Cells were cultured under conditions as described in the legend to Table 3and membranes were prepared from these cells. Membranes incubated with 1% Triton X-100 were subjected to high speed centrifugation, and the supernatant (S) and the precipitate (P) fractions were analyzed by Western blotting using anti-PLAP polyclonal antibody as described under ``Experimental Procedures.'' Lanes 1 and 2, CHO-K1/PLAP cell membranes; lanes 3-10, SPB-1/PLAP cell membranes. Molecular mass standards used are rabbit muscle phosphorylase B (97 kDa), bovine serum albumin (66 kDa), and hen egg white ovalbumin (45 kDa).
Figure 4: Western blotting of caveolin. Cells were cultured under conditions as described in the legend to Table 3. Membranes (5 µg of protein) prepared from these cells were analyzed by Western blotting using anti-caveolin antibody as described under ``Experimental Procedures.'' Lanes 1 and 2, CHO-K1/PLAP cell membranes; lanes 3-6, SPB-1/PLAP cell membranes. Molecular mass standards used are carbonic anhydrase (31 kDa) and soybean trypsin inhibitor (21 kDa).
We further examined whether there was a difference in the distribution of PLAP at the cell surface by indirect immunogold labeling electron microscopy. As shown in Fig. 5, A-C, PLAP molecules were almost randomly distributed on the cell surface, regardless of sphingolipid/cholesterol depletion, and no obvious difference in the distribution was observed between CHO-K1/PLAP, SPB-1/PLAP, and compactin-treated SPB-1/PLAP cells. Nontransfected SPB-1 cells showed no signal (Fig. 5D), confirming the specificity of the indirect immunogold labeling of PLAP.
Figure 5:
Distribution of PLAP at the cell surface.
Monolayers of CHO cells were grown in Nutridoma-BO with or without
compactin at 39 °C for 3 days. The monolayers were incubated with
rabbit anti-PLAP polyclonal antibody in Nutridoma-BO medium at 4 °C
for 1 h, rinsed with PBS, and fixed with PBS containing 0.5%
glutaraldehyde and 3% formaldehyde. After incubation with 0.1 M NHCl in PBS, the monolayers were incubated with
gold-conjugated protein A in Nutridoma-BO for 1 h. The samples were
fixed again with 1% formaldehyde and 3% glutaraldehyde, stained with
OsO
and uranyl acetate, dehydrated in ethanol, embedded in
Epon, sectioned, and viewed. Bar represents 0.5 µm. A, CHO-K1/PLAP cells; B, SPB-1/PLAP cells; C, compactin-treated SPB-1/PLAP cells; D, SPB-1 cells
(PLAP-negative control).
In this paper, we developed CHO cell systems to lower cellular sphingolipids and/or cholesterol, which should be useful for investigating the coordinate roles of these lipids in mammalian membranes. We demonstrated that deprivation of sphingolipids and cholesterol in CHO cell membranes enhanced the solubility of PLAP in Triton X-100 (Table 2), and this enhanced solubility was suppressed by metabolic complementation with exogenous sphingosine and cholesterol (Fig. 1). Moreover, determination of the mass levels of sphingolipids and cholesterol in the isolated membranes revealed independent changes in sphingolipid and cholesterol levels (Table 3), eliminating the possibility that deprivation of sphingolipids might induce a reduction of cholesterol and thereby enhance the solubility of PLAP and vice versa. It was unlikely that the enhanced solubility was due to structural changes in PLAP molecules since, regardless of sphingolipid and cholesterol depletion, almost all the PLAP molecules had the GPI-anchor moiety (Fig. 2) and there were no differences in the apparent molecular weight of the protein in the supernatant and precipitate fractions of the detergent-treated membranes (Fig. 3). Furthermore, our findings that sphingolipid and/or cholesterol depletion did not affect the solubility of total membrane proteins (Fig. 1) and that conversion of the GPI-anchor to a membrane-spanning domain abolished the insolubility of PLAP (Table 2) indicated that sphingolipids and cholesterol conferred the insolubility to a limited set of membrane proteins including GPI-anchored proteins, and therefore ruled out the possibility that the insolubility of PLAP resulted from nonspecific inclusion of membrane proteins into Triton X-100-insoluble sphingolipid/cholesterol aggregates. From these results, we conclude that both sphingolipids and cholesterol are involved in the insolubility of PLAP in Triton X-100 and suggest that these lipids may coordinately play a role in the formation of putative Triton X-100-resistant membrane microdomains where GPI-anchored proteins acquire their insolubility.
Both cholesterol and complex sphingolipids are enriched in the exoplasmic leaflet of the plasma membrane of intact cells(4, 5, 7) , and these lipids are preferentially recovered in Triton X-100-insoluble fractions obtained from cells(14) . Moreover, a strong interaction of cholesterol with sphingomyelin has been demonstrated in model membranes (reviewed in (8) ). The insolubility of GPI-anchored proteins is observed in glycosphingolipid-poor cells such as CHO cells (this study) as well as glycosphingolipid-abundant cells like Madin-Darby canine kidney cells(14) . These previous findings combined with the present study revealing participation of both membrane sphingolipids and cholesterol in the insolubility of PLAP lead us to suggest that an interaction between cholesterol and sphingolipids, especially sphingomyelin, may play a role in the initial formation for Triton X-100-resistant complexes, into which GPI-anchored proteins are subsequently integrated. However, it is still unknown whether additional membrane components are also involved in the formation of Triton X-100-resistant microdomains. Caveolin/VIP21 is an integral membrane protein, which associates with caveolae (noncoated invaginations at the plasma membrane) (37) and also with the trans -Golgi network(38) . Although caveolin might be necessary for GPI-anchored proteins to acquire Triton X-100 resistance (36) , the enhanced solubility of PLAP by sphingolipids and/or cholesterol depletion was not an indirect effect of reduced expression of caveolin since expression of caveolin was not significantly affected by sphingolipids and/or cholesterol depletion (Fig. 4). Recent findings that myristoylated/palmitoylated non-receptor kinases and multimeric GTP-binding proteins are also enriched in nonionic detergent-resistant complexes (39, 40, 41, 42) raise the intriguing possibility that putative cholesterol/sphingolipid-enriched membrane microdomains play an important role in signal transduction. Model cell systems such as the ones presented here will hopefully be useful in investigating this possibility in the future.