Article |
Address correspondence to Howard Riezman, Biozentrum, University of Basel, Klingelbergstr. 70, Basel CH-4056, Switzerland. Tel.: 41-61-267-2160. Fax: 41-61-267-2149. E-mail: howard.riezman{at}unibas.ch
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Abstract |
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Key Words: ceramide transport; sphingolipids; membrane contacts; secretion; Saccharomyces cerevisiae
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Introduction |
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In mammalian cells, ceramide is formed by decarboxylation and condensation of L-serine with palmitoyl-CoA, followed by reduction of 3-ketodihydrosphingosine to generate dihydrosphingosine (DHS),* N-acylation of DHS with a fatty acid, and finally desaturation of dihydroceramide (DHS-Cer) (Merrill and Jones, 1990). The biosynthesis of ceramide seems to occur at the cytosolic surface of the ER in mammalian cells (Mandon et al., 1992; Hirschberg et al., 1993), although the ceramide synthase has not been identified. Ceramide, a common precursor for all complex sphingolipids, is then transported to the Golgi apparatus for conversion to both sphingomyelin and glucosylceramide by yet-unknown mechanisms (van Meer and Holthuis, 2000). Recently, it has been shown by in vivo analysis of a mutant resistant to lysenin, an SM-directed cytolysin, that the transport of ceramide to the sites of sphingomyelin and glucosylceramide synthesis occurs via a mechanism that differs from the ER-to-Golgi vesicular transport of glycoproteins (Fukasawa et al., 1999).
In yeast cells, the steps in ceramide biosynthesis are similar to those in mammalian cells except that DHS-Cer is hydroxylated by Sur2p to give phytoceramide (Haak et al., 1997; Grilley et al., 1998), but the exact localization and topology of the reactions of ceramide biosynthesis have not been studied. In contrast to the situation in mammalian cells, it was assumed that ceramide is converted to inositolphosphorylceramide (IPC), one of three major classes of yeast sphingolipids, by addition of inositolphosphate from phosphatidylinositol (PI) in the ER. Subsequently, IPC was thought to be delivered to the Golgi apparatus where IPC is mannosylated to form mannosyl-IPC followed by mannosyl-diinositolphosphorylceramide (Dickson and Lester, 1999; Schneiter, 1999). This idea was based on the fact that IPC synthesis continued at nonpermissive temperature in sec mutants that block ER-to-Golgi vesicular transport of proteins (Puoti et al., 1991). Recently, Aur1p, a protein required for IPC synthesis, has been localized to the Golgi apparatus (Levine et al., 2000), implying that ceramide is delivered to the Golgi apparatus. This suggests that a nonvesicular transport mechanism exists for ceramide transport to the Golgi apparatus.
In vitro systems that reproduce transport events occurring in intact cells are powerful tools to understand molecular mechanisms (Wuestehube and Schekman, 1992; Moreau et al., 1993; Barlowe et al., 1994; Funakoshi et al., 2000). Therefore, we have developed a cell-free system with isolated donor and acceptor membranes that allows us to study the mechanisms of ceramide transport and ceramide synthesis. Here, we show that ceramide is transported from the ER to the Golgi apparatus via two distinct pathways, one vesicle dependent and the other vesicle and ATP independent. We provide biochemical evidence that ER-Golgi membrane contacts play an important role in nonvesicular ceramide transport. A cytosolic factor is required for transport after formation of ER-Golgi contacts.
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Results |
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This in vitro system using microsomal membranes showed that ceramide synthesis is temperature dependent and requires ATP, cytosol, and a membrane fraction (Fig. 3 B). When the liposomes containing hexacosanoic acid (C26) and PI were omitted from the reaction mixture, a small reduction (18%) in the synthesis of IPCs was found. We also found that N-ethylmaleimide (NEM) completely inhibited ceramide synthesis, suggesting the requirement of an NEM-sensitive protein or compound (unpublished data).
Ceramide and IPC synthase activities are localized in the ER and Golgi apparatus, respectively
In yeast, no evidence concerning the site of ceramide synthesis has been published. However, IPC synthase activity and Aur1p have been localized to the Golgi apparatus (Levine et al., 2000). Therefore, to investigate the transport of ceramide it was important to define where ceramide synthesis activity is located. Subcellular fractionation by sucrose density gradient of [3H]DHS-labeled membranes expressing Aur1p-hemagglutinin (HA) showed that the majority of synthesized ceramides and DHS and DHS-1-P behaved as the ER marker Wbp1p, whereas IPCs cosedimented with Aur1p-HA and Sed5, a marker for Golgi membranes (unpublished data; Banfield et al., 1994; Schröder et al., 1995; Levine et al., 2000). This result suggests the ER and Golgi localizations of ceramide and IPC synthesis, respectively.
Next, we investigated whether ceramide and IPC synthesis could be reconstituted using purified ER- and Golgi-enriched membranes. ER-enriched membranes were prepared as described previously (Wuestehube and Schekman, 1992), and Golgi-enriched membranes were isolated by differential centrifugation (see Materials and methods). Western blotting analysis showed that Wbp1p was found exclusively in P27 fraction and P45 fraction, resulting from further fractionation of S27, whereas Emp47p, a Golgi marker (Schröder et al., 1995) and Aur1p-HA were found mainly in the final supernatant (S45) (Fig. 4 A). Therefore, we used the S45 fraction as source of Golgi-enriched membranes even though a small amount of ER membranes remained in this fraction. Fractionation of the P27 fraction on a sucrose gradient resulted in further purification of ER membranes (at the 40/50% sucrose interface) as shown in Fig. 4 B. Fig. 4 C shows an experiment in which ceramide and IPC synthase activities were examined with the purified ER- and Golgi-enriched membranes. As expected, efficient synthesis and subsequent conversion of ceramide to IPC required the addition of both ER- and Golgi-enriched membranes. When only ER membranes were incubated, the amount of ceramide synthesized remained high (87% of complete incubation), but the level of IPC synthesis was low. This confirms that IPC synthase activity is localized in Golgi membranes. In contrast, addition of only Golgi membranes resulted in significant reductions of ceramide and IPC synthesis by 41 and 27%, respectively. Since a detectable amount of Wbp1p was found in the Golgi membrane fraction, the lack of complete loss of ceramide synthase activity is most likely due to ER contamination in the Golgi fraction. The above results are consistent with the localization of ceramide synthesis to the ER. Ceramide would then be transported to the Golgi apparatus for conversion to IPC.
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Depletion of ATP does not affect SEC18-independent ceramide transport in vivo
IPC synthesis still continues even though inhibited when ER-to-Golgi vesicular transport is blocked in sec mutant cells (Fig. 1, A and B). Our in vitro results suggest that SEC-independent IPC synthesis is mediated by ATP-independent transport of ceramide (Fig. 5, B and C, and Fig. 6 A). Since incorporation of [3H]myo-inositol into PI occurs at a lower temperature than the incorporation into IPC (Puoti et al., 1991), this difference allowed us to examine the effect of ATP depletion on SEC18-independent IPC synthesis in intact cells in an experiment that measures the synthesis of sphingolipids from endogenously synthesized ceramides. Mutant sec18 cells were labeled with [3H]myo-inositol at 13°C for 45 min and further incubated with NaF and NaN3 at 13°C for 10 min to allow depletion of ATP. Subsequently, the cells were chased at 37°C for 30 min. During the pulse at 13°C, very little IPC/C was synthesized. When sec18 mutant cells were chased at 37°C, approximately three times more IPC/C was produced. This increase in IPC/C synthesis was not affected by depletion of ATP (Fig. 8). We conclude that transport of ceramide from the ER to the Golgi apparatus by the nonvesicular (SEC18-independent) pathway in intact cells does not require ATP, and therefore this process reflects the ceramide transport we reconstituted in vitro.
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Discussion |
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Our study provides evidence that ceramide is transported from the ER to the Golgi apparatus by both vesicular and nonvesicular means in yeast. From in vivo experiments, IPC synthesis from radiolabeled DHS was reduced but still observed when ER-to-Golgi vesicular transport was blocked in temperature-sensitive secretion mutants even under the conditions where protein synthesis was also blocked in sec18 mutant cells. This SEC18-independent IPC synthesis was also not affected by ATP depletion. Consistent with this, IPC synthesis was inhibited partially by ATP depletion in wild-type cells (unpublished data). These results suggest that ceramide is transported from the ER to the Golgi apparatus via two distinct pathways in intact cells, one vesicle dependent and the other nonvesicular. Analysis using an in vitro assay demonstrated that transport of ceramide from the ER to the Golgi compartment was not sensitive to ATP depletion and was SEC18 independent, indicating a nonvesicular transport mechanism. The correlation between in vivo and in vitro experiments provides evidence that the in vitro assay for ceramide transport measures a physiologically relevant transport process.
Some previous studies have suggested that nonvesicular transport of ceramide in vitro does not require cytosol (Moreau et al., 1993; Kok et al., 1998). Recently a cytosolic protein requirement has been demonstrated using semiintact cells to measure ceramide transport from the ER to the site of synthesis of sphingomyelin in the Golgi compartment (Funakoshi et al., 2000). In vitro analysis with the LY-A mutant, displaying a specific defect in ER-to-Golgi trafficking of ceramide to the site of sphingomyelin synthesis, showed that LY-A mutant cells are defective in a cytosolic protein involved in the ceramide transport. Since ER-to-Golgi transport of GPI-anchored or transmembrane proteins in LY-A cells appeared to be normal, it was suggested that ceramide is delivered to the Golgi apparatus by a mechanism that differs from ER-to-Golgi vesicular transport of proteins. Although the delivery of ceramide from the ER to the Golgi apparatus is mediated by two pathways, ATP-dependent and ATP-independent (nonvesicular), the defect in LY-A cells is specific to ATP-dependent trafficking of ceramide (Fukasawa et al., 1999). It is not clear whether the ATP-dependent delivery of ceramide is via one of multiple vesicular pathways that is specific for ceramide or nonvesicular pathways. In this respect, evidence for the existence of two distinct vesicle populations upon budding from the ER was provided recently (Muñiz et al., 2001).
The dilution independence of ceramide transport after ER and Golgi membranes have been incubated together at 4°C, where transport does not occur, strongly suggests that stable membrane contacts are formed and that these are relevant to the nonvesicular transport process. The ER-to-Golgi membrane contacts are likely to be saturable and specific for ceramide transport, since microsomes from wild-type cells inhibited ceramide transport in vitro, whereas addition of purified mitochondria did not. Our data provide important biochemical evidence for a role of membranemembrane contacts in intracellular lipid transport and provide a system to begin to characterize this process.
Experimental evidence for an important function of membrane contacts has been found for ER-to-mitochondria trafficking of phosphatidylserine (Voelker, 1993; Achleitner et al., 1999). In addition, a close apposition of trans-ER with trans-Golgi cisternae has been reported by a high voltage electron microscope tomographic study (Ladinsky et al., 1999). Since the trans-ER lacks buds, it is most likely that this close apposition is associated with transport of lipids via nonvesicular rather than with vesicular transport of proteins.
ER-to-Golgi transport of ceramide for glucosylceramide synthesis was almost independent of ATP and cytosol (Funakoshi et al., 2000). In contrast, our experiments have shown that ATP-independent nonvesicular transport of ceramide requires a heat-labile and trypsin-sensitive cytosol protein(s). To our knowledge, this is the first clear evidence for a cytosolic protein requirement for nonvesicular transport of ceramide. What could be the function of this cytosolic factor?
Our dilution experiments have shown that the cytosol requirement is involved in the late step of ceramide transport after establishment of ER-Golgi membrane contacts. Thus, the cytosolic protein(s) seems to be required for transfer of ceramide from ER to Golgi membranes. It is unlikely that a cytosolic factor would be required for the transbilayer movement of ceramide after it has reached the Golgi compartment. Therefore, the late cytosolic requirement also makes it unlikely that ceramide is transferred from ER to Golgi during our short incubation at 4°C. The processes of membranemembrane contact and transfer could be obligatorily coupled because cytosol could not efficiently extract ceramide out of the ER without acceptor membranes (unpublished data).
Analysis using our in vitro transport assay also revealed that ER-to-Golgi transport of ceramide continues for >60 min with a lag period from 5 to 15 min. The rather long time requirement could be due to ceramide transport to the Golgi complex and/or reflect the need to translocate ceramide from the cytosolic to the lumenal side of Golgi apparatus. Based on the predicted topology of Aur1p and localization of its putative active site in the lumen of the Golgi compartment, this translocation step would be required in our assay (Levine et al., 2000). A lumenal active site is consistent with our findings that trypsin treatment of membranes did not destroy conversion of ceramide to IPC. The half times for spontaneous translocation of short acyl chain ceramide (C5-DMB-ceramide) across lipid bilayers has been reported to be 22 min (Bai and Pagano, 1997), but measurements for natural ceramides are not available. Since short acyl chain lipids appear to undergo rapid transbilayer movement, on the basis of the correlation between the hydrophobicity of acyl chain and the rate of transbilayer movement (Bai and Pagano, 1997), it would follow that the spontaneous translocation of natural ceramide would occur in the order of hours and that specific proteins must be involved in the movement of ceramide. This translocation should be ATP independent because nonvesicular transport of ceramide does not require ATP. Such specific lipid translocators or flippases exist in the ER, Golgi apparatus, and plasma membrane (Raggers et al., 2000). Most of them require energy (e.g., ATP), but some proteins such as scramblase are involved in ATP-independent transbilayer lipid movement. In addition, ATP-independent protein-mediated lipid movement has been postulated in the translocation of glucosylceramide from the cytosolic to the lumenal leaflet of the Golgi membranes.
Our in vitro transport assay seems to reconstitute only the nonvesicular ceramide transport pathway. The reasons for the lack of reconstitution of the vesicular pathway are not clear, but in yeast the Golgi apparatus is made up of apparently unconnected compartments. Aur1p is located primarily in the medial-Golgi but not cis-Golgi compartments (Levine et al., 2000). Therefore, it seems plausible that ceramide transported to the cis-Golgi compartment via vesicular mechanism could be not delivered to the IPC synthase compartment efficiently in our assay because it involves too many transport steps. This may help explain why GTP plus Sar1p inhibits ceramide transport in our assay. GTP and Sar1p stimulate vesicle budding (Barlowe et al., 1994), and ceramide could be sequestered in these vesicles. In any event, it seems likely that nonvesicular pathway is used directly to generate IPC. To show that the direct fusion of ER with Golgi membranes did not occur under our assay conditions, we assayed for the acquisition of -1,6 mannose modifications onto the ER form of the GPI-anchored protein, Gas1p (Muñiz et al., 2001). Less than 5% (background) of the Gas1p received this modification. Therefore, this cannot account for the >20% transport of ceramide that we see in our assay (unpublished data).
We postulate that the two different pathways of ceramide transport may have different functions. Vesicular-dependent transport would function to establish and maintain the lipid composition of ER and Golgi membranes during ER to early Golgi trafficking associated with possible functions of ceramide in regulation of cellular processes (Futerman et al., 1998; Hannun and Luberto, 2000) such as protein transport and signal transduction. For example, the sorting of GPI-anchored proteins from the ER is regulated by sphingoid base or ceramide synthesis (Horvath et al., 1994; Sütterlin et al., 1997). Nonvesicular transport would function to provide ceramide directly for de novo biosynthesis of sphingolipids for their important roles. These hypotheses will be addressed by further studies of the molecular mechanisms underlying the nonvesicular transport of ceramide.
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Materials and methods |
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In vivo labeling with [3H]DHS and [3H]myo-inositol
Cell cultures and labeling of lipids with [3H]DHS was performed as described previously (Zanolari et al., 2000). If present, Chx (200 µg/ml; Fig. 1, A and B) and AbA (20 µg/ml; Fig. 5 A) were added, and cells were incubated for 20 min at 24°C before preincubation and at the start of the preincubation, respectively.
For the energy deprivation experiments, sec18 cells were preincubated for 20 min at 13°C and labeled with [3H]myo-inositol at 13°C. After 45 min, myo-inositol (100 µg/ml) and NaF/NaN3 (each 10 mM) were added and incubated for 20 min at 13°C. The cells were then chased for 30 min at 37°C after the addition of 4 vol of SD medium containing myo-inositol.
After the incubations, cells were placed on ice and washed, and cell pellets were subjected to lipid extraction (Zanolari et al., 2000). If necessary, the extracted lipids were submitted to mild alkaline hydrolysis with NaOH. After neutralization with acetic acid, the lipids were desalted by partitioning between N-butanol and water. The pooled organic phases were dried under nitrogen. The lipids were dissolved in chloroform/methanol/water (CMW; 10/10/3 [vol/vol/vol]) for TLC on Kieselgel 60 plates (20 x 20; Merck) and developed in solvent I (chloroform/methanol/4.2 N NH4OH, 9/7/2 [vol/vol/vol]) or solvent II (chloroform/methanol/0.25% KCl, 55/45/10 [vol/vol/vol]). Radiolabeled lipids were visualized and quantified on a Cyclone Storage Phosphor System using a tritium-sensitive screen (Packard).
Cytosol and membrane preparations
Cytosols were prepared from RH981 and RH2043 strains as described (Salama et al., 1993). The preparation of microsomal membranes and ER-enriched membranes were performed as described (Baker et al., 1990; Wuestehube and Schekman, 1992) with a few modifications. In brief, spheroplasts (from 0.5 OD600 U of cells in log phase, total 4 x 1010 cells) were lysed in lysis buffer (0.1 M sorbitol, 20 mM Hepes, pH 7.4, 150 mM potassium acetate, 2 mM EDTA, 1 mM DTT, 1 mM PMSF, and 1 µg/ml protease inhibitor mixture [pepstatin, leupeptin, antipain]). Unbroken cells and cell debris were removed by centrifugation (3000 g, 10 min, 4°C), and the resulting supernatants were centrifuged (100,000 g, 1 h, 4°C) to collect the microsomal membrane fraction. The pellet was washed twice and resuspended in 1 ml (2550 mg protein) of B88 (20 mM Hepes-KOH, pH 6.8, 150 mM potassium acetate, 5 mM magnesium acetate, 250 mM sorbitol). Aliquots were frozen in liquid nitrogen and stored at -80°C. For ER- and Golgi-enriched membranes, supernatants containing microsomal membranes were subjected to two successive centrifugation steps (27,000 g, 10 min, 4°C) giving rise to pellet (P27) and supernatant (S27) fractions, and S27 was centrifuged (45,000 g, 15 min, 4°C), resulting in P45 and S45. S45 containing Golgi-enriched membranes was spun (100,000 g, 1 h, 4°C), washed twice, and resuspended in 1 ml (1020 mg protein) of B88. P27 was resuspended in lysis buffer and loaded onto a 7.5 ml sucrose density step gradient (2.5 ml each of 40, 50, and 60% sucrose in 10 mM Hepes, pH 7.4, 1 mM MgCl2). The gradient was centrifuged at 200,000 g for 2 h and 20 min at 4°C in TST41.14 rotor (Kontron Instruments). The ER-enriched membrane fraction at the 40/50% sucrose interface was collected, washed twice by centrifugation (27,000 g, 10 min, 4°C), and resuspended in 1 ml (24 mg protein) of B88. Aliquots of each membrane fraction were frozen in liquid nitrogen and stored at -80°C. Protein concentrations were determined using the protein assay kit from Bio-Rad Laboratories.
In vitro labeling with [3H]DHS
To assay for ceramide and IPC synthase activities, microsomal membranes (100200 µg) or membrane fractions (ER-enriched membranes, 510 µg; Golgi-enriched membranes, 2550 µg), cytosol (100 µg), ATP regenerating system (1 mM ATP, 40 mM phosphocreatine, 0.2 mg/ml creatine phosphokinase), GDP-mannose (50 µM), and a mix of [3H]DHS and unlabeled DHS (10 and 40 pmoles, respectively, 0.5 µCi) were first incubated (15 min, 10°C) for incorporation of DHS into membranes. Coenzyme A (50 µM) and liposomes containing hexacosanoic acid (C26) and PI (50 and 250 µM, respectively) prepared as described (Funato et al., 1992) were added, and the mixture was incubated (2 h, 24°C) in a final volume of 50 µl of B88. The reaction was stopped by adding 333 µl of chloroform/methanol (CM; 1/1 [vol/vol]). The organic phase was collected after centrifuging at 13,000 g for 5 min, and the pellet was reextracted with 250 µl of CMW. The extracted lipids were submitted to a mild alkaline treatment, N-butanol extraction, and analyzed by TLC with solvent I as described above. Radiolabeled ceramide was analyzed with solvent III (chloroform/acetic acid, 9/1 [vol/vol]) (Morell and Radin, 1970).
In vitro ceramide transport assay
Radiolabeled ceramide was synthesized in vitro from [3H]DHS using ER-enriched membranes (1020 µg) as described above except that GDP-mannose was omitted, AbA (0.2 µM) was included in the assay mixture, and liposomes containing hexacosanoic acid (C26) and PC were used instead of liposomes containing hexacosanoic acid (C26) and PI. The membranes were then recovered by centrifugation at 27,000 g for 10 min at 4°C, washed twice, resuspended in B88, and used as the donor membrane in the in vitro transport assay. The recovery of radiolabeled lipids was 5075% percent of the total. Subsequently, the donor membranes were incubated for 2 h at 24°C in the total reaction mixture (50 µl) containing cytosol (2 mg/ml), ATP regenerating system, GDP-mannose (50 µM), liposomes composed of PI (250 µM), FuB (200 µM), AbA (0.4 µM), and microsomal membranes (50100 µg) prepared from AbA-resistant strain (RH5255) as the acceptor membrane to allow transport to occur. The reaction was stopped by adding 333 µl of CM. The preparation of the lipid extracts and analysis were done as described above.
Lipid analysis
Radiolabeled lipids were collected from TLC plates by scraping and eluting with CMW. The isolated lipid extract was dried under nitrogen and partitioned between N-butanol and water. Lipid mixtures (Fig. 2 A) or isolated lipids (Fig. 2, BD) were subjected to phosphoinositide-specific PLC digestion as described previously (Hamburger et al., 1995). Strong HCl hydrolysis of lipids was performed as described (Puoti et al., 1991). After enzymatic and/or chemical treatments, lipids were dried under nitrogen and partitioned between N-butanol and water. After drying, the lipids were dissolved in CMW and analyzed by TLC as described above.
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Footnotes |
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Acknowledgments |
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This work was supported by a grant from the Swiss National Science Foundation and a European Community grant (HPRN-2000-00077) funded through the Bundesamt für Bildung und Wåssenschatt (to H. Riezman).
Submitted: 7 May 2001
Revised: 24 October 2001
Accepted: 24 October 2001
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References |
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