Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Tokyo 162-8640, Japan
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Abstract |
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LY-A strain is a Chinese hamster ovary cell mutant resistant to sphingomyelin (SM)-directed cytolysin and has a defect in de novo SM synthesis. Metabolic labeling experiments with radioactive serine, sphingosine, and choline showed that LY-A cells were defective in synthesis of SM from these precursors, but not syntheses of ceramide (Cer), glycosphingolipids, or phosphatidylcholine, indicating a specific defect in the conversion of Cer to SM in LY-A cells. In vitro experiments showed that the specific defect of SM formation in LY-A cells was not due to alterations in enzymatic activities responsible for SM synthesis or degradation. When cells were treated with brefeldin A, which causes fusion of the Golgi apparatus with the endoplasmic reticulum (ER), de novo SM synthesis in LY-A cells was restored to the wild-type level. Pulse-chase experiments with a fluorescent Cer analogue, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-D-erythro-sphingosine (C5-DMB-Cer), revealed that in wild-type cells C5-DMB-Cer was redistributed from intracellular membranes to the Golgi apparatus in an intracellular ATP-dependent manner, and that LY-A cells were defective in the energy-dependent redistribution of C5-DMB-Cer. Under ATP-depleted conditions, conversion of C5-DMB-Cer to C5-DMB-SM and of [3H]sphingosine to [3H]SM in wild-type cells decreased to the levels in LY-A cells, which were not affected by ATP depletion. ER-to-Golgi apparatus trafficking of glycosylphosphatidylinositol-anchored or membrane-spanning proteins in LY-A cells appeared to be normal. These results indicate that the predominant pathway of ER-to-Golgi apparatus trafficking of Cer for de novo SM synthesis is ATP dependent and that this pathway is almost completely impaired in LY-A cells. In addition, the specific defect of SM synthesis in LY-A cells suggests different pathways of Cer transport for glycosphingolipids versus SM synthesis.
Key words: sphingomyelin; ceramide; trafficking; mutant; Chinese hamster ovary cells ![]() |
Introduction |
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LIPIDS serve not only as essential constituents of the
fluid matrix of biological membranes, but also as
modulators regulating various cellular events. 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. In living cells, the membranes of different intracellular organelles have different
lipid compositions, and various biomembranes show an
asymmetric distribution of lipid types across the membrane bilayer (Alberts et al., 1994). Furthermore, various
steps in lipid biosynthesis can occur in different intracellular compartments; for example, ceramide (Cer)1 produced
in the ER is sorted to the Golgi apparatus for conversion to sphingomyelin (SM) or glucosylceramide (GlcCer) (reviewed in Hoekstra and Kok, 1992
; Rosenwald and Pagano, 1993
; van Meer, 1993
). Phosphatidylserine produced
in the ER is sorted to the mitochondria where phosphatidylserine is converted to phosphatidylethanolamine, which is translocated to the plasma membrane (reviewed
in Trotter and Voelker, 1994
). Thus, the synthesis, translocation, and sorting of lipids represent an important set of
problems relating to membrane biogenesis and homeostasis. However, little is known about the mechanisms underlying lipid transport and sorting, although various pathways for intracellular transport of newly synthesized lipids have been suggested (reviewed in Moreau and Cassagne,
1994
; Trotter and Voelker, 1994
).
Sphingolipid biosynthesis is initiated by condensation
of L-serine with palmitoyl CoA, a reaction catalyzed by
serine palmitoyltransferase to generate 3-ketodihydrosphingosine, which is then converted to dihydrosphingosine (see Merrill and Jones, 1990 for review of sphingolipid biosynthesis). Dihydrosphingosine is N-acylated to
produce dihydroceramide (DCer), followed by desaturation to form Cer. These early synthetic steps occur at the
cytosolic surface of the ER (Mandon et al., 1992
; Hirschberg et al., 1993
; Michel and van Echten-Deckert, 1997
).
Cer, a common intermediate for both SM and glycosphingolipid syntheses, is translocated from the ER to the Golgi
apparatus by yet-undefined mechanisms, and then converted to SM by the enzyme phosphatidylcholine:ceramide cholinephosphotransferase (SM synthase) in the lumenal side of the Golgi apparatus or to GlcCer on the
cytosolic surface of the Golgi apparatus (Coste et al., 1986
;
Futerman et al., 1990
; Futerman and Pagano, 1991
;
Trinchera et al., 1991
; Jeckel et al., 1992
). After translocation into the Golgi lumen, GlcCer is further converted to
lactosylceramide and more complex glycosphingolipids.
SM and glycosphingolipids produced in the Golgi lumen
are predominantly delivered to the plasma membrane,
most likely via the bulk flow of membranes from the Golgi
apparatus to the plasma membrane (reviewed in Hoekstra
and Kok, 1992
; Rosenwald and Pagano, 1993
; van Meer, 1993
), while a portion of newly synthesized GlcCer reaches
the plasma membrane by a non-Golgi pathway (Warnock
et al., 1994
).
A powerful approach towards understanding the metabolic mechanisms and biological functions of membrane
phospholipids is the biochemical and cell-biological characterization of cell mutants with specific defects in phospholipid metabolism (Nishijima et al., 1997). Lysenin, derived from the coelomic fluid of the earthworm Eisenia
foetida, is a cytolysin that binds to SM with a high affinity (Yamaji et al., 1998
). Recently, we isolated several types
of lysenin-resistant CHO cell mutants, two of which (designated LY-A and LY-B strains) are defective in SM production, and the LY-B strain has been found to lack the
LCB1 subunit of serine palmitoyltransferase (Hanada et al.,
1998
). In this study, we further characterize the LY-A
strain and find that this mutant is defective in an ATP-dependent trafficking pathway of Cer from the ER to the
Golgi apparatus for de novo SM synthesis. As far as we
are aware, this study is the first report providing genetic
evidence for the existence of specific machinery for intracellular trafficking of Cer. We also discuss models for ER-to-Golgi apparatus trafficking of de novo synthesized Cer
toward SM or glycosphingolipids.
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Materials and Methods |
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Materials
N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)- D-erythro-sphingosine (C5-DMB-Cer) and 6-[N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]caproyl-D-erythro-sphingosine (C6-NBD-Cer) were purchased from Molecular Probes. Brefeldin A, fatty acid-free BSA, and endoglycosidase H (Endo H) were from Sigma Chemical Co. L-[U- 14C]serine (5.85 GBq/mmol) was from Amersham Pharmacia Biotech. [Methyl-14C]choline chloride (2.04 GBq/mmol) and [3-3H]D-erythro-sphingosine (0.74 GBq/mmol) were from American Radiolabeled Chemicals Inc.
Cells and Cell Cultures
The CHO-K1 cell line was obtained from the American Type Culture
Collection (ATCC CCL 61), and LY-A strain, a CHO-K1-derived mutant
cell line, has been established previously by us (Hanada et al., 1998). Cells
were routinely maintained in Ham's F-12 medium supplemented with
10% NBS, penicillin G (100 U/ml), streptomycin sulfate (100 µg/ml), and
sodium hydrogen carbonate (1.18 g/liter) at 33°C (Hanada et al., 1990
).
Nutridoma medium (Ham's F-12 medium containing 1% Nutridoma-SP
[Boehringer Mannheim] and gentamicin [25 µg/ml]) was used as serum-free medium. Cells constitutively expressing a glycosylphosphatidylinositol (GPI)-anchored human placental alkaline phosphatase (PLAP)
(Berger et al., 1987
) or a membrane-spanning PLAP (PLAP-HA), which
is a chimera of PLAP with influenza hemagglutinin (Arreaza and Brown,
1995
), were established as described (Hanada et al., 1995
).
Metabolic Labeling of Lipids with [14C]Serine and [14C]Choline in Cells
Subconfluent cell monolayers in 6-cm dishes were incubated in 2 ml of
Nutridoma medium containing 37 kBq of [14C]serine or [14C]choline at
33°C for various times. After washing twice with 2 ml of cold PBS, cells
were lysed in 1 ml of 0.1% SDS at 4°C, and 0.1 and 0.8 ml of the cell lysate
were used for protein determination and lipid extraction, respectively.
Lipids extracted from the cells (Bligh and Dyer, 1959) were separated on
TLC plates with solvent of methyl acetate/n-propanol/chloroform/methanol/0.25% potassium chloride (25:25:25:10:9, vol/vol) in the [14C]serine-labeling experiments, or chloroform/methanol/acetic acid/water (25:15:4:2,
vol/vol) in the [14C]choline-labeling experiments. After collecting gels
from the plates by scraping, radioactivity in each lipid was determined by
liquid scintillation counting. The values were normalized to cell protein.
Since DCer and Cer were not separated by the TLC systems used, composition of metabolically labeled ceramides was determined after acid hydrolysis of them. Cells were labeled with [14C]serine for 2 h at 33°C and labeled lipids were separated by the TLC as described above. Lipids
containing both [14C]DCer and [14C]Cer were reextracted from the TLC
fractions, hydrolyzed in 0.5 ml of 10 N hydrochloric acid/water/methanol
(8.6:9.4:100, vol/vol) for 18 h at 70°C (Gaver and Sweeley, 1965), and then
the liberated sphingoid bases were separated on TLC plates with a solvent
of chloroform/methanol/2N ammonia solution (80:20:1, vol/vol) (Williams et al., 1984
). Radioactivity in separated dihydrosphingosine or sphingosine
was determined by liquid scintillation counting.
For determination of distribution of SM to the external surface of cells, cells were labeled with [14C]serine for 48 h at 33°C, fixed with 0.125% glutaraldehyde in PBS for 30 min at room temperature, and treated with or without 250 mU/ml of recombinant Bacillus cereus sphingomyelinase (Higeta Shoyu) for 30 min at 37°C. Radioactive SM extracted from the cells was analyzed as described above.
Metabolic Labeling of Lipids with [3H]Sphingosine in Cells
For preparation of a complex of 100 µM [3H]sphingosine (1.85 MBq/ml)
with 200 µM fatty acid-free BSA in PBS, a stock solution of [3H]sphingosine (370 KBq, 20 nmol) in ethanol was dried under a stream of nitrogen gas. The dried lipid was dispersed in 200 µl of PBS containing 200 µM
fatty acid-free BSA by vortex mixing and sonication. Subconfluent cell
monolayers were incubated in Nutridoma medium containing 1 µM
[3H]sphingosine complexed with BSA for various times. Cellular lipids were extracted under mild alkaline conditions for efficient recovery of the
sphingoid bases (Williams et al., 1984) or mild acidic conditions for efficient recovery of N-acetylneuramyl lactosylceramide (GM3), and were separated on TLC plates with a solvent of chloroform/methanol/water (65:25:4,
vol/vol). Radioactivity in each lipid was determined as described above.
In Vitro Enzyme Assays
Cell lysates were used as the enzyme source for assays. Subconfluent cells
were washed twice with PBS, harvested by scraping, and precipitated by
centrifugation (400 g, 5 min). The cells were washed with buffer A (10 mM
Hepes-Na, pH 7.5, containing 0.25 M sucrose and 5 mM EDTA), suspended in buffer A, and lysed with a probe-type sonicator. After removal
of unbroken cells by centrifugation (1,000 g, 5 min), cell lysates were
stored at 80°C until use.
An assay for SM synthase activity using C5-DMB-Cer was performed
as described (Hanada et al., 1991), except that C5-DMB-Cer was used instead of C6-NBD-Cer. GlcCer synthase activity was determined as described (Hanada et al., 1991
), except that 500 µM UDP-glucose was included in the assay mixture. Dihydrosphingosine N-acyltransferase (DCer
synthase) activity was determined as described (Morell and Radin, 1970
)
except for using [3H]sphingosine instead of [14C]acyl-CoA. Acid and neutral sphingomyelinase activities were determined as described (Schütze
and Krönke, 1995
).
Metabolism of Fluorescent Cer Analogues in Cells
Metabolism of C5-DMB-Cer and C6-NBD-Cer in cells was examined under the pulse-chase conditions as described (Pagano and Martin, 1988; Pagano et al., 1991
) with some modifications. In brief, subconfluent cell
monolayers were incubated in Ham's F-12 containing 1.25 µM C5-DMB-Cer complexed with 1.25 µM BSA, or 5 µM C6-NBD-Cer complexed with
5 µM BSA at 4°C for 30 min. Monolayers were washed three times with
Ham's F-12 and subsequently chased in Nutridoma medium containing
0.34 mg/ml fatty acid-free BSA at 33°C for various times. Lipids extracted
from the cells were separated on TLC plates with a solvent of chloroform/
methanol/15 mM calcium chloride (60:35:8, vol/vol). After collecting the
fluorescent spots by scraping, lipids were extracted with 0.5 ml of chloroform/methanol/0.1 M potassium chloride (1:2:0.8, vol/vol). NBD (excitation at 470 nm; emission at 530 nm) or DMB (excitation at 480 nm;
emission at 515 nm) fluorescence of each lipid was measured with a spectrofluorometer. Values are presented as percentages of total fluorescence
recovered (the sum of C5-DMB-Cer and C5-DMB-SM, or the sum of C6-NBD-Cer, C6-NBD-SM, and C6-NBD-GlcCer).
Fluorescence Microscopy
Cells grown on glass coverslips (22-mm diameter) were prelabeled with fluorescent lipids at 4°C and chased at 33°C under essentially the same conditions for the labeling of cells grown on culture dishes. After washing with
PBS, the cells were fixed with 0.125% glutaraldehyde solution in PBS for
5 min at 4°C. The fixed specimens were observed and photographed under
a fluorescence microscope. Note that the specimens were photographed
soon after fixation, because redistribution of fluorescent lipid analogues
might occur even in glutaraldehyde-fixed cells (Pagano et al., 1989).
Radiolabeling, Immunoprecipitation, and Endo H Treatment
CHO transfectants expressing PLAP or PLAP-HA were pulsed with 9.25 MBq/ml of EXPRESS 35S-protein labeling mix (DuPont-New England Nuclear) for 15 min at 33°C and chased for various times in Nutridoma medium containing 10 mM methionine. Radiolabeled cells were washed and lysed in 0.9 ml of lysis buffer, 50 mM Tris-HCl (pH 8.0) containing 150 mM NaCl, 0.1% SDS, and 1% Triton X-100. After addition of 100 µl of 10% BSA, the cell lysates were boiled for 5 min and clarified by centrifugation twice at 10,000 g for 10 min. The clarified extracts were incubated with 2 µl of rabbit antisera against human PLAP (Biomeda Corp.) at 4°C for 2 h. Resultant immunocomplexes were incubated with 50 µl of a 50% slurry of protein A-Sepharose CL-4B (Amersham Pharmacia Biotech) at 4°C for 2 h, pelleted, and washed once with lysis buffer containing 1% BSA and three times with lysis buffer at room temperature. Immunoprecipitates were resuspended in 50 mM sodium citrate (pH 5.5) containing 0.5% SDS, 1% 2-mercaptoethanol, and 1 mM 4-amidinophenylmethanesulfonyl fluoride, and boiled for 5 min. Immunoprecipitated proteins released from resin were incubated in the presence or absence of 6 mU of Endo H at 37°C for 16 h and separated by SDS-PAGE.
Protein Determination
Protein was determined according to Lowry et al. (1951) or with a BCA
protein assay reagent kit (Pierce Chemical Co.), using BSA as standard.
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Results |
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Metabolic Labeling of Lipids with [14C]Serine in LY-A Cells
We examined the effects of cell culture temperature on the activity of de novo SM synthesis in LY-A and wild-type cells by metabolic labeling of lipids with [14C]serine for 2 h, and found that the relative levels of SM synthesis in LY-A cells to that in wild-type cells at 33, 37, and 40°C were 9.5, 10, and 16%, respectively. Thus, further characterizations of LY-A cells were performed with cells cultured at 33°C, unless otherwise described.
Time courses of metabolic labeling of lipids with [14C] serine up to 12 h were compared between LY-A and wild-type cells. As shown in Fig. 1, the SM labeling in LY-A cells was <10% of the wild-type level throughout the incubation. In contrast, time courses for Cer and GlcCer labeling in LY-A cells were comparable to those in wild-type cells, although the early labeling rates of these lipids in LY-A cells were slower than wild-type cells. LY-A and wild-type cells were virtually identical in the labeling of phosphatidylserine or phosphatidylethanolamine, both of which incorporate serine via metabolic pathways distinct from the pathway for sphingolipids. These results confirmed that LY-A cells are defective in SM synthesis, but not in glycosphingolipid synthesis.
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Although DCer comigrated with Cer in the TLC system used, analysis of sphingoid base composition of metabolically labeled lipids in the TLC fraction containing both DCer and Cer showed that >80% of sphingoid bases derived from the fraction was sphingosine in both LY-A and wild-type cells, indicating that conversion of DCer to Cer is normal in LY-A cells.
For determination of the distribution of SM at the cell surface, cells were labeled with [14C]serine for 48 h, fixed with glutaraldehyde, and treated with or without B. cereus sphingomyelinase. Analysis of radioactive lipids extracted from the cells showed that the pool size of sphingomyelinase-sensitive SM among the total pool of cellular SM was 56% in wild-type cells and 50% in LY-A cells. Thus, SM appeared to be preferentially distributed in the outer leaflet of the plasma membrane bilayer in LY-A cells, as in wild-type cells.
Metabolic Labeling of Lipids with [14C]Sphingosine and [14C]Choline in LY-A Cells
A defect in SM formation might result in some repression
of de novo sphingoid base formation. Thus, to prevent
such possible feedback repression from affecting Cer-to-SM conversion by metabolic labeling, we used [3H]sphingosine as an alternative precursor for monitoring SM synthesis, since it has been shown that CHO cells are able to
utilize exogenously supplied sphingosine for synthesis of
Cer and complex sphingolipids (Hanada et al., 1992).
When cells were labeled with 1 µM [3H]sphingosine at
33°C for various times up to 2 h, wild-type and LY-A cells
similarly accumulated free [3H]sphingosine, with a plateau
level at 15-30 min after incubation, and showed almost
equal levels of the total radioactivity of cell-associated lipids (Fig. 2), indicating that the two cell types had an equal efficiency of sphingosine uptake. Under these conditions,
formation of radioactive SM in LY-A cells was <30% of
the wild-type level throughout the incubation, whereas
formations of Cer, GlcCer, and GM3 were slightly higher in
LY-A cells than in wild-type cells (Fig. 2). When [3H]dihydrosphingosine was used as another precursor, we obtained results similar to those obtained with [3H]sphingosine (data not shown).
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Synthesis of SM was also examined by metabolic labeling with [14C]choline. Incorporation of [14C]choline into SM in LY-A cells was ~20% or less of the wild-type level, while incorporation of [14C]choline into phosphatidylcholine (the phosphorylcholine donor for conversion of Cer to SM) was slightly higher in LY-A cells than in wild-type cells (Fig. 3), eliminating the possibility that LY-A cells had a defect in phosphatidylcholine synthesis.
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Cell-free Assays for Activities of Enzymes Involved in SM Metabolism
To identify the mechanism underlying the defect in conversion of Cer to SM in LY-A cells, we examined SM synthase activity in cell lysates by using C5-DMB-Cer, a fluorescent Cer analogue, as the substrate. LY-A cells showed essentially the same level of SM synthase activity as wild-type cells, as there was no difference in the dependence of C5-DMB-SM formation on C5-DMB-Cer concentration or in the time course of C5-DMB-SM formation between wild-type and LY-A cells (Fig. 4, A and B). Similarly, when C6-NBD-Cer or N-[14C]palmitoyl-sphingosine was used as the substrate, no difference in activity of SM synthase between the two cell types was observed (data not shown).
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We also measured in vitro activities of various other enzymes related to SM metabolism including serine palmitoyltransferase, dihydroceramide synthase, GlcCer synthase, and acid and neutral sphingomyelinases. However,
significant differences in these activities between wild-type
and LY-A cells were not detected (data not shown; see
also Hanada et al., 1998 for analysis of serine palmitoyltransferase activity). Collectively, the results of these in
vivo and in vitro experiments suggested that the specific
defect of SM formation in LY-A cells was not due to alterations in enzymatic activities responsible for SM synthesis
or degradation.
Effect of Brefeldin A on De Novo SM Synthesis in LY-A Cells
We next explored the possibility that LY-A cells had a defect in translocation of Cer from the ER to the Golgi apparatus where SM synthase exists. Treatment of CHO cells
with brefeldin A, leading to fusion of the Golgi membrane
with the ER (Klausner et al., 1992), enhances the rate of
conversion of Cer to SM, suggesting that in brefeldin
A-treated cells Cer is accessible to the normally Golgi apparatus-localized SM synthase without the ER-to-Golgi apparatus trafficking process (Warnock et al., 1994
; van
Helvoort et al., 1997
). Thus, if LY-A cells have a defect in
ER-to-Golgi apparatus trafficking of Cer, brefeldin A
treatment should restore SM synthesis in LY-A cells to the
wild-type level.
After pretreatment with 1 µg/ml brefeldin A for 30 min at 33°C, cells were labeled with [14C]serine in the presence of brefeldin A, and the labeling rates of lipids were compared between wild-type and LY-A cells. Under conditions without brefeldin A, the rate of SM synthesis in LY-A cells was only 10% of the wild-type level, while the labeling rates of other lipids in LY-A cells were comparable to the wild-type levels (Fig. 1). In contrast, under brefeldin A-treated conditions, the rate of SM synthesis in LY-A cells was quite similar to the wild-type level, which was about threefold higher than the rate in brefeldin A-untreated wild-type cells, while labeling rates of Cer, GlcCer, phosphatidylserine, and phosphatidylethanolamine were also similar between wild-type and LY-A cells (Fig. 5). In addition, we used [3H]sphingosine and [3H]dihydrosphingosine for monitoring the rate of Cer-to-SM conversion and observed restoration of SM synthesis in LY-A cells to the wild-type levels under brefeldin A-treated conditions (data not shown). These results demonstrated that LY-A cells had the Golgi apparatus-localized SM synthase normally and suggested that LY-A cells were defective in translocation of Cer from the ER to the Golgi apparatus.
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Metabolism of a Fluorescent Cer Analogue, C5-DMB-Cer, in LY-A Cells
To examine further whether LY-A cells were defective in
ER-to-Golgi apparatus trafficking of Cer, we used C5-DMB-Cer, a fluorescent Cer analogue, as a probe. Since
the critical micellar concentration of C5-DMB-Cer is
higher than that of natural Cer (Pagano et al., 1991), externally supplied C5-DMB-Cer can be readily transferred to
the plasma membrane and, after movement across the
plasma membrane, this probe fluorescently labels various
intracellular membranes including the ER membrane in
living cells at 4°C. In addition, when cells prelabeled with
C5-DMB-Cer are warmed up to physiological temperatures, C5-DMB-Cer is efficiently accumulated into the
Golgi region and metabolized to C5-DMB-SM (Pagano
et al., 1991
; Ktistakis et al., 1995
), indicating the possibility
that the ER-to-Golgi apparatus trafficking behavior of C5-DMB-Cer mimics that of natural Cer produced at the ER.
For testing whether the behavior of C5-DMB-Cer in cells reflected the defect of SM synthesis in LY-A cells, the metabolic rate of C5-DMB-Cer to C5-DMB-SM was compared between wild-type and LY-A cells. Cells were preincubated with C5-DMB-Cer for 30 min at 4°C for transferring the probe to intracellular membranes and, after washing the extracellular C5-DMB-Cer, cells were incubated at 33°C for various times to start conversion of intracellular C5-DMB-Cer to C5-DMB-SM. During the chase, C5-DMB-Cer was metabolized to C5-DMB-SM in a time-dependent manner, resulting in conversion of 50% of the total lipidic fluorescence to C5-DMB-SM in wild-type cells 30 min after the chase, while the metabolic rate of C5-DMB-Cer to C5-DMB-SM in LY-A cells was about half of the wild-type level (Fig. 6 A). In addition, the metabolic rate of C5-DMB-Cer to C5-DMB-SM in LY-A cells was restored to the wild-type level by brefeldin A treatment (Fig. 6 B). Note that the level of total lipidic DMB fluorescence associated with cells was identical in the two cell types during the pulse and chase (data not shown). These results indicated that the behavior of C5-DMB-Cer at least partly reflects that of natural Cer in cells.
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Intracellular Behavior of Pulse-labeled C5-DMB-Cer in LY-A Cells
Distribution patterns of intracellular DMB fluorescence
were compared between LY-A and wild-type cells under
the pulse-chase conditions. After prelabeling of cells with
C5-DMB-Cer at 4°C, intracellular fluorescence was distributed throughout intracellular membranes including the
ER in both wild-type and LY-A cells (Fig. 7, A and B).
After the chase at 33°C for 15-30 min, most of the intracellular fluorescence in wild-type cells accumulated in the perinuclear region corresponding to the Golgi complex
(Fig. 7, C and E), in agreement with previous studies (Pagano et al., 1991; Ktistakis et al., 1995
). Interestingly, accumulation of intracellular fluorescence in the Golgi region
was lower in LY-A cells than wild-type cells (Fig. 7, D and
F vs. C and E). The lower level of the Golgi fluorescence
in LY-A cells was not due to an enhanced secretion of intracellular DMB fluorescence to the medium, because there was no significant difference in the total amount of
DMB lipids associated with cells between the two cell
types during the pulse and chase periods (data not shown).
These observations also provided evidence that LY-A
cells are defective in ER-to-Golgi apparatus trafficking
of Cer.
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Energy Dependence of Redistribution of Intracellular C5-DMB-Cer to the Golgi Apparatus
For examination of the energy dependence of intracellular redistribution of C5-DMB-Cer, cells were pretreated with or without energy inhibitors (50 mM 2-deoxy-D-glucose and 5 mM NaN3) for 15 min at 33°C, incubated with C5-DMB-Cer for 30 min at 4°C, washed, and chased for 15 min at 33°C in the presence or absence of the energy inhibitors. Measurements of intracellular ATP showed that wild-type and LY-A cells had the same ATP levels under the inhibitor-minus control conditions, and ATP levels in both cell types were reduced by >95% in the presence of the inhibitors. ATP depletion did not affect the level and distribution of C5-DMB-Cer during the prelabeling of cells at 4°C; DMB fluorescence was distributed to various intracellular membranes (data not shown), as seen under the normal conditions (Fig. 7, A and B). However, after the chase at 33°C, the level of DMB fluorescence accumulated into the Golgi region in wild-type cells was strikingly lower under the ATP-depleted conditions than under the control conditions, while in LY-A cells ATP depletion did not appreciably affect the Golgi level of DMB fluorescence (Fig. 8, A-D). Consequently, ATP depletion reduced the DMB fluorescence level associated with the Golgi apparatus in wild-type cells to the level of LY-A cells.
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Biochemical analysis showed that the ATP-depleted conditions caused an ~50% reduction of the conversion rate of C5-DMB-Cer to C5-DMB-SM in wild-type cells (Fig. 9), indicating that the ATP-dependent redistribution of C5-DMB-Cer is responsible for synthesis of C5-DMB-SM in wild-type cells. The reduced rate in wild-type cells was almost identical to the rate in LY-A cells, which was not affected by ATP depletion (Fig. 9), being consistent with the similar level of DMB fluorescence in Golgi apparatus between ATP-depleted wild-type and normally cultured LY-A cells (Fig. 8, B and C). There was no difference in the total amount of lipidic DMB fluorescence associated with cells between the normal and ATP-depleted cells even after the 33°C chase (data not shown). Collectively, these results indicated that, in wild-type cells, redistribution of intracellular C5-DMB-Cer to the Golgi apparatus for C5-DMB-SM synthesis consists of an ATP-dependent pathway(s) and an ATP-independent (or less ATP-dependent) pathway(s), and that the ATP-dependent trafficking pathway of C5-DMB-Cer is almost completely impaired in LY-A cells.
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Metabolism and Intracellular Behavior of C6-NBD-Cer in Cells
While C5-DMB-Cer is a poor substrate for GlcCer synthase (Pagano et al., 1991; Paul et al., 1996
), C6-NBD-Cer,
another fluorescent analogue of Cer, serves as an efficient
substrate for both GlcCer and SM syntheses (Futerman
and Pagano, 1991
). C6-NBD-Cer has an ~10-fold faster
rate at spontaneous transfer between artificial membranes
than C5-DMB-Cer (Rosenwald and Pagano, 1993
; Bai and Pagano, 1997
), and C6-NBD-Cer rapidly targets to the
Golgi membrane even in glutaraldehyde-fixed cells, suggesting that no active process is required for ER-to-Golgi
apparatus transfer of C6-NBD-Cer (Pagano et al., 1989
).
Under normal culture or ATP-depleted conditions, wild-
type and LY-A cells were preincubated with C6-NBD-Cer
at 4°C to label intracellular membranes and, after washing
extracellular C6-NBD-Cer, cells were incubated at 33°C
for the chase. During the chase, C6-NBD-Cer was converted to C6-NBD-SM or C6-NBD-GlcCer in a time-dependent manner with redistribution of NBD fluorescence to
the Golgi apparatus, in agreement with a previous study
(Lipsky and Pagano, 1983). In contrast to the cases with
C5-DMB-Cer (Figs. 8, A-D, and 9 A), redistribution of C6-NBD-Cer to the Golgi region or conversion of C6-NBD-Cer to C6-NBD-SM was not affected by ATP depletion
even in wild-type cells, and C6-NBD-Cer behaved almost
identically between LY-A and wild-type cells (Figs. 8,
E-H, and 9 B). The conversion rate of C6-NBD-Cer to C6-NBD-GlcCer under the ATP-depletion conditions was
~70-75% of the normal level, probably due to a decrease
in the intracellular UDP-glucose level by ATP depletion. These results indicated that C6-NBD-Cer is efficiently redistributed to the Golgi apparatus not via the ATP-dependent pathway, which is involved in the redistribution of C5-DMB-Cer to the Golgi apparatus. However, this nature of
C6-NBD-Cer allowed us to demonstrate that the ATP-depleted conditions used did not cause an inhibition of SM
synthase activity itself or extensive destruction of the
Golgi apparatus.
Effects of ATP Depletion on SM and GlcCer Formation from [3H]Sphingosine
We next examined the effect of ATP depletion on conversion of natural Cer to SM by metabolic labeling with [3H]sphingosine. ATP depletion of wild-type cells resulted in the reduction of the [3H]SM formation to the level of LY-A cells, which was not affected by ATP depletion (Fig. 10), being consistent with the results of the pulse-chase experiments using C5-DMB-Cer (Fig. 9 A). Although the [3H]SM formation in wild-type cells under the ATP-depleted conditions was only 30% of the normal level, the [3H]GlcCer formation under the ATP-depleted conditions was ~65% of the normal level both in wild-type and LY-A cells (Fig. 10). Considering that GlcCer formation appeared to be partially inhibited by a reduction of the UDP-glucose level under the ATP-depletion conditions, effects of ATP depletion on accessibility of [3H]Cer to SM and GlcCer synthases could be estimated by the corrected values of [3H]SM/C6-NBD-SM and [3H]GlcCer/C6-NBD-GlcCer, respectively, although we did not eliminate the possibility that access of C6-NBD-Cer to SM and GlcCer synthases was not completely ATP independent. The corrected values suggested that the influence of ATP depletion on [3H]Cer trafficking caused ~70% and ~20% inhibition of [3H]SM and [3H]GlcCer formation, respectively (Fig. 10, bottom). ATP depletion did not reduce the level of [3H]sphingosine associated with cells and did cause an increase in the level of [3H]Cer along with the striking decrease in [3H]SM, ruling out the possibility that the decreased formation of [3H]SM under the ATP-depleted conditions was due to a decrease in cellular uptake of [3H]sphingosine or formation of [3H]Cer from [3H]sphingosine. These results demonstrated evidence that trafficking of natural Cer from the ER to the site for SM synthesis is mainly ATP dependent and that this ATP-dependent trafficking of Cer is defective in LY-A cells.
|
ER-to-Golgi Apparatus Trafficking of GPI-anchored and Membrane-spanning Glycoproteins in LY-A Cells
To address the question of whether ER-to-Golgi apparatus trafficking of proteins was affected in LY-A cells, we
determined the acquisition rate of Endo H resistance, a
well-accepted determinant for arrival in the medial Golgi
apparatus (Kornfeld and Kornfeld, 1985). For this, we
used wild-type and LY-A cells constitutively expressing
GPI-anchored alkaline phosphatase (PLAP) or chimeric alkaline phosphatase (PLAP-HA) having a membrane-spanning region in place of the GPI anchor. PLAP and
PLAP-HA in these cells were pulse-labeled with [35S]methionine/cysteine for 15 min and chased for various time at 33°C. Then, extracts from the cells were immunoprecipitated with anti-PLAP antibody, treated with Endo H,
and separated by SDS-PAGE for detection of the Endo
H-sensitive and -resistant forms. From the data shown in
Fig. 11, the acquisition rates of Endo H resistance of not
only PLAP but also PLAP-HA were quite similar (t1/2 = ~30 min) between wild-type and LY-A cells. Thus, it was
likely that the machinery for ER-to-Golgi apparatus trafficking of glycoproteins operated normally in LY-A cells.
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Discussion |
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LY-A Cells Have a Specific Defect in an ATP-dependent Trafficking Pathway of Cer from the ER to the Golgi Compartment for De Novo SM Synthesis
Previously, we isolated the LY-A strain, a CHO cell mutant resistant to an SM-directed cytolysin, and showed that
LY-A cells have only a 30% level of SM content, but
nearly a 100% level of glycosphingolipid content compared with wild-type cells (Hanada et al., 1998). In this
study, we attempted to identify a defective step causing
the phenotype of LY-A cells.
Metabolic labeling experiments with [14C]serine, [3H]
sphingosine, and [14C]choline indicated that conversion of
Cer to SM was impaired in LY-A cells, but that the formations of Cer and phosphatidylcholine, both of which are
immediate precursors for SM formation, are normal (Figs.
1-3). However, enzyme assays in cell lysates showed no
difference in the activities of SM synthase, neutral sphingomyelinase, or acid sphingomyelinase between LY-A
and wild-type cells. Although reduced SM synthesis along
with Cer accumulation was observed during the mitotic
phase of the cell cycle (Yokoyama et al., 1997), LY-A and
wild-type cells grown in Nutridoma medium displayed virtually identical patterns of the phase distribution of the
cell cycle; 30-32% of the total population of each cell type
was in the G1 phase, 50-54% in the S phase, and 15-17%
in the G2 plus M phases (data not shown). These in vivo
and in vitro experiments suggested that the defect of SM
formation in LY-A cells was not due to alterations in enzymatic activities responsible for SM metabolism nor to a secondary effect of cell-cycle phase on the lipid metabolism, leading us to the possibility that LY-A cells had a defect in translocation of Cer from the ER to the Golgi compartment where SM synthase exists.
Several lines of evidence support this conclusion. First,
when cells were treated with brefeldin A, SM synthesis
in LY-A cells was restored to the wild-type level (Fig.
5), in agreement with the expectation that in brefeldin
A-treated cells Cer is accessible to the normally Golgi apparatus-localized SM synthase without the ER-to-Golgi
apparatus trafficking process. Second, pulse-chase experiments with C5-DMB-Cer in intact cells demonstrated that redistribution of intracellular DMB fluorescence from intracellular membranes to the Golgi apparatus is strikingly
slower in LY-A cells than in wild-type cells (Fig. 7), along
with the defect of conversion of C5-DMB-Cer to C5-DMB-SM in intact LY-A cells (Fig. 6 A). Furthermore, ATP
depletion in wild-type cells inhibited the conversion of C5-DMB-Cer to C5-DMB-SM by 50% and also inhibited redistribution of C5-DMB-Cer to the Golgi apparatus,
whereas ATP depletion did not affect the behavior of C5-DMB-Cer in LY-A cells (Figs. 8, A-D, and 9 A). Although
conversion of C6-NBD-Cer to the SM metabolite may be
necessary for trapping NBD fluorescence in the Golgi apparatus (Pütz and Schwarzmann, 1995), at least a portion
of the Golgi apparatus-accumulated DMB fluorescence in
wild-type cells appears to be attributable to unconverted
C5-DMB-Cer, because the amount of C5-DMB-SM formed
in LY-A cells after a 30-min chase was equivalent to that
of C5-DMB-SM formed in wild-type cells after a 15-min
chase (Fig. 6 A), whereas DMB fluorescence in the Golgi
region was much denser in the latter cells than in the
former (Fig. 7, C and F). By contrast, intracellular labeling with C6-NBD-Cer, which rapidly transfers between membranes, displayed no difference in the Golgi redistribution
between LY-A and wild-type cells, or between normally
cultured and ATP-depleted cells (Fig. 8, E-H). Thus, the
impairment of the Golgi accumulation of C5-DMB-Cer in
LY-A cells reflects a defect in the energy-dependent transport of this probe from intracellular membranes to the
Golgi apparatus. In addition, the findings that intracellular
DMB fluorescence is initially distributed throughout intracellular membranes after prelabeling with C5-DMB-Cer at
4°C (Fig. 7, A and B; see also Pagano et al., 1991
) and that
>50% of the surface area of intracellular membranes is attributed to the ER membrane (Griffiths et al., 1989
) indicate that the energy-dependent redistribution of DMB fluorescence most likely represents an ER-to-Golgi apparatus transport of C5-DMB-Cer. Importantly, this energy-dependent trafficking of C5-DMB-Cer is relevant to
physiological trafficking of natural Cer for SM synthesis,
because ATP depletion in wild-type cells inhibited [3H]SM
formation from metabolically labeled [3H]Cer, whereas
[3H]SM formation in LY-A cells was not affected by ATP
depletion (Fig. 10). Collectively, we conclude that LY-A
cells are defective in ATP-dependent trafficking of Cer
from the ER to the Golgi compartment where SM synthase exists.
The defect in LY-A cells appears to be specific to Cer
transport. When cellular lipids were metabolically labeled
with [14C]serine, the formation rates of [14C]phosphatidylserine and [14C]phosphatidylethanolamine were identical between LY-A and wild-type cells (Fig. 1), indicating
that transport of phosphatidylserine from the ER to the
mitochondria proceeds normally in LY-A cells. Cholesterol metabolism in LY-A cells is also suggested to be normal, since the amount of free cholesterol in LY-A cells is
the wild-type level (Hanada et al., 1998). There was no appreciable difference in the acquisition rate of Endo H resistance of a membrane-spanning or a GPI-anchored protein between LY-A and wild-type cells (Fig. 11), indicating
that ER-to-Golgi apparatus trafficking of proteins is not
defective in LY-A cells. Both wild-type and LY-A cells display a similar preferential distribution of SM at the
plasma membrane (data not shown), suggesting that, once
SM is produced in the Golgi apparatus, SM is subsequently sorted to the plasma membrane in LY-A cells similar to wild-type cells.
Our findings that in wild-type CHO cells ATP depletion
inhibits conversion of C5-DMB-Cer to C5-DMB-SM by
50% concomitantly with inhibition of redistribution of intracellular DMB fluorescence to the Golgi apparatus (Figs. 8
and 9 A) suggest that a ~50% portion of ER-to-Golgi
apparatus transfer of C5-DMB-Cer is mediated via the
ATP-dependent pathway and that another 50% of the C5-DMB-Cer transfer is mediated by ATP-independent (or
less ATP-dependent) mechanisms. The ATP-independent
redistribution of C5-DMB-Cer to the Golgi apparatus may
be at least partly attributable to a nonphysiological spontaneous transfer of C5-DMB-Cer between membranes due
to less hydrophobicity of C5-DMB-Cer than natural Cer.
For natural Cer, ATP-dependent and -independent (or
less ATP-dependent) trafficking mechanisms were estimated to be responsible for 70-80% and 20-30%, respectively, of newly synthesized SM, since ATP depletion
caused a 70-80% inhibition of formation of [3H]sphingosine-derived SM without reduction of [3H]Cer formation (Fig. 10). In contrast, redistribution of intracellular C6-NBD-Cer to the Golgi apparatus or conversion of C6-NBD-Cer to C6-NBD-SM was not, or only slightly, affected by ATP depletion. The different trafficking behavior between C5-DMB-Cer and C6-NBD-Cer most likely
reflects the fact that C6-NBD-Cer has a much faster rate of
spontaneous transfer between artificial membranes than C5-DMB-Cer (Rosenwald and Pagano, 1993; Bai and Pagano, 1997
), implying that C5-DMB-Cer is embedded in
membranes more firmly and thus mimics natural Cer more
accurately, compared with C6-NBD-Cer.
Different Pathways of Cer Transport for SM versus GlcCer Synthesis
It has been postulated that a yet-undefined pathway(s)
mediates Cer trafficking from the ER to the Golgi apparatus and that, after arrival at the cytoplasmic surface of the
Golgi apparatus, Cer molecules are used for GlcCer synthesis and the residual Cer molecules are subsequently
used for SM synthesis after translocation to the lumenal
side of the Golgi apparatus (reviewed in Hoekstra and
Kok, 1992; Rosenwald and Pagano, 1993
; van Echten and
Sandhoff, 1993
; van Meer, 1993
). Collins and Warren
(1992)
have shown that newly synthesized Cer can be delivered to the sites of SM and GlcCer synthases by a different pathway from the major vesicular-mediated trafficking
pathway for newly synthesized proteins during the mitotic
phase in HeLa cells. Slomiany et al. (1992)
have demonstrated that Cer-containing vesicles derived from the ER
fuse with the Golgi membrane in an N-ethylmaleimide-
sensitive manner under cell-free conditions. On the other
hand, studies with another cell-free system and with a
semi-intact cell system to assay for the transfer of Cer between the ER and the Golgi membranes have suggested
that Cer could be transferred from the ER to the Golgi apparatus through a nonvesicular pathway in ATP-independent and N-ethylmaleimide-insensitive manners (Moreau
et al., 1993
; Kok et al., 1998
).
If ER-to-Golgi apparatus trafficking of Cer is mediated
by only one pathway, it would be a paradox that LY-A
cells synthesize GlcCer to the wild-type level or more, in
spite of the defect of the ATP-dependent ER-to-Golgi apparatus trafficking of Cer. A possible explanation for the
paradoxical phenotype of LY-A cells is that the putative
sole pathway for ER-to-Golgi apparatus Cer trafficking
might be narrowed in LY-A cells, but that the narrowed pathway might still supply enough Cer to produce a normal level of GlcCer in the Golgi apparatus, but not of SM.
However, this explanation is not consistent with the observation that formations of [3H]GlcCer and [3H]GM3 from
[3H]sphingosine were somewhat higher in LY-A cells than
in wild-type cells (Fig. 2). It is unlikely that the reduced
synthesis of SM results from an overconsumption of newly
synthesized Cer at the GlcCer synthesis step, since the
level of [3H]sphingosine-derived Cer was higher in LY-A
cells than in wild-type cells (Fig. 2) and there was no difference in GlcCer synthase activity between the two cell
types (data not shown). Another possibility is that a transbilayer transport of Cer across the Golgi membranes is impaired in LY-A cells. However, the observation that SM
synthesis in LY-A cells was restored to the wild-type level
after brefeldin A treatment (Fig. 5) favors the interpretation that the phenotype of LY-A cells is primarily due to a
defect in the interorganelle transport of Cer, although we
cannot completely exclude the possibility that brefeldin A
treatment facilitates a transbilayer transport of Cer across
the membrane where SM synthase exists, thereby restoring SM synthesis to LY-A cells. Considering that SM synthase as well as ganglioside synthases are suggested to
exist more distally in the Golgi complex than GlcCer synthase (Futerman and Pagano, 1991; Jeckel et al., 1992
; van
Echten and Sandhoff, 1993
), an intra-Golgi apparatus
transport of Cer might be specifically blocked in LY-A
cells. If so, one could expect accumulation of Cer in a pre-
or early Golgi region. However, there was no sign of such
specific redistribution of C5-DMB-Cer in LY-A cells (Fig.
7), although this observation does not conflict with the alternative expectation that a block of the intra-Golgi apparatus transport of Cer causes recycling of C5-DMB-Cer to
the ER instead of its accumulation in the early Golgi apparatus. Note that LY-A cells normally produce GM3 (Fig.
2), eliminating the possibility that intra-Golgi apparatus
transport pathways of various sphingolipid types are nonspecifically blocked in LY-A cells.
Therefore, we considered another possible mechanism that Cer, produced at the ER, is accessible to GlcCer synthase by different pathways from the ATP-dependent trafficking pathway which is impaired in LY-A cells. This mechanism appears to be consistent with the characteristics of LY-A cells; for example, the overflow of Cer to glycosphingolipids in LY-A cells could be mediated by the putative pathway responsible for GlcCer synthesis. The observation that an ATP depletion of wild-type cells caused ~70% inhibition of [3H]sphingosine-derived [3H]SM formation, but only ~20% inhibition of [3H]GlcCer formation (Fig. 10), may also be accounted for by the notion that Cer is efficiently accessible to GlcCer synthase by ATP-independent (or less ATP-dependent) trafficking, although there could be the alternative explanation that a minor population of GlcCer synthase is distributed to the ER in CHO cells, so that Cer produced at the ER is accessible to the ER-distributed GlcCer synthase without an interorganelle transport. Collectively, we propose that there are at least two pathways for Cer trafficking from the ER to the site of de novo SM synthesis: the ATP-dependent major pathway, which is impaired in LY-A cells, and the ATP-independent (or less ATP-dependent) minor pathway(s). This scenario appears to occur in various types of mammalian cells since depletion of intracellular ATP in HeLa cells and human skin fibroblasts affected redistribution of C5-DMB-Cer to the Golgi apparatus and conversion of C5-DMB-Cer to C5-DMB-SM (data not shown), as observed in wild-type CHO cells (Figs. 8 and 9 A).
Two hypothetical models may be conceivable to explain the preferential delivery of Cer to SM synthase by the ATP-dependent mechanism. In one model (Fig. 12 A), a portion of newly synthesized Cer molecules is rapidly internalized to the lumenal side of the ER, and the lumenal Cer is delivered to the Golgi apparatus via an ATP-dependent and vesicle-mediated pathway for SM synthesis, while Cer remaining at the cytosolic side of the ER is delivered to the cytosolic side of the Golgi apparatus via an ATP-independent (or less ATP-dependent) pathway for GlcCer synthesis. In another model (Fig. 12 B), regardless of the Cer topology in the ER membrane, a portion of the Cer molecules enters an ATP-dependent pathway, which is specifically directed to the Golgi subcompartment where SM synthase is localized, while Cer molecules associated with an ATP-independent (or less ATP-dependent) pathway have a broader specificity for their destination and are eventually accessible to both GlcCer and SM synthases. If LY-A cells have a deficiency in an intra-Golgi apparatus transport of Cer, it may be conceivable that the ATP-dependent step for specific delivery to the SM synthase- localizing compartment is located at the intra-Golgi apparatus transport of Cer.
|
Membrane proteins are delivered from the ER to the
Golgi apparatus by vesicular transport mechanisms (Rothman and Orci, 1990), and the same mechanisms for protein trafficking may be involved in the ATP-dependent
trafficking of Cer. However, since LY-A cells exhibit no
appreciable defect in ER-to-Golgi apparatus trafficking of proteins, we currently speculate that the machine defective in LY-A cells could be a specific device for Cer
transport. There might be multiple, independent vesicular
transport pathways from the ER to the Golgi apparatus,
one of which is exclusive for Cer. For further elucidation
of the molecular mechanism underlying the intracellular
trafficking of Cer, additional studies, including identification of the genes related to this function, will be necessary,
and the LY-A strain will be a useful tool for this purpose
as well.
![]() |
Footnotes |
---|
Address correspondence to Kentaro Hanada, Ph.D., Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. Tel.: 81-3-5285-1111, ext. 2126. Fax: 81-3-5285-1157. E-mail: hanak{at}nih.go.jp
Received for publication 24 September 1998 and in revised form 22 December 1998.
We thank Dr. Osamu Kuge of our laboratory for his helpful discussions and Dr. Richard E. Pagano (Mayo Clinic and Foundation, Rochester, MN) for his critical reading of the manuscript and helpful comments.
This work was supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan, CREST of Japan Science and Technology Corporation, and the Naito Foundation, and a Special Coordination Fund for Promoting Science and Technology from the Science and Technology Agency of Japan.
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Abbreviations used in this paper |
---|
Cer, ceramide; DCer, dihydroceramide; Endo H, endoglycosidase H; GlcCer, glucosylceramide; GPI, glycosylphosphatidylinositol; PLAP, human placental alkaline phosphatase; PLAP-HA, a chimera of PLAP with influenza hemagglutinin; SM, sphingomyelin; SM synthase, phosphatidylcholine:ceramide cholinephosphotransferase.
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