THEJOURNAL OF BIOLOGICAL CHEMISTRY
11144-1114S,1992
0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.
Subcellular Localization and Membrane Topology Serine
of
Palmitoyltransferase, 3-Dehydrosphinganine Reductase, and
Sphinganine N-Acyltransferase in Mouse Liver*
Elisabet C. Mandon$, Ingrid Ehses, Jiirgen Rother, Gerhild van Echten, and Konrad Sandhoffg
From the Institut fur Organische Chemie und Bwchemie, Universitat Bonn, Gerhurd-Domagk-StrasseI,
0-5300 Bonn, Germany
Serine palmitoyltransferase, 3-dehydrosphinganine
reductase and sphinganine N-acyltransferase are re-
sponsible for the first steps in sphingolipid biosynthesis
forming 3-oxosphinganine, sphinganine, and dihydro-
ceramide, respectively.We confirmed the localization
of these enzymes in the endoplasmic reticulum (ER)
using highly purified mouse liver ER and Golgi prep-
arations. Mild digestionofsealed “right-side out”
mouse liver ER derived vesicles with different prote-
olytic enzymes under conditions where latency of man-
nose-6-phosphatasewas 90%produced approximately
60430% inactivation of serine palmitoyltransferase,
3-dehydrosphinganine reductase,and sphinganine N-
acyltransferase activities.
Thesesphingolipidbiosynthetic activities (serine
palmitoyltransferase, 3-dehydrosphinganine reduc-
tase, and sphinganine N-acyltransferase) are not la-
tent, indicating that theyface the cytosolic sideof the
ER, so that substrates havefree access to theiractive
sites.
Moreover,themembrane-impermeablecompound,
4,4‘-diisothiocyanostilbene-2,2’-disulfonicacid, which
binds to a large number ofER proteins, inhibitsserine
palmitoyltransferase and sphinganine N-acyltransfer-
ase activities by 30-70%.
Sphingolipids are plasma membrane molecules, containing
a common hydrophobic portion (ceramide) which is anchored
in themembrane and a variable hydrophilic region composed
of phosphocholine for sphingomyelin or complex carbohy-
drate chains for glycosphingolipids (GSL).’ GSL play impor-
* This work was supported in part by the Bundesministerium fur
Forschungund Technologie and by Grant SFB-284 from the Deutsche
Forschungsgemeinschaft. The costs of publication of this article were
defrayed in part by the payment of page charges. This article must
therefore be hereby marked “aduertisement” in accordance with 18
U.S.C. Section 1734 solelyto indicate this fact.
$ A member of the Carrera del Investigador Cientifico, Consejo
Nacional de Investigaciones Cientificas y tbcnicas, Argentina. Sup-
ported by a research fellowship of the Alexander von Humboldt
Foundation in the Federal Republic of Germany. Present address:
Dept. of Biochemistry and Molecular Biology, University of Massa-
chusetts Medical Ctr., 55 Lake Ave. N., Worcester, MA 01655.
5 To whom correspondence should be addressed.
The abbreviations used are: GSL, glycosphingolipids;SPT, serine
palmitoyltransferase (EC 2.3.1.50); DSR, 3-dehydrosphinganine re-
ductase (EC 1.1.1.102); SpAT, sphinganineN-acyltransferase (EC
acid; CHAPS,
2.3.1.24); DIDS, diisothiocyanostilbene-2,2’-disulfonic
3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonic acid;
G-6-Pase, glucose-6-phosphatase; M-g-Pase, mannose-6-phospha-
tase; OGT, ovalbumin galactosyltransferase; 16OCoA, palmitoyl-
coenzyme A; ER, endoplasmic reticulum.
11144
This is an Open Access article under the CC BY license.
Vol. 267,No. 16,Issue of June 5, pp.
Printed in U.S.A.
(Received for publication, December 18,1991)
tant roles in many diverse biological functions including cell
differentiation and morphogenesis (Hakomori, 1984), onco-
genic transformation (Hakomori, 1981), and modulation of
membrane-bound receptors (Bremer et al., 1986).
Sphingosine, sphinganine, and phytosphingosine are the
most frequent long-chain bases found in cellular sphingolipids
(Karlson, 1970). Studies on the biological function of sphin-
gosine and related structures showed that these molecules are
reversible inhibitors of protein kinase C. They may act as
endogenous modulators of cell function and possibly as second
messengers (for review see Hannun and Bell, 1989; Merrill
and Stevens, 1989). Their intracellular levels are therefore
likely to be tightly regulated. The sphingolipid biosynthesis
starts with the condensation of serine and palmitoyl CoA
(16:OCoA) catalyzed by serine palmitoyltransferase (SPT),
yielding 3-dehydrosphinganine. It is immediately reduced by
a D-3-dehydrosphinganine-NADPHoxidoreductase (DSR) to
D-erythro-sphinganine. This long-chain base may be modified
to sphingosine or more likely is used directly in the synthesis
of dihydroceramide as catalyzed by sphinganine N-acyltrans-
ferase (SpAT), which condenses the long-chain base and fatty
acid via amide linkage (Merrill and Wang, 1986): Ceramide
formed by desaturation is then used either as a precursor for
sphingomyelin or glycosylated to glycosphingolipids (for re-
view see Kishimoto, 1983; Radin, 1984). Some evidence exists
that thefirst biosynthetic steps up to theformation of dihy-
droceramide occur in the ERwhereas later steps arelocalized
in the Golgi apparatus.
The sequence of biosynthetic steps is presumed to be ac-
companied by intracellular movement of the stepwise-growing
molecules either by lipid binding proteins and/or by vesicle-
bound membrane flow from the ER through the Golgi cister-
nae to the plasma membrane. The exact localization and
topology of the different enzymes of GSL biosynthesis in the
ER or in the individual Golgi compartments, however, remain
unclear (Schwarzmann and Sandhoff, 1990). Recently, we
have demonstrated that the addition of sphingosine to the
medium of primary cultured cerebellar cells produces down-
regulation of SPT activity in a time-andconcentration-
dependent manner without affecting other enzymatic activi-
ties in the pathway (van Echten et al., 1990; Mandon et al.,
1991). Thus,the down-regulation of SPT by sphingosine
seems to be specific and confirms the key role of this enzyme
in theregulation of sphingolipid biosynthesis. This possibility
has already been considered by others studying the effect of
lipoproteins (Verdery and Theolis, 1982; Chatterjee et al.,
1986) and serum (Merrill et al., 1988) on the levels of cellular
sphingolipids.
J. Rother, G . van Echten, G . Schwarzmann, and K. Sandhoff,
manuscript in preparation.
 Topography of Enzymes Involved in Dihydroceramide Biosynthesis 11145
To continue these studies it is necessary, however, to de- TABLE I
termine the localization and topology of the enzymes of this Localization of serine palmitoyltransferase, 3-dehydrosphinganine
metabolic pathway and to elucidate if the compartmentation reductase, and sphinganine N-acyltransferase in ER-and
Golgi-derived vesicles
plays a role in this down-regulation. We have studied there-
Mouse liver was fractionated into vesicles derived from ER and
fore the localization and topology of the first enzymes, SPT, Golgi according to the procedure described under “Materials and
DSR, and SpAT. Our results demonstrate that the catalytic Methods.” Total activities were obtained by multiplying the specific
centers of these three enzymes are at the cytosolic side of enzymatic activity of each fraction by the milligrams of protein
endoplasmic reticulum. recovered of each and dividing by the yield of corresponding marker
enzyme activity recovered for each fraction (20% of total homogenate
MATERIALS AND METHODS G-6-Pase activity for ER-derived vesicles and 13.6% of the total
homogenate ovoalbumin galactosyl transferase for Golgi).Values
Six-day-old NMRI mice were obtained from Prof. Dr. K. Karzel of shown are means f S.D. of three separate determinations.
the Pharmacological Institute in Bonn. ~-[3-’~C]Serine (55 Ci/mol) ER Golgi
was purchased from Amersham Corp. (Braunschweig, Germany). vesicles
vesicles
Thin layer Silica Gel 60 plates were supplied by Merck (Darmstadt,
Germany). Chymotrypsin (EC 3.4.21.1) from bovine pancreas, 40-60 Protein/fraction (mg) 75 6.5
units per mg protein; elastase (EC 3.4.21.36) from pancreas bovine, OGT Specific activity (pmol/min/ 8.2 -+ 2.1 377 f 11
type IV approximately 70 units per mgof protein; Pronase(EC mg)
3.4.21.19) from Staphylococcus aureus strain V8, 500-1000 units per Total activity (pmol/min) 31 189
mgof solid; trypsin(EC 3.4.21.4) from bovine pancreas, 10,000- Relative total activity (%) 14 86
13,000 benzoyl-L-arginine ethyl ester units per mg protein; and soy- G-6-Pase Specific activity (nmol/min/ 1280 f 75 154 & 14
bean trypsin inhibitor type I-S (1 mg inhibits 1.8 mg of trypsin with
mg)
10,000 benzoyl-L-arginineethyl esterunits per mg protein); and 4,4‘- Total activity (nmol/min) 4810 67
diisothiocyanostilbene-2,2’-disulfonic acid (DIDS) disodium salt were Relative total activity (%) 98 2
purchased from Sigma (Munchen, Germany). All other chemicals
were of analytical grade and obtained from Sigma (Munchen, Ger- SPT Specific activity (pmol/min/ 35.8 f 3.9 5.6 f 1.1
many) or Merck (Darmstadt, Germany). 3-Oxosphinganine was syn- mg)
thesized as described (Schmidt and Zimmermann, 1988).Sphinganine Total activity (pmol/min) 134 2.4
and 3-dehydrosphinganine were tritiated asdescribed (Schwarzmann, Relative total activity (%) 98 2
1978) and had specific activities of 750 and 40 Ci/mol, respectively. DSR Specific activity (pmol/min/ 121 f 5.0 32.5 f 1.3
Zsolutwn of ER and Golgi Vesicles-Mouse liver smooth and rough mg)
ER vesicleswere isolated as described previously by Fleisher and Total activity (pmol/min) 455 14.1
Kevina (1974). Both fractions werepooled and the vesicleswere Relative total activity (%) 97 3
resuspended in Tris-HC1 buffer, 50 mM, pH 7.2, sucrose 0.25 M at SPAT Specific activity (pmol/min/ 48.8 f 5.2 10.6 f 1.6
15-20 mg/ml of protein and frozen immediately in small aliquots at
mg)
-70 “C. Each aliquot was thawed only once. Total activity (pmol/min) 183 4.6
Smooth and rough endoplasmic reticulum-derived vesicles were Relative total activitv (%) 97 3
enriched 5-6-fold over homogenate in glucose-6-phosphatase activity
(10-20% yield of total homogenate activity), 0.9-fold in ovalbumin
galactosyltransferase (1% yield), and the contamination with other
cellular membranes was 2-fold in 5’-nucleotidase (2% yield) and 1-
fold in @-hexosaminidase(0.9% yield).
At least 95% of the vesicles weresealed and of the same membrane
topographical orientation as in vivo based on the latency of glucose- a *x)
P
6-phosphatase toward mannose 6-phosphate, a substrate which can-
not be transported across the liver ER membranes (Arion et al.,1976).
Latency at theend of each incubation is documented in the respective
tables. Golgi vesicles were prepared according to Sandberg et al. (1980)
as described in detail (Yusuf et al., 1983 a, 1983b). The vesicles were
enriched 1-fold in glucose-6-phosphatase (1%yield), 40-fold over
homogenate in ovalbumin galactosyltransferase (15% yield), and the
contamination with other cellular membranes was4.2-fold in 5’-
nucleotidase (1.5% yield) and 1.8-fold in &hexosaminidase (2.5%
yield).
Assays of Enzymatic Activities-The methods employed were sim-
ilar to those used previously to assay SPT and SPAT activities in
mouse cerebella (Mandon et al., 1991) and to measure DSR in rat
liver (Stoffel et al., 1968) except that detergent was excluded from
the standard reaction mixture in order to avoid interference with the
integrity of the vesicles. Solubilization of lipid substrates (labeled 0
sphinganine or 3-dehydrosphinganine) was achieved by sonication in 0 5 1 0 1 5 a o a m a 5
ice-cold bath during 1 min in the incubation medium. Under these paloltoylCaA [pU]
conditions solubilization of 75-80% was obtained, without any alter-
ation of the structure of the molecule. In all cases, the reaction FIG. 1. Dependence of enzymatic activity on palmitoyl-CoA
concentration. Assay mixtures contained all the components of
products were identified by comparing with authentic standards. SpAT, SPT, and M-6-Pase incubation medium. M-6-Pase activity
Disruption of ER-derived vesicles was performed by incubation for (m) was determined after 30 min a t 37 “Cas described under “Mate-
10 min a t 4 “C in the presence of 0.1% Triton X-100 (in case of SPT rials and Methods.” Serine palmitoyltransferase was measured in
and OSR) or alternatively 0.1% CHAPS (incase of SPAT). The final untreated ER vesicles (U) or vesicles preincubated 10 min a t 4 ‘C in
detergent concentration in the respective enzyme assay was 0.025% Tris-acetate 50 mM, pH 7.2, containing 0.1% (w/v) sucrose monolau-
and did not interfere with enzyme activity (see Tables I1 and IV). reate (e). In thepresence of this detergent M-6-Pase was not latent.
Ovalbumin galactosyltransferase (Briles et aL, 1977), glucose-6- Sphinganine N-acyltransferase was measured in untreated ER vesi-
phosphatase, mannose-6-phosphatase, and 5”nucleotidase (Aaron- cles (0)or vesicles preincubated 0.1% (w/v) CHAPS (A).Under these
son and Touster, 1974) and @-hexosaminidase(Storrie and Madden, conditions M-6-Pase was not latent. The experimental data ranged
1990) were measured according to previously described methods. within -+ 10%of the mean values of three experiments.
11146 Topography of Enzymes Involved in Dihydroceramide Biosynthesis
TABLE I1
Accessibility of serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase
on the cytosolic side of ER-derived vesicles
ER vesicles were incubated in the absence and presence of 0.1% Triton X-100 or CHAPS during 10 min a t 4 “C. SPT and SPAT were
determined using 10 p~ palmitoyl-CoA and DSR using 0.25 p~ 3-oxosphinganine. The dataare given as means f S.D. of three experiments.
G-B-Pm M-6-Pase SPT DSR SPAT
pmollminlmg
pmollminlmg
pmollminlmg
nmollminlmg
nmollminlmg
ER vesicles untreated 639 f 42 55 f 12 23 f 2.1 101 f 3 36 f 1.5
ER vesicles treated with 0.1%
962 f 79 872 f 36 28 f 5.1 110 f 7
Triton-X-100
ER vesicles treated 1095
with 0.1% f 76 921 f 91 39 f 2.1
CHAPS

TABLE
I11 and totalactivites of glucose-6-phosphatase (ER marker) and
Effect of different protease treatments on serine palmitoyl transferase, the first enzymes of sphingolipid biosynthesis demonstrates
3-dehydrosphinganine reductase, and sphinganine N-acyltransferase that therelative enrichment of ER vesicles in these activites
activities in intact ER vesicles from m u s e liver was similar. As these values were obtained independently
ER vesicles were preincubated with proteases in 50 mM Tris-HC1 from one another, these resultsstrongly suggest that, for this
buffer (pH 7.4), 50 mM KCl, 0.25 M sucrose at 30 ‘C for 20 min.
Different concentrations of proteases were added in a total volume of particular subcellular fractionation procedure, the activities
400 pI with an ER protein concentration of 10 mg/ml. At the end of of SPT, DSR, SpAT, and glucose-6-phosphatase were copu-
the experiment, the membranes were diluted with 35 ml of ice-cold rifying in similar yield.
50 mM Tris-HC1 buffer, 50 mM KCl, and 0.25 M sucrose, with 4-fold Topology of Serine Palmitoyltransferase, 3-Dehydrosphin-
excess of soybean trypsin inhibitor in the experiment with trypsin, ganine Reductase, and Sphinganine N-Acyltransferase-Al-
and vesicles were isolated by centrifugation for 1 h a t 105,000 X g. though uncharged molecules oflow molecular weight (less
The resulting pellets were resuspended in 100 pl of ice-cold buffer
and assayed immediately. The latency of M-6-Pase remained un- than 600-1000) readily penetrate microsomal vesicles, most
changed (90%)after proteolytic treatment (M-6-Pase activity in charged molecules like palmitoyl-CoA, one of the substrates
intact vesicles 87 nmol/min/mg, in disruptedvesicles 853 nmol/min/ for SPT and SpAT, do not cross the membrane freely (De
mg). However, in vesicles, disrupted by 0.1% Triton X-100 at 4 ‘C Pierre and Dallner, 1975; De Pierre and Ernster, 1977). Fur-
for 10 min, M-6-Pase was susceptible to proteolysis by all proteases thermore, since 16OCoA is an amphipathic molecule, it could
used, as shown before by Waddell and Burchell (1991). Addition of act asa detergent as well as a substrate. A possible lag in the
the assay Components to intact vesicles did not alter the membrane
integrity (M-6-Paselatency remained at approximately 90%). Control palmitoyl-CoA dependence of SPT or SPAT activities may be
values are: SPT, 27 pmol/min/mg; DSR,110 pmol/min/mg; SpAT, caused by the necessity of destroying the permeability barrier
40 pmol/min/mg. The average results of four experiments areshown. of the ER vesicles for expression of the latent enzymatic
activity (Williams et al., 1984).When the 16:OCoA dependence
Protease SPT DSR SPAT
of SPT and SPAT were investigated, no lag in the activities
% of inactivation were observed (Fig. 1).Moreover, the activities do not change
Trypsin 1000 85 ND” ND by pretreating the ERvesicles with detergent.
100 ND
45 ND The hypothesis that theSPT and SPAT activities are latent
50 0 70 50 was tested additionally by examining the effects of 16OCoA
Pronase 50 70 60 ND on ER vesicle integrity and theeffects of detergent disruption
ND 40
40 50 on these vesicles. We have found in agreement with other
25 20 15 60 authors (Polokoff and Bell, 1976)that thelatency of mannose-
10 0 0 30 6-phosphatase does not change after incubation of ER vesicles
Chymotrypsin 200 70 0 ND during 30 min in presence of 10 p~ 16OCoA (Fig. 1). This
100 40 0 60 shows that theintegrity of the ERmembrane was not altered;
Elastase 60 10060 65 thus at10 p~ palmitoyl-CoA only acts as a substrate.
‘ND, not determined. We have performed the SPT and SPAT test using 10 p~ of
16:OCoA in sealed “right-side out” (96% latent toward man-
nose-6-phosphate) mouse liver ER vesicles. Addition of all
RESULTS AND DISCUSSION
other assay components did not alter themembrane integrity
Subcellular Distribution of SPT, DSR, and SPAT-Previous either (e.g. sphinganine, 15 p ~ 3-dehydrosphinganine,
; 5 pM).
studies from different laboratories (Stoffel et al.,1968; Morrell Vesicles werepretreated with 0.1% Triton X-100 or CHAPS
and Radin, 1970; Williams et al., 1984) showed that the and SPT or SPAT were assayed, respectively. As shown in
activities of SPT, DSR, and SPAT are in the microsomal Table I1 no difference was observed in the SPT and SPAT
fraction which represented a mixture of vesicles derived from activities; whereas, under identical conditions, maximal ac-
the smooth and rough endoplasmic reticulum and from the tivities of mannose-6-phosphatase and glucose-6-phosphatase
Golgi apparatus. It was, therefore, important to determine were found only in the disrupted ER vesicles.
whether enzymatic activities were occurring in vesicles de- These data clearly demonstrate that latency does not exist
rived from the ER and/or from the Golgi. Fractions highly in theexpression of S P T or SPAT activities.
enriched in vesicles from the above organelles were obtained We also did not find latency in the expression of DSR
(see “Materials and Methods”). activity in an assay where the concentration of 3-dehydros-
As shown in Table I the ERvesicles are highly enriched in phinganine does not destroy the ER membrane barrier, and
the enzymatic activities. Almost 97%of the enzymatic activity no changes in the activity were observed when sealed and
of the total homogenate was in the ERfraction and approxi- disrupted vesicles wereused (Table 11).
mately 3% in the Golgi. Moreover, a comparison of specific Another approach to determine on which side of the ER
 Topography of Enzymes Involvedin Dihydroceramide Biosynthesis 11147
TABLE IV
Effect of DIDS on glucose-6-phosphutase, serine palmitoyltransferase, and sphinganine N-acyltransferase
in sealed and disrupted ER vesicles from mouse liver
The vesicles were preincubated with 20 PM DIDS for 15 min in 2 ml of 50 mM Tris-HC1 buffer (pH 7.0),50 mM KCl, and 0.25 M sucrose.
The suspension was then diluted to 35 ml with the same buffer a t 4 "C and bovine serum albumin was added (to adsorb DIDS) to a final
concentration of 0.5% following centrifugation at 105,000X g for 1 h. The pellet was resuspended in 100 pl of buffer and assayed immediately.
Vesicles weredisrupted by addition of 0.1% Triton X-100or CHAPS at 4 "C before G-6-Pase and SPTor SPAT were measured, respectively.
Latency of M-6-Pase remained unchanged a t approximately 90% (M-6-Pase activity in intact vesicles was 78 nmol/min/mg, in disrupted
vesicles activity was 807 nmol/min/mg). Values represent means f S.D. of three separate experiments.
G-6-Pase SPT SPAT
Disrupted Sealed Disrupted Sealed
nml/rnin/mg pmol/rnin/mg pml/rnin/mg
Control 307 f 822
53 f36.4
22 f 1.6 32.3 f 2.1
122 f 12.7
110 f 8.5
DIDS 20 IIM 178 f 16
803 f 16 11.2 f 9
3.6
5 It 2.8 31 f 2.8
33 f 0.3
% inhibition
70 75 42 302 30

membrane the catalytic site of these enzymes is located, is to
determine whether transport of the substrate into thelumen
of ER-derived vesicles is a prerequisite for the formation of
the respective product. No transporter for 16:OCoA orfor
other CoA esters has been describedso far. Moreover, studies
on other acyltransferase activities have provided strong evi-
dence that 16OCoA cannot readily penetrate ER vesicles
(Polokoff and Bell, 1978).
Another approach to determine the topology of an enzy-
matic active center is to use mildproteolytic treatment of ER
vesicles. Taking into account the different specificities of
proteases, we have used one of broad (Pronase) and three
with diverse specificity: trypsin (carboxyl side of Arg, Lys),
chymotrypsin (carboxyl side of Tyr, Trp, Phe, Leu) and
elastase (carboxyl side of Gly, Ala, Val, Leu, Ile). Protease
treatment of intact ER vesicles had little effect on mannose-
6-phosphatase activity (Table 111) and did not affect mannose-
6-phosphatase latency. Since this enzyme is a lumenal pro-
tein, these data demonstrate that the ER vesicles remained
intact during protease treatment.
Under these conditions, the enzymatic activites of SPT,
DSR, and SPAT were inactivated between approximately 50
and 80% with the different proteases tested (Table 111). This
considerable loss of enzymatic activities suggests that SPT,
DSR, and SPAT contain residues important for the catalytic
sites exposed onthe cytoplasmic surface of ER vesicles.
The possibility that proteolysis of possible transporter pro-
teins for thesubstrates of these enzymes resulted in an
apparent loss of sphingolipid synthetic activities was elimi-
nated by demonstrating that protease-treated ER vesicles did
not contain latent synthetic activities (data notshown).
Another approach to determine the topology of active sites
of enzymes isto test the effect of the anion-specificinhibitor
DIDS, a nonpenetrating reagent on the enzymatic activity.
It was shownto interact with the cytoplasmaticallyoriented
anion binding sites of different enzymes and transporters
(Cabantchik et al., 1978; Zoccoli and Karnovsky, 1980; Spiro
and Spiro, 1985). We have tested the possibility of DIDS
interaction with residues of SPT and SPAT exposed on the
cytosolic side of ER membranes. The activities of these en-
zymes were found to be affected by the addition of DIDS in
the incubation medium (Table IV), whereas mannose-6-phos-
phatase activity remained 90-92% latent. The glucose-6-phos-
phatase activity was inhibited by 55%. This effect is due to
blocking the transport of glucose-6-phosphate across the ER
vesicles to the lumenal membrane surface where the hydro-
lytic site of glucose-6-phosphataseis exposed. This inhibition
can be reversed by disruption of the ER membrane by deter-
gent (Table IV). In contrastwe did not observe a reversibility
of DIDS inhibition on SPT and SPAT activities in disrupted
ER membranes (Table IV). This fact rules out thepossibility
of a block in thehypothetic 16OCoA transport across the ER
vesicles.
We conclude from our experiments that the enzymes which
synthesize 3-dehydrosphinganine,sphinganine, and dihydro-
ceramide act at the cytoplasmatic side of the endoplasmic
reticulum. This implicates a transport of dihydroceramide
and/or of ceramide to the cytosolic and the lumenal sites of
the early Golgi compartments where the biosynthesis of glu-
cosylceramide (Coste et al., 1986) and sphingomyelin (Futer-
man et al., 1990),respectively, occurs.
The degradation of sphingoid bases released after break-
down of sphingolipids starts in the cytosol withthe formation
of sphingosine 1-phosphate (Hirschberg et al., 1970) and
continues at the cytosolic sideof the ER with the cleavage by
sphingosine-1-phosphate-lyase(van Veldenhoven and Man-
naerts, 1991). This suggests that the intracellular levels of
sphingoid bases could be regulated very fast within the same
compartment by degradation or by N-acylation catalyzed by
SpAT.
Acknowledgments-We thank Prof. A. H. Merrill, Jr. (Emory Uni-
versity School of Medicine, Atlanta, GA) and Prof. C. Hirschberg and
Dr. C. Abeijon (University of Massachusetts Medical Center, Worces-
ter, MA) for critical discussions. Bernd Liessem's advice concerning
presentation of data is gratefully acknowledged.
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