Download The ins and outs of sphingolipid synthesis

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TRENDS in Cell Biology
Vol.15 No.6 June 2005
The ins and outs of sphingolipid
synthesis
Anthony H. Futerman1 and Howard Riezman2
1
2
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel
Department of Biochemistry, University of Geneva, 30 quai E. Ansermet, CH-1211 Geneva 4, Switzerland
Sphingolipids are ubiquitous components of eukaryotic
cell membranes, where they play important roles in
intracellular signaling and in membrane structure. Even
though the biochemical pathway of sphingolipid synthesis and its compartmentalization between the endoplasmic reticulum and Golgi apparatus have been
known for many years, the molecular identity of the
enzymes in this pathway has only recently been
elucidated. Here, we summarize progress in the identification and characterization of the enzymes, the transport
of ceramide from the endoplasmic reticulum to the Golgi
apparatus, and discuss how regulating the synthesis of
sphingolipids might impact upon their functions.
Introduction
The past few years have seen an upsurge of interest in
sphingolipids (SLs) – membrane lipids containing a
ceramide backbone – resulting in a vast increase in our
understanding of the roles that they play in signaling
events and in membrane lipid rafts. In addition, there has
been remarkable progress recently in identifying the
enzymes involved in the de novo synthesis of SLs, and a
more-or-less comprehensive picture of the route taken by a
SL can now be delineated from initiation of its biosynthesis on the cytosolic leaflet of the endoplasmic reticulum
(ER), through its transport from the ER to the Golgi
apparatus, and its metabolism in the Golgi apparatus.
Previous reviews have focused on a particular enzyme
[1], transport step [2,3], or a particular aspect of SL
function [4,5], whereas the current review presents an
overview of all steps of SL synthesis and transport
through the early compartments of the secretory pathway.
To this end, we systematically discuss the enzymes of the
SL synthetic pathway (Figure 1), what is known about the
topology of the reactions, and the mechanisms of transport
of ceramide from the ER to the Golgi apparatus.
Sphingolipid structure, synthesis and transport
SLs consist of three main structural elements (see insert
of Figure 2). The basic building block of a SL is the
sphingoid long-chain base (lcb), normally sphingosine,
sphinganine (dihydrosphingosine) or 4-hydroxysphinganine (phytosphingosine). A fatty acid is attached to
carbon-2 (C-2) of the lcb via an amide bond, yielding
ceramide, and attachment of hydrophilic head groups to
Corresponding author: Futerman, A.H. ([email protected]).
Available online 30 April 2005
the OH-group at C-1 yields complex SLs. The head group
can be a sugar, in the case of glycosphingolipids (GSLs),
or phosphorylcholine in the case of sphingomyelin (SM).
At least five different lcbs are known in mammalian cells,
O20 species of fatty acid (varying in chain length, degree
of saturation, and degree of hydroxylation) can be
attached to the lcb, and hundreds of different carbohydrate structures have been described in GSLs. The
possible relevance of this complexity is discussed elsewhere [6], but a central question in SL cell biology is how
the synthesis of these different SL species is regulated,
and how changing their levels, through regulation of
de novo synthesis, might impact upon the various cellular
events in which SLs play key roles.
Unlike synthesis of the other major membrane lipids,
sterols and glycerolipids, which takes place mainly in the
ER, the initial steps of SL synthesis take place in the ER but
later steps take place for the most part in the Golgi
apparatus; moreover, specific steps of SL synthesis occur
on opposite surfaces of these organelles (lumenal versus
cytosolic). Of the 11 enzymes in the SL biosynthetic pathway
discussed below, eight are located in the ER and three in the
Golgi apparatus; of these, the active site of most appears to
face the cytoplasm, but some face the lumen. This implies
significant transbilayer and interbilayer movement of SLs
during the biosynthetic process, and, as these lipids do not
appear, on the whole, to spontaneously undergo such
movements [2], facilitated mechanisms are probably
involved in their transport. The reasons for these complex
topological relationships are not entirely clear, although, in
some cases, such as SM and GSLs, synthesis in the Golgi
lumen can be rationalized by the topological equivalence of
the lumenal leaflet of the Golgi apparatus with the outer
(extracellular) leaflet of the plasma membrane, where SM
and GSLs mainly reside. By contrast, the reason that
glucosylceramide (GlcCer) is synthesized on the cytosolic
leaflet of the Golgi apparatus, whereas all other GSLs are
synthesized on the lumenal leaflet of the ER or Golgi
apparatus, is less easy to rationalize.
We now discuss each of the SL biosynthetic enzymes in
their order of action during SL trafficking along the
biosynthetic pathway. A relatively simple biochemical
overview of SL biosynthesis in given in Figure 1, and
another level of complexity is introduced in Figure 2, in
which the location and topology of each of these reactions
in the ER and Golgi apparatus, together with the transport mechanisms of SLs, is shown.
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Review
TRENDS in Cell Biology
Vol.15 No.6 June 2005
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Palmitoyl CoA + Serine
Serine
(i)
palmitoyltransferase
(v)
3-ketosphinganine
(iii)
Sphingosine-13-ketosphinganine
Sphingosine
phosphate
(ii)
reductase
kinase
Sphinganine- lyase Hexadecanal +
Sphinganine
ethanolamine
Sphingosine-1- 1-phosphate
Dihydroceramide
phosphate
(vi)
phosphate
synthase
Dihydroceramide phosphatase
(iv)
Dihydroceramide
(vii)
desaturase
Ceramide
(x) GlcCer
CGalT
SM synthase
synthase
(viii)
(ix)
Sphingomyelin
Glucosylceramide Galactosyl(xi)
ceramide
Complex GSLs
TRENDS in Cell Biology
Figure 1. The biosynthesis of sphingolipids (SLs) and glycosphingolipids (GSLs). The biochemical pathways of SL biosynthesis are shown, with enzymes in italics. The
numbers in boxes refer to the enzymatic reactions, as in Figure 2, and are referred to throughout the text when describing each specific reaction.
Serine palmitoyl transferase (SPT) (Reaction i)
SL synthesis begins with the conversion of serine and
fatty acyl CoA into 3-ketosphinganine, CoA and CO2 by
serine palmitoyl transferase (SPT) (Figure 1). The
predominant fatty acid used in mammals and yeast is
palmitoyl CoA, but stearyl CoA can also be used. SPT
belongs to a family of pyridoxal 5 0 -phosphate-dependent
a-oxoamine synthases, and is composed of a heterodimer
of two similar, but non-identical, subunits, named Lcb1p
(long chain base 1) and Lcb2p, which were first identified
in yeast by genetic methods [7,8].
Eukaryotic SPTs are membrane bound, with the active
site facing the cytoplasm (Figure 2). Studies of the
mammalian enzyme originally suggested a single-pass
transmembrane protein, with the C-terminus in the cytoplasm [9], but these data are not inconsistent with recent
studies in yeast suggesting that the enzyme actually
traverses the membrane three times [10], as also predicted
for an Arabidopsis plant SPT [11]. These data suggest that
the SPT1 and SPT2 subunits of the SPT heterodimer
might interact in the cytosol, as well as within the
membrane and/or the lumen of the ER [10].
In addition to Lcb1p and Lcb2p, another small protein,
Tsc3p, binds to yeast SPT and is required for optimal
enzyme activity and for cell growth at 37 8C [12]. The
growth requirement could be due to a need for increased
sphingolipid synthesis at the higher temperature. However Tsc3p is not indispensable for SPT activity, and
dominant mutants in LCB2 have been identified that
eliminate the need for TSC3 [13], suggesting that Tsc3p is
a regulator of SPT. No similar subunit has been detected
thus far in other cell types.
3-Ketosphinganine reductase (3KSR) (Reaction ii)
3-Ketosphinganine reductase (3KSR) reduces 3-ketosphinganine to dihydrosphinganine in an NADPH-dependent manner. Yeast 3KSR is predicted to have two
transmembrane domains near the C-terminus, but the
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human ortholog, follicular lymphoma variant translocation-1 (FVT-1), has an additional transmembrane domain
near the N-terminus [14]. The catalytic site is sensitive to
proteases in intact microsomes and thus probably exposed
to the cytosol [15]. Thus, no topological problems exist for
the first two enzymes of the pathway as SPT uses soluble
precursors to form 3-ketosphinganine on the cytoplasmic
leaflet of the ER, and the active site of 3KSR also faces the
cytosol.
Sphingosine kinase (SK) (Reaction iii)
Subsequent to its formation by 3KSR, sphinganine can
either be acylated by dihydroceramide synthase (DHCerS,
see Reaction vi below), or phosphorylated by sphingosine
kinase (SK) to form sphingosine 1-phosphate (S1P), an
important bioactive lipid mediator [16]. SKs were originally identified in yeast as products of two genes, LCB4
and LCB5, required for the efficient utilization of exogenously added sphingoid bases [17]. Lcb4p is principally
required for this process and is found on the cytoplasmic
face of internal membranes, including the ER, Golgi and,
probably, endosomes [18,19]. Two mammalian isoforms,
SK1 and SK2, have been identified that differ in their
substrate specificity and possibly also in other functional
aspects [20]. SK1 seems to be a soluble enzyme that
translocates to membranes upon interaction with phosphatidic acid, which binds near to the C-terminus of the
enzyme [21]. SK2 has been reported to have a nuclear
targeting signal, although the relevance of this is
currently unknown [22].
S1P phosphatase (SPP) (Reaction iv) and S1P lyase (SPL)
(Reaction v)
Once formed in the cytoplasm, S1P can be degraded by
either of two enzymes, S1P phosphatase (SPP), which
removes the phosphate head group from sphinganine, or
S1P lyase (SPL), which yields the cleavage products,
hexadecanal and ethanolamine phosphate, the major exit
Review
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Vol.15 No.6 June 2005
OH
SPT2
CH2
+
N
(i)
SPT1
NH
H0
O
N
C
N
3KSR
(ii)
GCFlip
C
SK
(x)
GCS
DHCerS
(iii)
C
(vi)
P
P-
GalT1 (xi)
N
(vii)
SPP
(iv)
Vesicular
transport
CERT
DHCD
CERT
N
SMS (ix)
SPL
P
+
(v)
CGalT
(viii)
CERT
C
C
ER
Golgi
TRENDS in Cell Biology
Figure 2. The sites and topology of sphingolipid (SL) metabolism in the endoplasmic reticulum (ER) and Golgi apparatus. Enzymes, transporters and steps of SL synthesis or
transport are abbreviated as in the text and numbered as in Figure 1: SPT, serine palmitoyl transferase; 3KSR, 3-ketosphinganine reductase; SK, sphingosine kinase; SPP,
sphingosine 1-phosphate phosphatase; SPL, sphingosine 1-phosphate lyase; DHCerS, dihydroceramide synthase; DHCD, dihydroceramide desaturase; CGalT,
UDP-galactose:ceramide galactosyltransferase; CERT, ceramide transfer protein; SMS, SM synthase; GCS, GlcCer synthase; GCflip, GlcCer flippase; GalT1, LacCer synthase.
FAH and ACB1p are not shown. Solid arrows indicate enzymatic reactions, and dashed arrows indicate transport steps. The topologies of the reactions, where known, are
shown in green circles, and steps for which the topology is not known, or the protein not unambiguously identified, as green rectangles. The number of transmembrane
domains of integral membrane proteins is shown when it has been determined or predicted, together with the putative cytosolic or lumenal loops, but is not to scale.
Likewise, the C- and N-termini of enzymes for which the topology has been established are indicated, and not shown for those for which the topology is still unclear. The
insert shows the structure of ceramide, and is color-coded according to the SLs shown in the scheme: Blue, sphingoid long chain base; thin blue line, 3-OH (as in
3-ketosphinganine); black, fatty acid; white line, 4,5-double bond in sphingoid base; black circle and line, sugar head group (i.e. glucose, galactose, or GlcGal in the case of
LacCer); purple rectangle, phosphorylcholine (as in SM); P, phosphate. Further details regarding each step are given in the text.
route of metabolites from the SL biosynthetic pathway.
Similar to many other components of this pathway, two
SPP isozymes, which both contain a typical lipid phosphatase motif, were initially identified in yeast and, more
recently, in mammals [16]. The major yeast SPP, Lcb3p, is
an ER integral membrane protein that probably spans the
membrane eight or nine times, with its active site
proposed to be on the lumenal side of the ER [23]. Gene
fusions with invertase were used to determine the
topology of the enzyme and, although the data seem to
be internally consistent, it is impossible to know whether
the topology predictions are entirely accurate because the
methodology used leads to the loss of enzyme activity. The
active site of SPL, which is also an integral ER membrane
protein, faces the cytoplasm [24].
Dihydroceramide synthase (DHCerS) (Reaction vi)
Dihydroceramide synthase (DHCerS) acylates sphinganine to form dihydroceramide, but can also acylate
sphingosine to ceramide; sphinganine is produced through
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the biosynthetic pathway, and sphingosine mainly
through SL degradation [25]. In contrast to yeast, whose
SLs contain only one kind of fatty acid, mammalian
(dihydro)ceramides contain a wide spectrum of fatty acids
[6,25], and it was formerly assumed that this was due to a
lack of specificity of DHCerS with respect to the use of
fatty-acyl CoAs. However, recent studies have shown that
this is not the case. Rather, there is a family of mammalian
DHCerS genes, and each displays a remarkably high
specificity in utilizing acyl CoAs (Figure 3).
Clues to the identity of DHCerS were originally
obtained in yeast, in which it was shown that the two
proteins Lag1p and Lac1p were required for the synthesis
of the very long-chain (C26) ceramides found in yeast
[26,27]. Subsequent to this, tomato (asc1) [28] and mammalian [29] homologs were identified, some of which
contain a Hox domain (Figure 3), a transcription factor
involved in developmental regulation; although there is
currently no evidence to support this possibility, it would
be surprising if the Hox domain did not play an important
Review
Current
name
Old
name
TRENDS in Cell Biology
Fatty acids Hox
in ceramide domain
% Identity
Lag1/Lac1
LASS1
UOG1
LASS4
TRH1
C26
C18
C18/C20
Absent
Absent
74–132
versus
Lag
72 (lac)
27
30
LASS3
T3l
Unknown
73–131
24
LASS2
TRH3
Unknown
71–132
24
LASS6
T1l
Unknown
74–131
27
LASS5
TRH4
C16
82–140
25
??
Absent
0
CLN8
TRENDS in Cell Biology
Figure 3. Phenogram showing the relationship between LASS genes. The new
LASS terminology is listed next to the original terminology [29–32]. The fatty acids
used for dihydroceramide synthesis by each of the genes are indicated where
known. Amino acid residues encompassing the Hox domain are shown for mouse
homologs. The percent amino acid identities of the mouse homologs compared
with the yeast Lag gene product are also shown. CLN8 is a member of the same
family, but it is not known whether this protein is involved in SL synthesis.
role in one or other aspect of the regulation of SL
synthesis, perhaps similar to the role of the sterol
regulatory element binding protein (SREBP), in which a
transcriptional activation domain is released by proteolysis when sterol levels are low [30]. Quite unexpectedly,
overexpression of one of the homologs, upstream of growth
and differentiation factor 1 (UOG1), increased the amount
of dihydroceramide containing only one type of fatty acid,
namely stearic acid [29]. Other family members were then
identified [30,31] [recently renamed as LASS genes
(Longevity assurance gene homologs) (Figure 3)], and
overexpression of two of these [32] also led to an increased
amount of dihydroceramide synthesis, with a distinct fatty
acid profile. Thus, to date, three of the six mammalian
LASS genes have been characterized, and each synthesizes dihydroceramide containing distinct fatty acids
(Figure 3). When some of these genes were expressed in
yeast defective in LAG1 and LAC1, they were able to
reconstitute ceramide synthesis [33].
Ceramide synthase was recently purified in active form
from yeast, and a new subunit of the enzyme, Lip1p, was
identified [34]. Lip1p is a single-span ER membrane
protein required for ceramide synthesis activity in vivo
and in vitro. No mammalian homolog of Lip1p has been
found, and it is not known whether mammalian LASS
homologs require additional subunits. Although (dihydro)
ceramide synthesis has been assumed to occur on the
cytosolic surface of the ER [2], based on protease protection
experiments [15], the Lip1p regions required for (dihydro)
ceramide synthesis are localized within the ER membrane
or lumen. Topology predictions suggest that Lag1p and
the LASS proteins span the ER membrane multiple (seven
or eight) times, but the topology of the catalytic site of
DHCerS has not been unambiguously identified.
Interestingly, sphinganine can be phosphorylated by
SK on the cytoplasmic leaflet (Reaction iii), and, when
exogenously added sphinganine is fed to cells or incubated
in vitro with ER membranes in yeast, it is phosphorylated
by SK (Lcb4p) [18,35] before its acylation, and then
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dephosphorylated by SPP (Lcb3p), presumably on the
lumenal side of the ER membrane, as discussed above.
Therefore, as endogenous sphinganine is produced on the
cytosolic face and is acylated without being first metabolized to SPP, either DHCerS can utilize sphinganine pools
generated on either side of the ER membrane or the
hypothesis for the topology of the SPP is incorrect. The
rationale for the phosphorylation/dephosphorylation of
exogenously added sphinganine is uncertain, but it is
probably used to differentiate sphinganine arising from
different pools, presumably for regulation of SL synthesis
or signaling.
Fatty acid 2-hydroxylase (FAH) and acyl-CoA-binding
protein (Acb1p)
Two other proteins involved in fatty acid supply and
modification are important for SL synthesis, at least in
yeast. In yeast, most SLs are hydroxylated on the 2 position of the fatty acid by a process requiring the ceramide
2-hydroxylase gene (FAH1). The protein encoded by this
gene, Fah1p, has a consensus desaturase/hydroxylase
motif and a cytochrome b5 domain, and membrane
topology predictions suggest that these motifs are on the
cytoplasmic side of the ER membrane. A human ortholog
encodes a 2-hydroxylase that can work on free fatty acids,
probably of various chain lengths, whereas the yeast
enzyme appears to work on fatty acids only once they are
attached to ceramide [36]. The essential fatty acyl CoA
binding protein, Acb1p, is also required for efficient
ceramide synthesis in yeast [37], and the mammalian
Acb1p homolog is essential for the viability of mammalian
tissue-culture cells [38]. Acb1p is most likely essential for
efficient delivery or presentation of very long-chain fatty
acids to the ceramide synthase in yeast.
Dihydroceramide desaturase (DHCD) (Reaction vii)
The enzyme responsible for the formation of the 4,5-double
bond of the sphingoid lcb, dihydroceramide desaturase
(DHCD), was identified by a bioinformatics approach [39],
and its identity was subsequently confirmed by expression
studies. Protease digestion studies and its being part of a
cytochrome b5 electron transport system suggest that the
active site of this enzyme faces the cytosol [40], which is
consistent with topology predictions implying three
transmembrane domains [41,42].
UDP-galactose:ceramide galactosyltransferase (CGalT)
(Reaction viii)
Once formed in the ER, ceramide can be removed from the
ER by active transport mechanisms (see below), or
metabolized to galactosylceramide (GalCer). The enzyme
responsible for this reaction, UDP-galactose:ceramide
galactosyltransferase (CGalT), is located in the ER, and
is a type 1 membrane protein, with the N-terminus and
active site in the lumen [43]. The UDP-galactose transporter, which is required for GalCer synthesis, might form
a molecular complex with CGalT [44].
SM synthase (SMS) (Reaction ix)
Sphingomyelin synthase (SMS) transfers the phosphorylcholine head group from phosphatidylcholine to ceramide,
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to yield SM and diacylglycerol. A major advance over the
past year was the molecular identification of SMS. Based
on a functional cloning strategy in yeast [45], and an
expression cloning strategy in WR19L cells [46], two SM
synthases were identified, which have different locations;
SMS1 is in the Golgi apparatus and SMS2 on the PM. The
active site of SMS1 faces the lumen of the Golgi apparatus,
and topology predictions suggest that this protein spans
the membrane four [46] or six [45] times, with the N- and
C-termini facing the cytosol.
GlcCer synthase (GCS) (Reaction x)
Glucosylceramide synthase (GCS) was first identified in
1996 [47], and subsequent work demonstrated that it is a
type III membrane protein, with the C-terminus located in
the lumen of the Golgi apparatus. Its active site faces the
cytosol, which is consistent with biochemical analyses
performed before the enzyme was cloned [1]. Drosophila
[48] and plant [49] homologs of GCS have also been
identified. Expression of the Drosophila homolog in GM95
cells (mammalian cells deficient in GCS) restores GlcCer
synthesis and the synthesis of downstream complex GSLs,
but the Drosophila enzyme is surprisingly localized to
both the Golgi and the ER [48].
For both SMS and GCS, ceramide must be transported
from the ER to the Golgi apparatus. One of the most
exciting developments in this field over the past couple of
years was the identification of a protein, CERT, which
mediates the transport of ceramide from the ER to the
Golgi apparatus in a non-vesicular manner [50]. CERT is a
cytoplasmic protein with a phosphatidylinositol-4-monophosphate-binding domain and a putative domain for
catalysing lipid transfer. This protein specifically extracts
ceramide from phospholipid bilayers and shows very little
activity towards other related SLs or glycerolipids. CERT
also extracts dihydroceramide but displays a relatively
restricted fatty acid specificity, efficiently mediating the
transfer of ceramides containing C14–C20 fatty acids but
is less efficient towards ceramide containing longer-chain
fatty acids [51]. CERT is involved in the delivery of ceramide for SM synthesis [52], but not for GlcCer synthesis,
which seems to depend on the supply of ceramide through
a distinct ATP- or cytosol-independent (or less dependent)
pathway [53]. Thus, at least two mechanisms exist for
delivering ceramide from the ER to the Golgi apparatus.
Once delivered to the Golgi apparatus, ceramide needs
to be translocated to the lumen for SM synthesis. Studies
with ceramide analogs suggest that its rate of spontaneous
transbilayer movement is significantly quicker than that
of structurally related lipids [2], but a direct measurement
of the rate of transbilayer movement of natural, long-acylchain ceramide is not available. However, recent studies
have demonstrated that the asymmetric addition of ceramide to one side of a lipid bilayer promotes transbilayer
movement [54,55]. These observations are likely to result in
re-evaluation of traditional views concerning lipid bilayer
stability and imply that changes in the lipid composition
itself, without the need for membrane proteins, might be
sufficient to drive lipid transbilayer movement.
Although spontaneous transbilayer movement or
‘scrambling’ might be sufficient to account for ceramide
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Vol.15 No.6 June 2005
flip-flop, it is definitely not sufficient to account for GlcCer
translocation. GCS is unique among glycosyltransferases
in that its active site faces the cytosol, and thus GlcCer
must be translocated across the Golgi membrane for
subsequent metabolism to lactosylceramide (LacCer) by
GalT1 (Reaction xi), which is found on the lumenal leaflet
of the Golgi apparatus [56,57]. Biophysical studies
demonstrate a rapid, protease-sensitive transbilayer
movement of GlcCer in rat liver ER and Golgi membranes,
whereas transbilayer movement is much slower at the PM
[58]. This movement occurs in both directions (i.e. from
the cytosolic leaflet to the lumenal leaflet, and vice versa),
implying that GlcCer exists with a symmetric distribution
in the Golgi apparatus. As no significant transbilayer
movement of LacCer occurs [58], the metabolism of GlcCer
to LacCer on the lumenal leaflet would act to trap LacCer
on this surface and make it available for subsequent
glycosylation steps.
There is some controversy about the molecular identification of the flippase responsible for transbilayer movement of GlcCer [59]. It has been suggested that the
multiple drug resistance pump, MDR1, is the GlcCer
flippase in the Golgi apparatus, but other proteins might
also be involved. However, inhibition of MDR1 by
cyclosporin A inhibits LacCer and globotriaosyl ceramide
synthesis, but not ganglioside synthesis [59]. This
suggests that neutral and acid GSLs are synthesized
from distinct precursor GSL pools. Nothing is currently
known about putative distinct GlcCer pools in the Golgi
apparatus.
Moving forward
The molecular identification of most of the components in
the SL synthetic pathway is a major achievement and
illustrates that the description of a biochemical pathway
by itself (as in Figure 1) is not sufficient to understand, or
even to ask the correct questions, about how a pathway is
regulated; for this, the cellular context must also be
known (as in Figure 2). With these tools in hand, it is now
possible to turn to questions that, to date, have been
inaccessible. Central among these is why the location and
topology of SL synthesis is so complex, with different
reactions occurring in either the ER or Golgi apparatus,
and with distinct topologies. One way to address this
would be to alter the location of specific enzymes, and then
determine the effect, first, on levels of SL synthesis, and
then on downstream events in which they are involved.
For instance, what would happen if SMS was targeted to
the ER or if DHCerS was targeted to the Golgi apparatus?
In the case of the latter, how would the activities of SMS
and GCS be altered by alleviating the need for ceramide
transport through either CERT or vesicular transport?
Would DHCerS be accessible to the same pool(s) of
sphinganine if it were located in the Golgi apparatus?
How would this impact upon the supply of sphinganine for
SK? Although answers to this type of question would not
by themselves provide an unambiguous answer to the
question of why the cellular pathways of SL synthesis are
so complex, it would allow us to begin to unravel the
relationships between the different enzymes, and the
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TRENDS in Cell Biology
relationships between different substrate pools, a required
first step in addressing this question.
Does the description of the location and topology of the
enzymes in the SL biosynthetic pathway also provide clues
as to how this pathway is regulated? Intuitively, the
answer to this question is ‘yes’, but, when pressed for
specifics, it becomes harder to provide firm answers as to
how regulation might be achieved. Clearly, multiple levels
of regulation exist, which would not be the case were the
enzymes all located in the same compartment and with
the same topology. For instance, altering the activity of
CERT would directly impact upon SM synthesis and
would therefore change the balance between GSL and SM
levels. Likewise, altering the activity of the GlcCer
flippase, or of ceramide transbilayer movement, would
have major consequences for their supply to upstream
enzymes. Little experimental evidence exists at present
for these modes of regulation, but it seems inevitable that
discoveries concerning the regulation of each step are just
around the corner, particularly in the light of recent
studies showing that de novo synthesis of SLs, particularly ceramide, is regulated in various signaling paradigms. One candidate for this kind of regulation is
DHCerS, which is the only enzyme in which multiple
isoforms (the LASS genes) exist (Figure 3); this presumably indicates that the type of fatty acid added to the
sphingoid long-chain base is of great importance in the
intracellular signaling pathways in which ceramide is
involved, and evidence is slowing accumulating that this
is indeed the case [6]. Different LASS genes display
different tissue distributions, and the presence of a Hox
domain in five of the mammalian homologs potentially
provides an important regulatory mechanism.
Concluding remarks
In summary, the molecular identification of the enzymes
involved in SL synthesis allows both the formulation of
relevant questions, mainly concerning regulation, and will
also provide the tools to answer them. The availability of
yeast mutants and the possibility of up- or down-regulating
expression of mammalian homologs of the enzymes will
permit new approaches to study the function of SLs. Thus,
this is the beginning of an exciting new era in the study
and discovery of the intricacies of SL function.
Acknowledgements
Tony Futerman is the Joseph Meyerhoff Professor of Biochemistry at the
Weizmann Institute of Science, and his work was supported by the Israel
Science Foundation. Howard Riezman’s work was supported by grants
from the Swiss National Science Foundation. A.H.F. and H.R. were
supported by an EC network grant HPRN-2000-00077.
References
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