REVIEWS

THE SREBP PATHWAY — INSIGHTS

FROM INSIGS AND INSECTS
Robert B. Rawson
Animal cells coordinate lipid homeostasis by end-product feedback regulation of transcription.
The control occurs through the proteolytic release of transcriptionally active sterol regulatory
element binding proteins (SREBPs) from intracellular membranes. This feedback system has
unexpected features that are found in all cells. Here, we consider recently discovered
components of the regulatory machinery that govern SREBP processing, as well as studies in
Drosophila that indicate an ancient role for the SREBP pathway in integrating membrane
composition and lipid biosynthesis.

SREBP
(Sterol regulatory element
binding protein). A transcription
factor in animal cells that is
necessary for the transcriptional
activation of genes required for
lipid synthesis.
RIP
(Regulated intramembrane
proteolysis). The cleavage of a
protein within a membrane-
spanning helix to release a
functional domain. This
cleavage usually follows an
initial, regulated cleavage that
occurs in the extracytoplasmic
domain of the substrate.
S1P
(Site-1 protease). A membrane-
anchored subtilisin-like serine
protease of the secretory
pathway, the active site of which
is in the lumen.
Department of Molecular
Genetics, University of Texas
Southwestern Medical
Center, 5323 Harry Hines
Boulevard, Dallas,
Texas 75390, USA.
e-mail: Rob.Rawson@
UTSouthwestern.edu
doi:10.1038/nrm1174
In the decade since the discovery of the sterol regulatory
element binding proteins (SREBP ), a powerful combina-
S the transcriptionally active amino-terminal domain2.
tion of genetics, biochemistry and molecular biology
has exposed fascinating details of how lipid homeostasis
is regulated in animal cells. These studies have revealed
a beguilingly complex signalling mechanism for the
end-product feedback regulation of gene transcription.
In addition to providing the first example of intracellu-
lar trafficking under explicit metabolic control, these
studies have also uncovered a surprising feature of the
SREBP pathway that is broadly used in cellular sig-
nalling by a wide range of organisms — regulated
intramembrane proteolysis (RIP)1.
The defining feature of the SREBP pathway is the
proteolytic release of a membrane-bound transcrip-
tion factor, SREBP, freeing it to move to the nucleus
and upregulate the transcription of target genes (FIG. 1).
The SREBP precursor protein (which has a molecular
mass of ~120 kDa) is anchored in the membranes of
the endoplasmic reticulum (ER) and nuclear enve-
lope by its two membrane-spanning helices. This pre-
cursor protein adopts a hairpin orientation in the
membrane, so that both the amino-terminal tran-
scription-factor domain and the carboxy-terminal
regulatory domain face the cytoplasm (FIG. 1). The two
membrane-spanning helices are separated by a loop
of ~30 amino acids that faces the lumen of the ER.
Two separate cleavages, which are carried out by
two distinct proteases — site-1 protease (S1P) and
site-2 protease (S2P) — are necessary for the release of
In addition to S1P and S2P, the regulated release of
transcriptionally active SREBP also requires SREBP-
cleavage-activating protein (SCAP), which forms a com-
plex with the SREBP precursor protein owing to an
interaction between their respective carboxy-terminal
domains. FIGURE 2 shows a diagram of the predicted
topology of these proteins.
The control of hydrolytic activity presents cells with
specific challenges; at the right time and place it might
be absolutely essential, but inappropriate activity can
kill the cell. Regulation of SREBP cleavage makes use of
a notable feature of eukaryotic cells — subcellular com-
partmentalization delimited by the endomembrane
system — to ensure that cleavage occurs only when it
is needed. Feedback regulation of SREBP processing is
best understood in the case of cholesterol. When the
cellular demand for cholesterol rises, the SREBP–SCAP
complex leaves the ER by inclusion in COPII vesicles3,4
and travels to the Golgi apparatus where the
SREBP–SCAP complex encounters active S1P. Here,
S1P cleaves the SREBP precursor protein into two
halves, but because each half contains a membrane-
spanning helix, each remains bound to the membrane
(FIG. 1). The newly generated amino-terminal half of
SREBP, termed the intermediate form, is then cleaved at
a site within its membrane-spanning helix by an
unusual metalloproteinase, S2P. This releases the

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REVIEWS

SREBP mechanisms by which the composition of cellular mem-

bH
LH g branes influences the transcription of biosynthetic genes.
-z
ip Re

Cytoplasm Components of the SREBP pathway

bHLH-zip
The SREBPs were identified by biochemical methods,
ER Nucleus whereas complementation and expression cloning in
Lumen mutant cells with global defects in cholesterol home-
ostasis enabled the identification of other components
of the processing machinery7. The use of mutant cells
Sterols has also been crucial in the subsequent functional analy-
sis of these proteins. Recently, biochemical approaches
bH
bH bH
LH have returned to the fore with the identification of the
-z
LH LH ip
-z Re
g
-z Re
g Insig proteins.
ip ip
Cytoplasm
SREBP. Wang and colleagues in the Brown and
S1P S1P Site-2 S2P Goldstein laboratory purified SREBPs from HeLa cell
Golgi nuclei, and identified them as proteins that bind to the
Lumen sterol regulatory element (SRE)8,9. The SREs are DNA
Site-1 motifs that are found in the promoters of genes, such as
those encoding 3-hydroxy-3-methylglutaryl coenzyme A
Figure 1 | Two-step processing of SREBP. The sterol regulatory element binding protein
(HMG CoA) synthase and the low-density lipoprotein
(SREBP) precursor is inserted into membranes of the endoplasmic reticulum (ER). Both the
amino-terminal transcription-factor domain (bHLH-zip) and the carboxy-terminal regulatory (LDL) receptor, the transcription of which is highly
domain (Reg) are located in the cytoplasmic compartment76. When the cellular demand for responsive to cholesterol levels10,11. The purified binding
sterols rises, the SREBP precursor protein travels to the Golgi apparatus77,78, where the site-1 activity contained two distinct SREBP proteins.
protease (S1P) cleaves at site-1 in the luminal loop (red line), producing the membrane-bound Cloning of the corresponding cDNAs revealed that the
intermediate form31. The intermediate form is the substrate for the site-2 protease (S2P), which SREBPs are basic helix–loop–helix leucine-zipper
cleaves the intermediate at site-2 (double red line), which is located three amino acids into the
(bHLH-zip) transcription factors. They differ from
membrane-spanning helix79. This second cleavage releases the transcription-factor domain from
the membrane, freeing it to enter the nucleus and direct the increased transcription of target other bHLH-zip proteins in that they are able to bind
genes. bHLH-zip, basic helix–loop–helix leucine-zipper. the typical E-box inverted DNA repeat (5′-CANNTG-3′),
as well as the direct DNA repeat of the SRE (for example,
5′-TCACNCCAC-3′)12.
transcriptionally active, amino-terminal cytoplasmic The SREBPs also have an unusually long carboxy-
S2P portion of the SREBP, which can then travel to the terminal extension compared with other bHLH-zip
(Site-2 protease). An unusually nucleus where it can activate the transcription of target proteins, that includes two membrane-spanning
hydrophobic metalloproteinase genes (FIG. 1). helices13,14. These anchor the SREBPs to the membranes
that cleaves its substrates within
Mammalian (and perhaps all chordate) genomes of the ER and nuclear envelope15. When the cellular
membrane-spanning helices.
include two separate SREBP genes (BOX 1) that demand for cholesterol increases, the SREBP is prote-
SCAP encode three different isoforms of SREBP, known as olysed to release the transcription-factor domain from
(SREBP-cleavage-activating SREBP-1a, -1c and -2. The SREBP-1a and -1c alleles the membrane16 (FIG. 1).
protein). A large, polytopic differ in their first exons owing to the use of different Studies of mutant Chinese hamster ovary (CHO) cells
membrane protein that senses
cellular sterol concentrations
transcriptional start sites for the SREBP-1 gene5. confirmed the crucial role of the proteolytic release of the
and escorts SREBP from the ER Tontonoz et al. identified the SREBP-1c protein in rats SREBPs in governing cholesterol homeostasis. In these
to the Golgi apparatus. as ADD1 (REF. 6). In discussing SREBP processing in cells, known as SRD1, a mutation in the gene encoding
this review, the three isoforms of SREBP are referred SREBP-2 led to a sterol-resistant phenotype in which
COPII
to without distinction as the mechanisms described the genes of both the cholesterol synthesis and uptake
Coatamer protein complex II,
which is required for here, derived from the study of animal cells in culture, pathways were transcribed at high levels, even in the
intracellular vesicle formation. seem to apply equally to all SREBP isoforms. At pre- continued presence of sterol concentrations that would
sent, the basis of the differential processing of SREBP normally suppress this transcription17. The mutation
INSIG
isoforms that is observed in mammalian cells treated caused a rearrangement within the SREBP-2 gene and
A membrane protein of the
endoplasmic reticulum (ER)
with unsaturated fatty acids is unknown5. The three created a new transcript that encoded a fusion between
that interacts with the isoforms will be discussed in more detail below. another soluble protein and SREBP-2 amino-terminal to
sterol-sensing domain of SCAP This review presents a current model of the prote- its first membrane-spanning helix. The resultant protein
and serves to retain the olytic activation of the SREBP pathway and describes its did not insert into membranes and was therefore free to
SCAP–SREBP complexes within
components and their functions. For a recent discussion travel to the nucleus, bypassing the regulated processing
the ER.
of the complexities of transcriptional regulation of the machinery, thereby explaining the sterol-resistant pheno-
HMG CoA genes encoding the various SREBP isoforms, see REF. 5. In type of the SRD1 cells, as well as that of two similar
(3-hydroxy-3-methylglutaryl this review, I will emphasize the discovery of two new mutant cell lines18.
coenzyme A). The activated components of the SREBP pathway in mammalian cells As mentioned above, mammalian cells express three
species that is reduced to form
mevalonate, the precursor of
— INSIG-1 and -2. Recent work on the role of the SREBP isoforms of SREBP: SREBP-1a, -1c and -2. SREBP-1a
cholesterol and isoprenoid pathway in Drosophila will be considered, along with the has a longer acidic motif at its amino-terminal
compounds. implications of these results for understanding the domain compared with SREBP-1c, and is a more

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 REVIEWS

SREBP SCAP in sterol metabolism, including the Niemann–Pick

N bH type C disease protein 1 (NPC1; REFS 22,23) and HMG
LH g
-z
ip Re WD CoA reductase, the rate-limiting enzyme of cholesterol
SSD biosynthesis24. More direct support for the functional
Cytoplasm N importance of the sterol-sensing domain comes from
studies on one class of mutant cells that are defective
in cholesterol-mediated feedback regulation of SREBP
ER
processing. In these cells, point mutations that alter
conserved residues within the sterol-sensing domain
Lumen
of one of the two SCAP alleles present confer sterol
resistance in a dominant fashion24–26.
S1P S2P
The carboxy-terminal domain of SCAP consists of
N multiple WD repeats (so called because they usually end
Cytoplasm HEXXH with a tryptophan-aspartate motif) that mediate pro-
tein–protein interactions27. This domain of the protein
LDG
interacts with the carboxy-terminal regulatory domain
of SREBP28. This interaction between SREBP and SCAP
Golgi
is not regulated by sterols but is absolutely required
Lumen for normal SREBP processing29,30. Mutant cells that
D lack SCAP require the addition of free cholesterol to
S the growth medium, as SREBP is unable to reach the
H nucleus to upregulate the transcription of cholesterol
biosynthetic genes30.
N
S1P. The first of two sequential cleavages that result in
Figure 2 | Components of the SREBP pathway in mammals and insects. In the presence of the release of the transcription-factor domain of SREBP
sterols, sterol regulatory element binding protein (SREBP) and SREBP-cleavage-activating protein from the membrane occurs at a Leu-Ser bond in the
(SCAP) are found in the membranes of the endoplasmic reticulum (ER). Site-1 protease (S1P) and middle of the ~30-amino-acid luminal loop that sepa-
site-2 protease (S2P) reside in the Golgi membranes, where they also process other proteins in
rates the two membrane-spanning helices31. This cleav-
addition to SREBP, such as ATF6 (activating transcription factor 6). The membrane topology data
for SREBP76, SCAP80, S1P32 and S2P47 are derived from protease protection and glycosylation age requires a 1,052-amino-acid serine protease, termed
studies. Membrane-spanning helices 2–6 of SCAP comprise the sterol-sensing domain (SSD). S1P (which is also known as subtilisin/kexin isozyme-1,
The carboxy-terminal domain contains multiple copies of a WD protein–protein interaction motif. SKI-1)32,33. Anchored to the membrane by a single
This portion of the molecule (WD) binds to the regulatory domain (Reg) of SREBP28. S1P is a membrane-spanning helix, the bulk of the protease
subtilisin-like serine protease and the Asp-Ser-His (DSH) residues of the catalytic triad are faces the lumen of the ER (FIG. 2). The carboxy terminus
necessary for S1P function32. S2P is an exceptionally hydrophobic metalloproteinase containing
is a short stretch of basic amino acids that face the cyto-
an HEXXH active-site motif in an otherwise hydrophobic portion of the protein (where H is
histidine, E is glutamate and X can be any amino acid). Both histidine residues and the glutamate
plasm, which makes S1P a type-1 membrane protein.
residue of the active site (indicated in bold) are necessary for S2P function43, as is a distal The luminal location of the active site is sensible as the
aspartate residue (D; bold), found within the LDG motif. This residue, which also lies within a substrates of S1P (for example, the luminal loop of
hydrophobic portion of the protein, is believed to provide an additional ligand for the active-site SREBP) are also found in the lumen of the ER. Like
metal atom47. N, amino-terminal. other proteases of the secretory pathway that are dis-
tantly related to the bacterial serine protease subtilisin,
S1P is synthesized as a proenzyme that undergoes auto-
potent transcriptional activator of their target genes13,19. catalytic cleavage of its prosegment to yield the active
SREBP-1c and SREBP-2 are the predominant isoforms enzyme34,35.
in animal livers, which is the main site of lipid synthesis. In addition to SREBP, S1P is also necessary for the
These proteins are approximately 80% identical in their processing of another membrane-bound transcription
DNA-binding domains but have a considerable selectiv- factor, ATF6 (activating transcription factor 6), that is
ity difference in their target genes20,21. SREBP-1c pre- involved in the UNFOLDED PROTEIN RESPONSE36,37. S1P also
dominantly governs the upregulation of genes that are processes a number of viral proteins38–40, and probably
involved in fatty-acid biosynthesis, whereas SREBP-2 is has other endogenous substrates33,41. Mutant cells that
STEROL-SENSING DOMAIN more selective for the genes of cholesterol biosynthesis lack S1P are cholesterol auxotrophs42.
A motif of five transmembrane and the LDL receptor (BOX 2). So, together, the SREBPs
helices that is found in SCAP coordinate the synthesis of the main membrane compo- S2P. The most hydrophobic protease described so far,
and HMG CoA reductase where
nents in eukaryotic cells — cholesterol and fatty acids. S2P contains an HEXXH zinc-binding motif 43,which is
it mediates protein movement or
stability through interaction characteristic of the active site of many families of met-
with the Insig proteins. Similar SCAP. SCAP is a 1,276-amino-acid integral membrane alloproteinases44. The unusual location of this motif
domains are present in other protein that consists of two distinct domains (FIG. 2). within an otherwise hydrophobic region of the protein
proteins associated with The amino-terminal half of the protein has eight might be due to the nature of its substrates  as
cholesterol metabolism, such as
the Niemann–Pick C disease
membrane-spanning helices, five of which (helices hydrophobic membrane-spanning helices are thought
protein and the Hedgehog 2–6) comprise the STEROL-SENSING DOMAIN12. Similar to lie within the plane of the lipid bilayer1. S2P is suffi-
receptor, Patched. domains are present in other proteins that are involved ciently different from all other metalloproteinases to be

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Box 1 | SREBPs, sterols and evolution
So far, all metazoan genomes sequenced have been found to contain SREBP
homologues. The limited data available at present indicate an interesting correlation:
chordate genomes have two SREBP homologues, whereas the invertebrate genomes
that have been examined have only one. This difference is also in agreement with
another; that is, the invertebrates considered (for example, flies and worms) cannot
synthesize sterols de novo. Chordates, on the other hand, are competent to synthesize
their own sterols. For example, experimental evidence indicates that tunicates can
synthesize sterols de novo from acetate73. Fragmentary sequences from the genome
sequencing of Ciona (see Genome sequence of Ciona in online links box) indicate that
this basal chordate/urochordate might also have two distinct SREBP genes
(R. B. R., unpublished observations). An additional correlation is the absence of clear
homologues of the Insig proteins in the genomes of animals with only a single SREBP.
In flies and worms, SREBP is intimately involved in non-sterol lipid synthesis63,74.
In Drosophila, cleavage of dSREBP is regulated by phosphatidylethanolamine, rather
than by the concentration of cellular cholesterol as in mammals71. These data indicate
that in its ancestral role, SREBP integrated signals that originated from the composition
of the membrane with the transcriptional control of cellular lipid synthesis. Therefore,
the best known role of SREBP — in regulating cholesterol metabolism — might have
been acquired subsequent to gene duplication in the lineage leading to chordates once it
had diverged from arthropods and nematodes.
As more complete genome sequences become available, it will be interesting to see if
this two-SREBPs:one-Insig:de novo sterol synthesis correlation holds up. In particular,
de novo sterol synthesis has been reported for echinoderms75, another major
deuterostome lineage. At present, it is not known whether echinoderms have any SREBP
or Insig proteins.
included in its own family, M50, which consists of at
least 68 proteins found in bacteria, archaea, plants and
animals45.
S2P was identified using human genomic DNA to
complement mutant cells that were unable to cleave
SREBPs at site-2 (REF. 43). These cells, which were
termed M19 cells, were originally isolated in the labo-
ratory of T. Y. Chang, where the complementation
approach was initiated 46. In the absence of S2P, the
intermediate form of SREBP (FIG. 1) cannot be
released from the membrane and the transcription of
target genes cannot be upregulated. As a result, cells
that lack S2P, like cells that lack S1P or SCAP, are also
UNFOLDED PROTEIN RESPONSE auxotrophic for cholesterol43,46.
An intracellular signalling Experiments with mutant versions of S2P showed
pathway that connects the that its ability to restore proteolytic processing of
endoplasmic reticulum (ER)
SREBPs in mutant M19 cells requires an intact HEXXH
with the nucleus. Under stress
conditions (when unfolded motif 43. A distal aspartate residue, Asp467, is believed to
proteins accumulate in the ER), provide an additional coordinating ligand for the active-
cells react by the increased site metal atom47. It is found in the sequence LDG that is
transcription of chaperone highly conserved among homologues of S2P (REF. 48).
genes. These chaperones are
required for the maintenance of
Like S1P, S2P is required for the processing of ATF6
protein folding. in response to ER STRESS37. Although mutant cells that lack
S2P have a profound deficit in an important branch of
ER STRESS the unfolded protein response pathway, under normal
A cellular response to an
culture conditions, addition of cholesterol to the growth
accumulation of unfolded
proteins in the endoplasmic medium is sufficient to enable these mutant cells to
reticulum (ER). Although a grow at wild-type rates.
normal physiological
phenomenon, it can also be New components of the SREBP pathway
provoked by several conditions,
such as viral infections and
When Hua et al. first cloned SCAP, they found that its
mutations that impair protein overexpression could abolish sterol suppression of
folding. SREBP cleavage24. Competition experiments using
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constructs that encode the amino-terminal membrane-
spanning domain of SCAP indicated the existence of a
factor, which, on interaction with SCAP, serves to
retain the SREBP–SCAP complex in the ER in the
presence, but not in the absence, of sterols49. The over-
expression of a construct encoding helices 1–6 of
SCAP (SCAP TM1–6, which includes the entire sterol-
sensing domain) in human or hamster cells, disrupted
the normal sterol-regulated processing of SREBP. This
indicated competition between SCAP TM1–6 and full-
length, wild-type SCAP for the binding of a factor that
retains the SREBP–SCAP complex in the ER in the
presence of sterols. When mutations known to disrupt
sterol sensing by SCAP were introduced into SCAP
TM1–6, no disruption of regulation was observed,
indicating that point mutations in SCAP that abolish
sterol regulation act by blocking the interaction with
the retention factor.
This retention factor has now been identified as
either of two highly similar membrane proteins —
Insig-1 and Insig-2 (REFS 50,51; FIG. 3). Diamond et al. first
identified insig-1 as a gene of unknown function, the
transcription of which was especially sensitive to insulin
in cells of regenerating liver52, and insig-2 was identified
on the basis of its high sequence identity51.
Insig1. To identify the ER retention factor that interacts
with the SREBP–SCAP complex, Yang et al. used co-
immunoprecipitation to biochemically isolate proteins
that interact with the SCAP TM1–6 construct50. As a
control for the specificity of the interaction with this
membrane-bound protein domain, they conducted par-
allel experiments with a construct that encodes helices
1–5 (SCAP TM1–5). This construct does not interfere
with the sterol regulation of SREBP processing and was
therefore not expected to interact with the putative ER
retention factor. Using tandem mass spectrometry of
proteins that co-immunoprecipitated with SCAP
TM1–6, but not with SCAP TM1–5, two short amino-
acid sequences were identified that corresponded to
Insig-1 (REF. 50). Although Insig-1 was one of several pro-
teins identified, it was chosen for further study because
it is a transcriptional target of SREBP53.
Analysis of the Insig-1 sequence predicts a hydropho-
bic protein with multiple membrane-associated
domains. Immunofluorescence data show that epitope-
tagged Insig-1 is located in the ER membrane in both the
presence and the absence of sterols50. Membrane local-
ization of the retention factor was expected on the basis
of previous results, and in vitro vesicle-formation assays
indicated that sterol-dependent retention of SCAP
requires a membrane-associated factor3. Overexpression
of Insig-1 blocked SREBP cleavage even under condi-
tions that normally promote high levels of cleavage.
Insig-1 prevented the exit of SREBP–SCAP complexes
from the ER, further supporting its identification as the
retention factor. SCAP and Insig-1 form a complex in
the presence, but not in the absence, of sterols and inter-
act in a stoichiometric manner. Taken together, these
observations identify Insig-1 as a sterol-regulated ER
retention factor for SREBP–SCAP complexes50.
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Box 2 | Targets of SREBPs sterol levels51. The relative affinities of Insig-1 and Insig-2
for SCAP are as yet unknown.
Mammalian and Drosophila genes that encode acetyl coenzyme A (CoA) synthetase, The identification of insig-2 as a gene with a product
acetyl CoA carboxylase and fatty acid synthase are targets of mammalian and Drosophila that is involved in the regulation of SREBP cleavage
sterol regulatory element binding proteins (SREBPs), respectively (see table). Genes that could help explain the mechanisms by which insulin
encode long-chain fatty acyl elongase, stearoyl CoA desaturase, glycerol-3-phosphate regulates SREBP activity (for a recent discussion of the
acyl-transferase, ATP citrate lyase, 3-hydroxy-3-methylglutaryl (HMG) CoA synthase interaction between insulin and the SREBPs, see REF. 5).
and HMG CoA reductase are targets for SREBPs in mammalian cells, but not in
The insig-2 gene produces two distinct mRNAs that dif-
Drosophila. Genes that encode squalene synthase, squalene epoxidase, lanosterol
fer in their first, noncoding exons54. One mRNA, desig-
synthase, lanosterol 14α-demethylase, lathosterol oxidase and 7-dehydrocholesterol
nated insig-2b, is produced throughout the body in
reductase are targets of SREBPs in mammalian cells, whereas insects have no clear
orthologues of these genes, as determined by BLAST searches of insect genomes. This
mice. The second, insig-2a mRNA, is liver specific and is
absence is likely to reflect the fact that, in insects, de novo synthesis of sterols is blocked at highly responsive to insulin concentrations.
multiple steps81. When insulin concentrations are high, insig-2a
In mammals, the genes of fatty acid synthesis are preferentially activated by SREBP-1 mRNA levels are low. When insulin concentrations fall,
and the genes of sterol synthesis and uptake are preferentially activated by SREBP-2. the levels of insig-2a mRNA increase. This contrasts with
Consistent with the similarity of their gene targets, the DNA-binding domain of human insig-2b mRNA, the abundance of which is unaffected by
SREBP-1 is 82% identical to the DNA-binding domain of Drosophila SREBP. By contrast, the concentration of insulin. The levels of insig1 mRNA
the DNA-binding domain of human SREBP-1 has only 80% identity to the DNA-binding are regulated in a manner that is reciprocal to insig-2a
domain of human SREBP-2. mRNA in the liver. For example, the highest levels of
insig-1 mRNA are expressed in the presence of insulin,
Mammalian SREBPs Drosophila SREBPs
and this expression declines in the absence of insulin54.
Fatty acids Yabe et al. speculated that insulin could regulate the pro-
Acetyl CoA synthetase Acetyl CoA synthetase duction of insig-2a at the level of transcription and that
Acetyl CoA carboxylase Acetyl CoA carboxylase low levels of insig-2a mRNA might correlate with lower
Fatty acid synthase Fatty acid synthase
Long-chain fatty acyl elongase Fatty acyl CoA synthetase concentrations of the Insig-2 protein in the liver. A
Stearoyl CoA desaturase decrease in the concentration of the Insig-2 protein
Glycerol-3-phosphate acyl-transferase might then allow SREBP-1c to exit the ER under condi-
Sterols tions in which cleavage of SREBP-2 is suppressed
ATP citrate lyase through a continued interaction with the Insig-1
Acetoacetyl CoA thiolase protein54. This, in turn, would allow SREBP-1c to enter
HMG CoA synthase the nucleus and direct the increased transcription of the
HMG CoA reductase
Mevalonate kinase genes of fatty-acid biosynthesis that is observed in
Mevalonate pyrophosphate decarboxylase response to insulin signalling in the liver. However,
Geranylgeranyl pyrophosphate synthase future experiments will be needed to test this hypothesis.
Isopentenyl pyrophosphate isomerase
Farnesyl pyrophosphate synthase
Squalene synthase Insig and SCAP interactions. The basis of the domi-
Squalene epoxidase nant, gain-of-function sterol resistance observed in
Lanosterol synthase
Lanosterol 14α-demethylase
cells that contain one mutant allele of SCAP, which has
Lathosterol oxidase the effect of making SREBP cleavage insensitive to
7-dehydrocholesterol reductase sterols, is explained by the identification of the Insig
LDL receptor proteins as the ER retention factor24,25,55. Three such
LDL, low-density lipoprotein. Mammalian SREBP targets are reviewed in REF. 5 and Drosophila point mutations, each within the sterol-sensing domain
data are from REF. 63.
of SCAP, are now known. Yabe et al. show that all three
SCAP mutations disrupt the normal interaction
Insig-2. This protein has 59% amino-acid sequence between SCAP and the Insig proteins26. As a result, the
identity to Insig-1 and performs a similar role in regulat- SREBP–SCAP complex exits the ER even in the pres-
ing cholesterol homeostasis51. However, unlike Insig-1, ence of sterol concentrations that completely suppress
Insig-2 is not a transcriptional target of SREBP. Another SREBP processing in cells expressing only wild-type
important distinction is that when Insig-1 is expressed at SCAP. These studies confirm the crucial role of the SCAP
high levels, the SCAP–SREBP complex is retained in the sterol-sensing domain and its interaction with the Insig
ER even in the complete absence of sterols50. By contrast, proteins for the maintenance of cellular cholesterol
Insig-2 requires that at least some sterols be present51. homeostasis.
Transcription of the gene encoding Insig-1, which can When cholesterol is added to membrane vesicles
act in the absence of sterols, rises in the absence of sterols, in vitro, SCAP undergoes a conformational change as
whereas transcription of the gene encoding Insig-2, revealed by its differential sensitivity to proteolytic diges-
which is absolutely dependent on sterols for binding, is tion56. This change is much less pronounced in mutant
unaffected by sterol levels. Together, these properties SCAP that contains either the Tyr298Cys or the
allow the Insig proteins to modulate the feedback regula- Asp443Asn mutation. The correlation between this cho-
tion of cholesterol by controlling the exit of SREBP from lesterol-induced conformational change in vitro, and the
the ER (and therefore, ultimately, to regulate the tran- binding of SCAP and the Insig proteins in vivo, indicates
scription of target genes) over a broad range of cellular the possibility that the interaction between the two

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REVIEWS

a +Sterols of HMG CoA reductase has also been found to occur in

SREBP SCAP yeast60. The sterol-regulated stability of the enzyme
bH requires the presence of its amino-terminal sterol-sens-
LH g
-z
ip Re WD SSD ing domain61.
Sever et al. demonstrated that Insig-1 interacts with
Cytoplasm N the amino-terminal sterol-sensing domain of HMG
CoA reductase and mediates the increased degradation
Insig of this protein, which is observed in the presence of
ER sterols62. When HMG CoA reductase is overexpressed in
Lumen CHO cells, the normal sterol-induced increase in degra-
dation is impaired. Regulated degradation of the
enzyme can be restored to these cells by the additional
b –Sterols
expression of Insig-1 (REF. 62). Moreover, expression of
bH wild-type SCAP TM1–6 blocked the increased degrada-
LH g
-z
ip Re WD tion conferred by Insig-1, whereas no effect was seen
with the expression of a SCAP TM1–6 construct that
Cytoplasm
contained the Tyr298Cys mutation that disrupts the
interaction between SCAP and the Insig proteins. These
Insig results show that the sterol-sensing domains of SCAP
ER and HMG CoA reductase compete for binding to Insig-1.
Lumen Co-immunoprecipitation studies have shown that the
binding of Insig-1 to HMG-CoA reductase occurs in its
sterol-sensing domain, and that binding is dependent
Golgi on sterols in the same way as is observed for the
Insig–SCAP interaction62.
Figure 3 | Sterol-regulated interaction between Insig and SCAP. In the presence of sterols,
The interaction of two sterol-sensing domains with
complexes of sterol regulatory element binding protein (SREBP) and SREBP-cleavage-activating
protein (SCAP) remain in the endoplasmic reticulum (ER). Retention in the ER requires interaction Insig-1 is intriguing, especially as the immediate conse-
between the sterol-sensing domain (SSD) of SCAP and Insig-1 or -2. This interaction is strongly quences of these interactions are so different. As a result
enhanced by cholesterol (in the case of Insig-1), or is absolutely dependent on cholesterol of its interaction with Insig-1, HMG CoA reductase is
(in the case of Insig-2)50,51. In the absence of sterols, SCAP and the Insig proteins do not interact. degraded. By contrast, the SREBP–SCAP complex that
The SREBP–SCAP complex is then free to be packaged into COPII vesicles and travel to the is retained in the ER seems quite stable. However, the
Golgi apparatus to be processed. The membrane topology of the Insig proteins is unknown.
long-term consequences of the Insig–sterol-sensing
bHLH-zip, basic helix–loop–helix leucine-zipper; Reg, regulatory; SSD, sterol-sensing domain;
WD, aspartate-tryptophan motif.
domain interactions are more similar. Following the
interaction between HMG CoA reductase and Insig-1,
cholesterol synthesis falls owing to the increased degra-
dation of this rate-limiting enzyme. The interaction
proteins requires SCAP to be in a conformation that between SCAP and Insig-1 also reduces cholesterol syn-
depends on the presence or absence of specific lipids in thesis, in this case by retaining the SREBP–SCAP com-
the bilayer. The conformational change in SCAP could be plex within the ER, thereby preventing the release of
secondary to the cholesterol-induced interaction between transcriptionally active SREBP.
the Insig proteins and the SCAP sterol-sensing domain. Insig-1 is one of several proteins identified by Yang
et al. that specifically co-precipitate with SCAP TM1–6
Insig-1 and HMG CoA reductase, another sterol sensor. (REF. 50). Some or all of these proteins might also prove to
Recent studies have revealed an additional role for Insig-1 be involved in feedback regulation of cholesterol synthe-
in the maintenance of cholesterol homeostasis. HMG sis. For example, the Insig proteins lack any obvious ER
CoA reductase is the rate-limiting enzyme of cholesterol localization signal and might need to interact with an
biosynthesis and, like SCAP, it has an amino-terminal additional factor to be retained in the ER. The study of
sterol-sensing domain24. Its carboxy-terminal domain one or more of these additional proteins might provide
catalyses the production of mevalonate (through one of insights into the mechanisms that govern the differential
a series of enzymatic reactions that are known as the fates of SCAP and HMG CoA reductase following bind-
MEVALONATE PATHWAY), which is a substrate for isoprenoid ing to Insig-1. The identification of Insig-1 and -2 as
and sterol biosynthesis57. The activity of this crucial proteins that interact with the sterol-sensing domains of
enzyme is regulated at many levels: it is activated tran- SCAP and HMG CoA reductase provides a significant
scriptionally by SREBPs (BOX 2), and it is also regulated at advance in the understanding of cholesterol homeosta-
MEVALONATE PATHWAY the level of protein stability. In the presence of sterols, sis in mammalian cells.
The series of enzymes, and the HMG CoA reductase is rapidly degraded, whereas in
reactions they catalyse, that their absence, it is much more stable58. Degradation of The SREBP pathway in insects
convert acetyl coenzyme A to the enzyme can be blocked with inhibitors of the pro- The genome of Drosophila melanogaster encodes a single
many different hydrophobic
products such as farnesol,
teasome such as MG132 or ALLN, indicating a role for homologue of SREBP, as well as homologues of SCAP, S1P
geranylgeranol, dolichol, and, in the proteasome in its degradation59. In addition to and S2P (REF. 63). This is also true for Anopheles gambiae,
vertebrate animals, cholesterol. mammalian cells, the ubiquitin-dependent degradation the only other insect for which the complete genome

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 REVIEWS

O O
(CH2)NCH3 (CH2)NCH3
N N
HO (CH2)12CH3 HO (CH2)12CH3

OH OH
Dihydroceramide Ceramide

NH3+ NH3+
HO (CH2)12CH3 HO (CH2)12CH3

OH Sphingosine OH
Sphinganine

Sphingosine-1-
NH3+ 3-Oxosphinganine phosphate NH3+
HO (CH2)12CH3 O– O (CH2)12CH3
P
O O O OH

NH3+
O– O
P
NH3+ Palmitoyl-CoA Hexadecanal O O
HO O– CoAS (CH2)14CH3 (CH2)14CH3 Phosphoethanolamine
O Serine O O
Palmitate
Cytosine-ribose- O O
–O (CH2)14CH3 P NH3+
O O O
O O
O P
O (CH2)14CH3 O O
R′ O
OH CDP-ethanolamine

Diacylglycerol
O
O
O (CH2)14CH3
R′ O
O O
P NH3+
O O

Phosphatidylethanolamine

Figure 4 | Dual roles of palmitate in the suppression of dSREBP cleavage. In Drosophila cells, the suppression of Drosophila
sterol regulatory element binding protein (dSREBP) cleavage by palmitate requires its incorporation into sphingolipids71.
Other compounds, such as sphinganine, ceramide and hexadecanal, can also suppress cleavage when added to cells in culture.
The suppressive effects of these products require the activity of phosphoethanolamine cytidylyltransferase, which produces cytidine
5′-diphosphate (CDP)-ethanolamine from phosphoethanolamine and cytidine 5′-triphosphate (CTP). When progression through this
cycle is blocked by specific inhibitors or RNA-mediated interference (RNAi) against the various enzymes involved, palmitate no
longer suppresses cleavage. Under these conditions, the addition of exogenous ethanolamine (which can be converted to
phosphoethanolamine by ethanolamine kinase) restores suppression of dSREBP cleavage. This indicates a second role for
palmitate, in addition to the production of phosphoethanolamine. This second role can also be partially satisfied by fatty acids other
than palmitate, such as oleate, which reflects the nature of these compounds in the synthesis of diacylglycerol. These results
pointed to phosphatidylethanolamine, the synthesis of which requires both phosphoethanolamine and diacylglycerol, as the
compound that suppresses dSREBP cleavage in Drosophila cells71. The synthesis of phosphatidylethanolamine by decarboxylation
of phosphatidylserine or by base exchange does not seem to be quantitatively important in Drosophila cells. R′, fatty acid, typically
an unsaturated fatty acid; N represents the number of (CH2) groups. The pathway shown is based on REF. 71 and references
cited therein.

sequence has been reported64. The presence in insects of a
pathway that is so well known for its role in cholesterol
homeostasis seems surprising, as insects are cholesterol
auxotrophs because they lack several enzymes in the cho-
lesterol biosynthesis pathway, from squalene synthase
onwards (BOX 2). However, in addition to cholesterol syn-
thesis, SREBPs also have an important role in fatty-acid
synthesis5,65,66 (BOX 2) and in the synthesis of the main
phospholipid of mammalian cells, phosphatidylcholine67.
Seegmiller et al. explored the notion that
Drosophila SREBP (dSREBP, also known as HLH106;
REF. 68) might mediate fatty-acid biosynthesis by tran-
scriptional regulation. These authors showed that in
Drosophila S2 cells, nuclear dSREBP is necessary for
increased transcription of several genes of fatty-acid
synthesis, such as fatty-acid synthase63. The table in
BOX 2 lists Drosophila genes, the transcription of which
depends on nuclear dSREBP.

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REVIEWS
RNAi
RNA-mediated interference of
gene expression. The process
whereby the introduction of
double-stranded RNA into a cell
elicits a response that
catalytically destroys single-
stranded RNAs that contain the
cognate sequence.
PHOSPHATIDYLETHANOLAMINE
A phospholipid with the
unusually small headgroup
ethanolamine attached to its
diacylglycerol backbone.
HII PHASE
Lipids that form tubular
structures following hydration,
wherein the hydrophilic moieties
are facing the inner surface of a
tube, and the hydrophobic
moieties face outward. As a
result, multiple tubes associate
through hydrophobic
interactions, yielding a structure
that seems hexagonal in cross
section.
638 | AUGUST 2003 | VOLUME 4
In S2 cells, dSREBP cleavage occurs in a two-step
manner and requires Drosophila SCAP (dSCAP) and pre-
sumably also Drosophila S1P and S2P (dS1P and dS2P)
on the basis of the requirement for their cognate cleavage
sites in dSREBP. Despite these strikingly conserved simi-
larities in organisms, the evolutionary history of which
diverged perhaps a billion or more years ago, in
Drosophila, dSREBP processing is not suppressed by
sterols63, nor is it suppressed by unsaturated fatty acids
(as seen in mammalian cells)69,70. Instead, dSREBP cleav-
age is fully suppressed by the addition of the 16-carbon-
saturated fatty acid, palmitate. This effect is highly
specific for palmitate as 14- and 18-carbon-saturated
fatty acids show little effect, and neither does palmi-
toleate, an unsaturated fatty acid containing 16 carbon
atoms and 1 unsaturated bond63.
Regulation by phospholipid, not sterols. The specificity
of palmitate was an important clue in deducing the
nature of the species that suppresses dSREBP cleavage
in insect cells. Palmitate has many metabolic fates, but
one biosynthetic pathway that uses palmitate to the
exclusion of other fatty acids is the formation of
sphingolipids (FIG. 4). Through the action of serine
palmitoyltransferase, palmitoyl CoA and serine are
condensed to yield sphinganine. Sphinganine is then
converted into ceramide and then to sphingosine.
Using RNA-mediated interference of gene expression
(RNAi) and specific enzyme inhibitors, Dobrosotskaya,
et al. systematically eliminated the activities of succes-
sive key enzymes in the sphingolipid biosynthetic
pathway71. These studies revealed that the suppressive
effects of palmitate require its conversion, through sev-
eral intermediates, to sphingosine-1-phosphate. This
compound, itself a well-known cellular signal, is not,
however, the end product that mediates the suppres-
sion of dSREBP cleavage. Eliminating the activity of
sphingosine-1-phosphate lyase, which cleaves sphin-
gosine-1-phosphate to produce trans-2-hexadecenal
and phosphoethanolamine, blocks the suppression of
dSREBP cleavage by palmitate. Additional RNAi exper-
iments revealed that palmitate was needed in a catalytic
cycle that converts serine (plus ATP) into phospho-
ethanolamine (plus ADP; FIG. 4).
The importance of phosphoethanolamine was con-
firmed by blocking the biosynthesis of sphingolipids at
several different points using RNAi. In these circum-
stances, the suppression of dSREBP cleavage by palmi-
tate could be restored by adding ethanolamine to the
culture medium71. Exogenous ethanolamine can be
phosphorylated by ethanolamine kinase to yield
phosphoethanolamine, thereby bypassing the sphin-
golipid biosynthetic pathway. These experiments
revealed a requirement for palmitate, even when it
was not needed for the production of phospho-
ethanolamine.
Phosphoethanolamine is attached to cytidine 5′-
diphosphate (CDP) by the enzyme phospho-
ethanolamine cytidylyltranferase (PECT) to produce
CDP-ethanolamine. Elimination of PECT by RNAi
blocked the suppression of dSREBP cleavage by
© 2003 Nature Publishing Group
palmitate. Another role for palmitate is in the produc-
tion of diacylglycerol, which is used in the synthesis of
all phospholipids. CDP-ethanolamine and diacylglyc-
erol yield PHOSPHATIDYLETHANOLAMINE, the principal
phospholipid in Drosophila cell membranes.
In the presence of exogenous ethanolamine, other
fatty acids, such as oleate (which contains 18 carbon
atoms and 1 unsaturated bond) could suppress
dSREBP cleavage. This result further indicates that
palmitate has two roles in the suppression of dSREBP
cleavage: first, a specific requirement for palmitate in
producing phosphoethanolamine through the sphin-
golipid pathway; and second, a less stringent require-
ment in the formation of diacylglycerol. These findings
provide indirect evidence that the compound that
mediates feedback regulation of dSREBP processing is
phosphatidylethanolamine.
Phosphatidylethanolamine and SCAP. The sterol-sensing
domain of dSCAP shares 47% amino-acid sequence
identity with its human homologue. The role of the
mammalian sterol-sensing domain in the regulation of
cholesterol synthesis is well supported (see above).
Nevertheless, the presence of a sterol-sensing domain in
dSCAP, in a pathway that is insensitive to sterols, indi-
cates that its role is not strictly limited to sensing sterol
concentrations. The sterol-sensing domain might be a
more general monitor of membrane lipid composition.
In mammals, cholesterol induces a conformational
change in SCAP56 and promotes interactions between
SCAP and the Insig proteins26,50,51. The Drosophila
genome encodes no obvious homologue of the Insig
proteins and dSREBP cleavage is unresponsive to sterols.
Nevertheless, based on the extensive similarities between
the mammalian and insect SREBP pathways, it seems
likely that some, as yet unidentified, protein might func-
tion to retain a dSREBP–dSCAP complex within the ER
in insect cells. The ‘sterol-sensing domain’ of dSCAP
might also undergo a conformational change in
response to changes in the membrane lipid composi-
tion. In this light, the unusual behaviour of cholesterol
and phosphatidylethanolamine indicates a plausible
common mechanism of action. Unlike other phospho-
lipids, phosphatidylethanolamine adopts an HII PHASE,
hexagonal, non-lamellar structure in solution.
Cholesterol confers similar tendencies on lipid bilayers72.
It is possible that the SCAP sterol-sensing domain is, in
fact, sensing local changes in the physical properties of
the ER membrane that are induced by phos-
phatidylethanolamine or cholesterol, rather than inter-
acting with a particular molecule71.
It is also possible that the Insig proteins (or an analo-
gous protein) could function as sensors of sterols or
phospholipids, and interact with the sterol-sensing
domain only in the presence of the relevant lipid. In this
case, the sterol-sensing domain could be thought of as an
interaction domain for proteins that themselves sense
lipid levels either through direct binding or through con-
formational changes that are induced by alterations in the
membrane composition. Conversely, the interaction
between a sterol-sensing domain and the Insig proteins
www.nature.com/reviews/molcellbio
 REVIEWS

might be needed to create a sensor from proteins that are
unable to function in this manner by themselves.
Conclusion
Together, the identification and characterization of
the Insig proteins and the discovery that phos-
phatidylethanolamine is the feedback suppressor in
Drosophila provide a better understanding of the role of
SCAP in lipid homeostasis. These results indicate a mech-
anism by which a conformational change in the sterol-
sensing domain of SCAP converts information about the
physical properties of membranes into an action that
governs the transcription of genes with end products
that ultimately alter the membrane composition.
The findings discussed here raise new questions.
What keeps the Insig proteins in the ER? Do fatty
acids affect Insig–SCAP interactions or is another
retention factor involved in this aspect of the regula-
tion? Which protein has an analogous role to the
Insigs in Drosophila? Is the conformation of SCAP
important to both systems? The answers to these
questions should lead to further advances in our
understanding of the mechanisms by which cells
maintain lipid homeostasis. These advances are
important for devising additional interventions that
are aimed at restoring desirable lipid concentrations
in people with hypercholesterolaemia and other
lipid metabolism disorders. Moreover, in the past 25
years, studies of cholesterol homeostasis have led to
the recognition and understanding of widespread
cellular phenomena such as receptor-mediated
endocytosis and RIP.

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Acknowledgements
I am grateful to R. A. DeBose-Boyd, J. D. Horton and members of
the Rawson laboratory for comments on this manuscript. This work
is supported by grants from the American Heart Association, the
National Institutes of Health and the Perot Family Foundation.
Online links
DATABASES
The following terms in this article are linked online to:
LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/
Insig-1 | Insig-2 | NPC1 | SREBPs
Swiss-Prot: http://www.expasy.ch/
HMG CoA reductase | S1P | S2P | SCAP
FURTHER INFORMATION
Genome Sequence of Ciona:
http://genome.jgi-psf.org/ciona4/ciona4.home.html
Robert B. Rawson’s laboratory:
http://swnt240.swmed.edu/gradschool/webrib/rawson.htm
Access to this interactive links box is free online.
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