Drosophila NPC1b Promotes an Early Step
in Sterol Absorption from the Midgut Epithelium
Stephen P. Voght,1Megan L. Fluegel,1,2Laurie A. Andrews,1and Leo J. Pallanck1,*
1Department of Genome Sciences
2Molecular and Cellular Biology Graduate Program, University of Washington, Seattle, WA 98195, USA
The NPC1 family of proteins plays crucial roles
in the intestinal absorption and intracellular
trafficking of sterols. The Drosophila genome
encodes two NPC1 homologs, one of which,
NPC1a, is required for intracellular sterol traf-
ficking in many tissues. Here we show that
the other Drosophila NPC1 family member,
NPC1b, is expressed in the midgut epithelium
and that NPC1b is essential for growth during
the early larval stages of development. NPC1b
mutants are severely defective in sterol absorp-
tion, and the midgut epithelium of NPC1b mu-
tants is deficient in sterols and sterol trafficking
intermediates. By contrast, NPC1a mutants ab-
sorb sterols more efficiently than wild-type ani-
mutants absorb sterols as efficiently as wild-
type animals. Together, these findings suggest
that NPC1b plays an early role in sterol absorp-
tion, although sterol absorption continues
at high efficiency through an NPC1a- and
NPC1b-independent mechanism under condi-
tions of impaired intracellular sterol trafficking.
Vertebrates acquire cholesterol from their diet and
through the synthesis of cholesterol from acetate. Al-
though much is known of the enzymology of cholesterol
biosynthesis (Brown and Goldstein, 1986), far less is
known of the molecular mechanisms by which dietary
cholesterol is absorbed by the intestine. However, recent
genetic and pharmacological work indicates that the Nie-
mann-Pick C1-like 1 (NPC1L1) protein plays an important
role in the intestinal absorption of dietary sterols. In partic-
ular, cholesterol absorption is largely inhibited by the drug
ezetimibe, which binds to and presumably inactivates the
NPC1L1 protein (Garcia-Calvo et al., 2005). Moreover,
NPC1L1 knockout mice absorb dietary cholesterol ineffi-
ciently and show no response to ezetimibe (Altmann
et al., 2004; Davis et al., 2004). While these findings clearly
demonstrate that NPC1L1 promotes dietary cholesterol
absorption, the specific mechanism by which it does so
The NPC1L1 gene is homologous to the Niemann-Pick
type C1 (NPC1) gene, loss-of-function mutations of which
cause Niemann-Pick type C disease, an autosomal reces-
sive lipid-storage disorder. The NPC1 and NPC1L1 gene
products are both 13-pass transmembrane proteins with
a putative sterol-sensing domain, a cysteine-rich domain,
broad homology to the Patched morphogen receptor, and
structural similarity to the RND family of prokaryotic
permeases (Carstea et al., 1997; Davies et al., 2000a,
2000b). NPC1 also contains a lysosomal targeting motif
and localizes to an endosomal compartment where it pro-
motes intracellular trafficking of cholesterol and sphingo-
lipids, perhaps by directing the movement of sterol-rich
transport vesicles to their proper locations (Ko et al.,
2001) or by facilitating the export of sterols from the lyso-
some (Ioannou, 2000).
Although the structural similarity of NPC1L1 to NPC1
suggests that these proteins play conserved biochemical
roles, many important questions concerning the function
of NPC1L1 remain unanswered. For example, it remains
unclear whether NPC1L1 specifically promotes the ab-
sorption of sterols orwhether this protein also participates
in the absorption of other dietary factors. The identities of
key factors that regulate NPC1L1 and that function in
concert with NPC1L1 to promote cholesterol absorption
in the intestine also remain unknown. Whether NPC1L1
promotes an early step in sterol acquisition and/or, like
NPC1, a later step in intracellular trafficking of cholesterol
in the intestinal epithelium also remains controversial
(Sane et al., 2006; Yu et al., 2006). Finally, the precise mo-
lecular functions of both NPC1L1 and NPC1 remain un-
knownand the topic of muchdebate(Daviesand Ioannou,
2006; Garver and Heidenreich, 2002; Karten et al., 2006;
Malathi et al., 2004; Mellon et al., 2004).
To study the functions of the NPC1 gene family and the
mechanisms of sterol absorption and intracellular sterol
trafficking, we used a genetic approach in the fruit fly
Drosophila melanogaster. Although insects lack the ability
to synthesize sterols, dietary sterols are required for
proper insect development, and previous work suggests
that sterol trafficking mechanisms are highly conserved
in insects and vertebrates (Rodenburg and Van der Horst,
2005). The Drosophila genome contains two NPC1 homo-
logs, designated NPC1a and NPC1b. The NPC1a and
NPC1b genes encode polypeptides with 42% and 35%
Cell Metabolism 5, 195–205, March 2007 ª2007 Elsevier Inc. 195
amino acid identity, respectively, to the human NPC1 pro-
NPC1L1 protein. We and others have previously demon-
strated that NPC1a is ubiquitously expressed and is re-
quired for efficient intracellular sterol trafficking in many
tissues, including the ring gland, where the major insect
steroid hormone ecdysone is synthesized (Fluegel et al.,
2006; Huang et al., 2005).
In our current work, we subjected the NPC1b gene to
mutational analysis and found that NPC1b functions in
a role homologous to that of mammalian NPC1L1 in the
regulation of dietary sterol uptake from the gut epithelium.
Our results suggest that NPC1b promotes an early step in
sterol absorption and may participate in the absorption of
other essential dietary factors. Our findings also suggest
the existence of an NPC1a- and NPC1b-independent
mechanism of sterol absorption.
Expression Analysis of NPC1b
To determine where NPC1b is expressed, we generated
a reporter construct consisting of an NPC1b promoter-
containing fragment placed upstream of the yeast GAL4
protein coding sequence. Transgenic flies bearing this
ing a UAS-GFP transgene. Larval progeny of this cross
that bear both the NPC1b-GAL4 and UAS-GFP trans-
genes express GFP in two distinct areas, corresponding
to compartments m2 and m10–m12 of the midgut (Fig-
ure 1A; see also Figure S1 in the Supplemental Data avail-
able with this article online; Murakami et al., 1999). GFP
expression diminishes during pupation and then reap-
pears in the adult midgut approximately 2 days posteclo-
sion (Figure 1B). Higher magnification of the larval midgut
revealed that the NPC1b-GAL4 transgene drives expres-
sion specifically in the midgut epithelium and not in sur-
rounding muscle tissue (Figure 1C). GFP fluorescence
was not detected in any tissues outside of the midgut at
any stage of development, suggesting that NPC1b ex-
pression is restricted to the midgut epithelium throughout
the Drosophila life cycle. Control animals bearing only the
NPC1b-GAL4 or UAS-GFP transgene failed to produce
detectable GFP fluorescence (data not shown), demon-
strating that the NPC1b promoter is directly responsible
for GFP expression in the midgut.
Identification of NPC1b Mutants
To investigate the functional role of NPC1b, we subjected
the NPC1b gene to mutational analysis. An NPC1b mutant
allele was created using a gene-targeting construct that
included a nonsense mutation in the first exon of NPC1b
at codon 392, several nucleotide alterations in the first in-
site, and other alterations required for gene targeting
(Rong and Golic, 2000). This construct was used to gener-
ate mutations as detailed in the Experimental Procedures
section and outlined schematically in Figure 2A. From
a screen of 35,000 flies, we recovered two independent
lines in which a targeting event occurred. Both lines car-
ried a wild-type copy of the NPC1b gene and a tandem
copy bearing the inactivating mutations (Figure 2A). Sub-
sequent generation of a double-stranded break between
the mutationally altered and wild-type copies of the
NPC1b gene led to the recovery of 33 recombinant lines
bearing only the mutant copy of NPC1b. Loss of wild-
type NPC1b and retention of the mutation were verified
by PCR across the diagnostic restriction site introduced
Approximately 800 bp of sequence upstream
of the NPC1b start codon was joined to the
GAL4 transcription factor. Larvae bearing this
transgene and a GAL4-responsive GFP trans-
gene express GFP in tissues in which the
NPC1b promoter is active.
(A) Dissected third-instar larvae, showing two
distinct areas of GFP expression within the
midgut. Dotted line shows approximate loca-
tion of cuticle prior to dissection.
(B) Isolated midgut from 3-day-old adult fly.
One area of GFP expression is detected in
(C) Close-up of isolated GFP-expressing larval
midgut cells, showing hexagonal morphology
characteristic of epithelial cells. Scale bars in
all images = 50 mm.
Drosophila NPC1b Is Involved in Sterol Absorption
196 Cell Metabolism 5, 195–205, March 2007 ª2007 Elsevier Inc.
developing our filipin methodology, respectively. The authors also
thank K. Golic for helpful discussions on the gene-targeting methodol-
ogy and for providing reagents, J. Carlson for the pG4PB vector, and
all members of the Pallanck lab for insightful input on this project.
This work was made possible by grants from the Jim Lambright Nie-
mann-Pick Foundation, the Ara Parseghian Medical Research Foun-
dation, the American Heart Association (AHA 0655717Z to L.J.P. and
AHA 0515513Z to M.L.F.), and the National Institutes of Health (F31
GM70186 to S.P.V.).
Received: August 8, 2006
Revised: December 4, 2006
Accepted: January 23, 2007
Published: March 6, 2007
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Drosophila NPC1b Is Involved in Sterol Absorption