1 Structural Biology Center, National Institute of Genetics, and Department of
Genetics, The Graduate University for Advanced Studies, Mishima 411-8540,
Japan
2 Department of Molecular Oncology, Graduate School of Medical and Dental
Sciences, Kagoshima University, Sakuragaoka 8-35-1, Kagoshima 890-8544,
Japan
Author for correspondence (e-mail:
ikatsura{at}lab.nig.ac.jp)
Accepted 18 May 2005
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SUMMARY |
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Key words: C. elegans, Multidrug resistance-associated protein, mrp-1, ABC transporter, Dauer larva
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Introduction |
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The nematode C. elegans also has a diapause stage, called a dauer
larva. In the normal life cycle, C. elegans grows to adulthood
through four larval stages (L1 to L4) in 2-3 days at 20°C. But under
inadequate conditions for growth, that is, under reduced food availability,
crowding or high temperature, the animal arrests development and forms the
dauer larva, corresponding to the third larval stage
(Cassada and Russell, 1975;
Golden and Riddle, 1984
). When
the environmental conditions are improved, the dauer larva molts to a normal
L4 larva and resumes the life cycle. The dauer larva has a characteristic
shape and common features of dormant animals, i.e. low metabolism, no feeding,
no aging, accumulation of fat and resistance to stress
(Cassada and Russell, 1975
;
Klass and Hirsh, 1976
;
Riddle, 1988
;
Wadsworth and Riddle, 1989
).
Because many molecular biological and genetic techniques are available, C.
elegans is a good model organism for studying the regulation of
transition into the diapause stage at both molecular and cellular levels.
Genes that regulate dauer larva formation have been studied by the
isolation and characterization of mutants that show abnormality in this
function. These mutants, called daf (dauer larva formation abnormal),
consist of two groups: dauer-constitutive (daf-c) mutants, which form
dauer larvae even under non-crowding and well-fed conditions, and
dauer-defective (daf-d) mutants, which do not form dauer larvae, even
under conditions of crowding and starvation. The genetic pathways of dauer
larvae formation have been revealed by epistasis tests between daf-c
and daf-d mutations, and by molecular cloning of the mutant genes. At
least four signal transduction pathways control dauer larva formation: the
cGMP, TGF-ß, insulin and steroid hormone signaling pathways
(Riddle and Albert, 1997;
Gerisch et al., 2001
;
Jia et al., 2002
).
In addition to mutations that show an abnormality in dauer larva formation
by themselves, mutations that show Daf-c phenotypes only in the background of
another mutation have been discovered and called synthetic Daf-c mutations. A
great majority of them were isolated by other phenotypes, mostly those in
neuronal functions, and were later found to show this phenotype when double
mutants of these mutations were constructed. For example, unc-3,
unc-31 and aex-3 single mutants produce no or few dauer larvae
under favorable conditions, but the double mutants unc-31;unc-3 and
unc-31;aex-3 produce many dauer larvae
(Bargmann et al., 1990;
Iwasaki et al., 1997
). The
unc-3 gene encodes an OLF-1/EBF (O/E) family transcription factor
(Prasad et al., 1998
). The
unc-31 gene encodes a homolog of CAPS (calcium-activated protein for
secretion) (D. Livingstone, PhD thesis, University of Cambridge, 1991)
(Ann et al., 1997
), which is
required for the exocytosis of dense core vesicles
(Berwin et al., 1998
).
unc-31 mutants show slow locomotion
(Brenner, 1974
) and a Daf-c
phenotype in the wild-type background at a very high temperature (27°C),
at which C. elegans cannot reproduce
(Ailion and Thomas, 2000
). The
aex-3 gene encodes a guanine nucleotide-exchange factor for Rab3
GTPase, which is required for intraneuronal transport
(Iwasaki et al., 1997
).
Although daf-c mutations have already been saturated
(Malone and Thomas, 1994
),
many synthetic Daf-c mutations remain to be identified.
To identify new genes regulating dauer larva formation and to discover new
mechanisms, we isolated 44 synthetic Daf-c mutants in the
unc-31(e169) background and named them sdf (synthetic
abnormality in dauer larva formation) mutants (N.S., T.I. and I.K.,
unpublished). Of these mutants, the genes for sdf-9 and
sdf-13 have been cloned. The sdf-9 gene encodes a protein
tyrosine phosphatase-like molecule. It is expressed in a pair of
neuron-associated cells called XXXL/R, and regulates dauer larva formation in
the steroid hormone signaling pathway
(Ohkura et al., 2003).
sdf-13 encodes a homolog of the transcription factor Tbx2/Tbx3, and
is expressed in AWB, AWC and ASJ sensory neurons, and in many pharyngeal
neurons. It controls olfactory adaptation in AWC and dauer larva formation in
cells other than AWC (possibly ASJ)
(Miyahara et al., 2004
).
In this report, we describe the cloning, expression and functional analyses of another sdf gene, sdf-14, in which three mutant alleles, ut151, ut153 and ut155, were isolated. sdf-14 is allelic to mrp-1, which encodes an MRP (multidrug resistance-associated protein) homolog. It acts in multiple tissues to regulate dauer larva formation. Human MRP1 can substitute for C. elegans MRP-1 in dauer larvae regulation, for which the transport activity of MRP1 is required. We discuss the mechanism of dauer larva regulation by MRP-1.
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Materials and methods |
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Dauer larva formation assay
Three to eight adult hermaphrodites were transferred onto 35-mm plates
seeded with Escherichia coli OP50, and allowed to lay eggs for 3-8
hours at 18, 20, 23.5, 25.5, 26.5 or 27°C. The parents were then removed,
and the progeny were cultured at the same temperatures. The numbers of dauer
larvae and non-dauer animals (L4/adults) among the progeny were scored after
4-5 days at 18°C and 20°C, 3-4 days at 23.5°C to 26.5°C, and
3-5 days at 27°C. At 27°C, the growth rates were variable from animal
to animal.
In the experiments for testing the effect of drugs, PAK-104P, agosterol A (AG-A), MK571 and sodium arsenite were dissolved in dimethyl sulfoxide (PAK-104P), ethanol (AG-A) or H2O (MK571, sodium arsenite), and added to the NGM agar medium for plates, when it was cooled to 60°C, to give appropriate concentrations. Dauer larva formation was assayed with these plates after E. coli OP50 was grown. PAK-104P, AG-A, MK571 and sodium arsenite were obtained from Nissan Chemical Industries (Chiba, Japan), Dr Shunji Aoki (Graduate School of Pharmaceutical Sciences, Osaka University), Cayman Chemical Company (Ann Arbor, Michigan, USA), and Wako Pure Chemical Industries (Osaka, Japan), respectively.
Mapping of sdf-14 mutations
sdf-14 mutations were mapped near the left end of the X chromosome
by STS mapping with the strain RW7000
(Williams et al., 1992). The
map position was determined more precisely by using single-nucleotide
polymorphisms (SNPs) (Wicks et al.,
2001
). For SNP mapping, CB4856 males were crossed to
unc-31(e169);sdf-14(ut153) double mutant hermaphrodites at 20°C.
Then, individual F2 animals that showed the Unc-31 phenotype were picked and
cultured separately at 25.5°C for the test of dauer larva formation.
Genomic DNA was prepared from bulk of F3 self-progeny, and SNPs were detected
by RFLPs or DNA sequencing. In addition to the SNP data provided by Dr Wicks,
we also found some SNPs by sequencing the CB4856 genomic DNA and used them for
mapping. The results showed that sdf-14 mutations are located between
the cosmid clones M02E1 and F02G3.
Rescue of sdf-14 mutations for positional cloning
The cosmid clones between M02E1 and F02G3, either separately or as
combinations of two adjacent clones, were injected into the germline of the
unc-31(e169);sdf-14(ut153) double mutant
(Mello et al., 1991). Of all
the injected clones, only the combination of F57C12 and F55H6 rescued the
Daf-c phenotype, which suggested that the sdf-14 gene was identical
to F57C12.5. We confirmed this by injecting various genomic DNA clones that
contained only the F57C12.5 gene. One of these clones, pSDF14, was made by the
integration of the following three fragments into the
HincII-XmaI site of pBluescript II KS(): (1) the
SphI-ClaI fragment of the cosmid F57C12; (2) the
ClaI-Aor51HI fragment of the cosmid F57C12; and (3) the
XmaI-SphI fragment corresponding to the promoter and N
terminus of F57C12.5 gene. Fragment 3 was made by PCR amplification of genomic
DNA using the F57C12.5-4 primer GCT GGA TGA TTT GCA CTT CGA GTA GTT GGC and
the F57C12.5-35 primer GCC GAA CAT CAA TTT GAC GG, cloning of the PCR fragment
into the XbaI-SphI site of pUC119, and excision of the
XmaI-SphI fragment from the clone.
In the rescue experiments, the genomic DNA clones were injected into unc-31(e169);sdf-14(ut153) animals at concentrations of 5-10 ng/µl, together with injection marker DNA (gcy-10::GFP; 25-30 ng/µl). Dauer larva formation of the transformants was assayed at 25.5°C.
Rescue of the dauer-constitutive phenotype with mrp-1 isoforms
The DNA constructs of b-, c- or e-type isoforms for transformation
experiments were made from cDNA clones, yk1067b09(b), yk46e1(c), yk494b1(e)
and yk831b09(e), kindly provided by Y. Kohara. The cDNA sequences were
confirmed by sequencing. Then, the exon 3-19 region of pSDF14::GFP was
replaced by the corresponding part of each cDNA, flanked by BstEII
and SapI sites. The resulting isoform constructs (pSDF14b::GFP,
pSDF14c::GFP and pSDF14e::GFP) were injected at a concentration of 10 ng/µl
together with the gcy-10::GFP injection marker (25-30 ng/µl) into
unc-31(e169);mrp-1(ut153) animals, respectively. Dauer larva
formation of the transformants was assayed at 25.5°C.
Analysis of expression pattern
To examine the expression pattern of the mrp-1 gene, we made a
GFP-tagged mrp-1 gene (pSDF14::GFP) as follows. The region containing
the GFP-coding sequence and the unc-54 3' UTR sequence of
pPD95.79 (gift from A. Fire) was amplified by PCR with the
pPD95.75-AgeI primer GAG GGT ACC GGT AGA AAA ATG AGT AAA GGA GAA GAA
CTT TTC ACT GGA G and the AMP3 primer CTC AAC CAA GTC ATT CTG AGA ATA GTG.
Then, the AgeI-ApaI fragment of the PCR product was inserted
at the end of the mrp-1 coding sequence of pSDF14 at which point an
AgeI site was made in advance.
pSDF14::GFP and the rol-6(dom) marker DNA were co-injected into wild-type animals at concentrations of 40 ng/µl and 50 ng/µl, respectively. The expression pattern of the transformants was observed under a fluorescence microscope. To check whether pSDF14::GFP was functional, pSDF14::GFP was injected into unc-31(e169);mrp-1(ut153) animals together with the gcy-10::GFP marker at concentrations of 10 ng/µl and 12 ng/µl, respectively. Dauer larva formation of the transformants was assayed at 25.5°C.
Rescue by tissue-specific expression with extrinsic promoters
The construct for pharyngeal muscle expression (pMyo2p::SDF14b::GFP) was
made by inserting the following two fragments of pSDF14b::GFP into the
KpnI-ApaI site of pPD30.69 (gift from A. Fire, containing
the myo-2 promoter): (1) the KpnI-SphI fragment of
the PCR product amplified from pSDF14b::GFP with the KpnI/5up primer CGG GGT
ACC AAT TAA GAA ATG TTC CCG TTA G and the F57C12.5-36-1 primer CGT TCA ACC TTC
GTC AAC TGC; and (2) the SphI-ApaI fragment of pSDF14b::GFP.
For intestinal and neuronal expression, the constructs were made in the same
way except that the myo-2 promoter in pPD30.69 was replaced by the
ges-1 promoter or the H20 promoter, respectively (pGes1p::SDF14b::GFP
and pH20p::SDF14b::GFP). These constructs were injected either separately or
in combination into unc-31(e169);mrp-1(ut153) animals together with
the gcy-10::GFP injection marker. The concentration of
gcy-10::GFP was 25-30 ng/µl, and that of the tissue-specific
expression constructs was 10 ng/µl. Dauer larva formation of the
transformants was assayed at 25.5°C.
Human MRP1 cDNA experiments
The construct of wild-type human MRP1 cDNA with the C. elegans
mrp-1 promoter (pSDF14p::hMRP1) was made by inserting the mrp-1
promoter into the XbaI-XmaI site of MCSI (multi-cloning site
I), and human MRP1 cDNA into the KpnI-SacI site of
MCSII in pPD49.26 (gift from A. Fire). The mrp-1 promoter (about 3.7
kb) was amplified by PCR, by which an XbaI site and an XmaI
site were made at the 5' and 3' end, respectively. The full-length
human MRP1 cDNA was manipulated as the 5' half
(SalI-EcoRI fragment) and the 3' half
(EcoRI-NotI fragment). A KpnI site was made by PCR
upstream of the initiation codon in the 5' fragment, and a SacI
site downstream of the stop codon in the 3' fragment. The construct of
the dmL0MRP1 mutant cDNA driven by the C. elegans
mrp-1 promoter (pSDF14p::mhMRP1) was made by replacing the
DraIII-EcoRI region of pSDF14p::hMRP1 with that of
dmL0MRP1 cDNA. The PCR primers used were as follows. For
the addition of XbaI and XmaI sites at the ends of the
mrp-1 promoter fragment, 3707/XbaI-FW primer GCT CTA GAA TTA
TAT CAC TTT TCG and 3707/XmaI-RV primer TCC CCC CGG GTT CTT AAT TGG CTC
GGT TCG G. For introducing a KpnI site upstream of the initiation
codon of human MRP1 cDNA, hmrp1/KpnI-1FW primer CGG GGT ACC AAT TAA
GAA ATG GCG CTC CGG GGC TTC TG and hmrp1-121RV CCC ACA CGA GGA CCG TG. For
introducing a SacI site downstream of the stop codon of human
MRP1 cDNA, hmrp1/3881FW primer GCT GGT TCG GAT GTC ATC TG and
hmrp1/SacI-4739RV primer GAT GCG GAG CTC TAT CAC ACC AAG CCG GCG TC.
To make transformants, pSDF14p::hMRP1 (40 ng/µl) and pSDF14p::mhMRP1 (100 ng/µl) were injected into unc-31(e169);mrp-1(ut153) animals together with the gcy-10::GFP (25-30 ng/µl) marker. Dauer larva formation of the transformants was assayed at 25.5°C.
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Results |
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We then cloned the sdf-14 gene by positional cloning, using the
synthetic Daf-c phenotype (see Materials and methods for details). The mutant
phenotype was rescued by a genomic DNA fragment containing only the F57C12.5
gene (pSDF14; Fig. 2). The
C. elegans database WormBase
(http://www.wormbase.org/;
Release WS136) indicated that F57C12.5 encodes a homolog of the multidrug
resistance-associated proteins (MRPs) belonging to the ATP-binding cassette
(ABC) transporter superfamily. Sheps et al.
(Sheps et al., 2004) reported
that F57C12.5 belongs to the same group as human MRP1, MPR2, MPR3 and possibly
MRP6. The amino acid sequence of SDF-14 showed about 60% homology to that of
human MRP1, and the homology extended all over the primary structure. Like
human MRP1, SDF-14 seemed to have three membrane-spanning domains (MSDs) and
two nucleotide-binding domains (NBDs). As human MRP1 was shown to have 17
transmembrane regions by experiments (Bakos
et al., 1996
; Kast and Gros,
1998
), SDF-14 also seemed to have 17 transmembrane regions, based
on the homology.
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C. elegans MRP-1 has multiple isoforms
WormBase suggested that the mrp-1 gene (F57C12.5) produces three
types of cDNAs encoding apparently functional isoforms (a-, b- and c-type;
Fig. 2A), as well as those
encoding short polypeptides (d.1, d.2 and d.3-type; not shown). Because the
diversity of the former isoforms originates from differences in exon 13, we
sequenced the exon 13 region of all of the nine cDNA clones kindly donated by
Y. Kohara. The results showed that four clones (yk15b10, yk131e9, yk892h09 and
yk1067b09) belonged to the b-type, two (yk46e1 and yk1289f01) belonged to the
c-type, and two (yk494b1 and yk831b09) belonged to a new type, which we call
the e-type. The remaining one clone (yk1240d1), a partial-length cDNA, had the
a-type sequence in exon 13, but retained intron 13, which caused a frame shift
that resulted in a stop codon in exon 14. Thus, we could not confirm the
presence of the a-type cDNA. One of the e-type clones, yk831b09, which
contained full-length cDNA, was sequenced completely (DDBJ/EMBL/GenBank
Accession Number AB199793).
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To learn in which tissue MRP-1 acts in the regulation of dauer larva formation, we investigated the phenotype of unc-31(e169);mrp-1(ut153) in which MRP-1 was expressed in various tissues by using extrinsic promoters. Expression of MRP-1 in only one tissue (neurons, intestinal cells or pharyngeal muscles) rescued the dauer larva formation abnormality only weakly. By contrast, the abnormality was rescued efficiently by expressing MRP-1 in two or three tissues (Fig. 6). The results show that there is no specific tissue in which MRP-1 acts to prevent dauer larva formation, and that MRP-1 molecules in neurons, intestinal cells and pharyngeal muscles act together for this function.
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We then carried out two experiments to investigate whether the transport activity of human MRP1 is required for this function. We first examined whether dmL0MRP1 mutant cDNA, driven by the C. elegans mrp-1 promoter, can rescue the Daf-c phenotype of unc-31(e169);mrp-1(ut153). The dmL0MRP1 protein has multiple amino acid substitutions in the L0 region and cannot transport the known substrates leukotriene C4 (LTC4) and 17ß-estradiol 17ß-(D-glucuronide) (E217ßG) (T. Noguchi, X.-Q. Ren and T.F., unpublished). Although as many as 19 transformant lines were examined, the dauer larva formation abnormality of unc-31(e169);mrp-1(ut153) was not rescued in any of them (data not shown). Thus, the amino acid residues of human MRP1 that are essential for its function as a transporter in human cells are also essential for dauer larva regulation in C. elegans.
Next, we investigated the effects of human MRP1 inhibitors on the dauer
larva formation of unc-31(e169);mrp-1(ut153) mutants carrying the
extrachromosomal array of human MRP1 cDNA connected to the C.
elegans mrp-1 promoter. We used three inhibitors, PAK-104P
(Shudo et al., 1990),
agosterol A (AG-A) (Aoki et al.,
1998
) and MK571 (Jones et al.,
1989
), each of which was added to the agar plates for the assay of
dauer larva formation. The results showed that PAK-104P, but not AG-A (0-200
µM) or MK571 (0-200 µM), enhanced the dauer larva formation of the
strain expressing human MRP1 (Fig.
7B and data not shown). None of the inhibitors had any effect on
unc-31(e169);mrp-1(ut153) carrying the extrachromosomal array of the
C. elegans mrp-1 gene (Fig.
7C). These experiments indicate that PAK-104P acts specifically on
human MRP1 and that the transporter activity of human MRP1 is required for the
suppression of dauer larva formation.
Position of the mrp-1 gene in the genetic pathways of dauer larva formation
To obtain information on the mechanism of dauer larva regulation by MRP-1,
we investigated the position of the mrp-1 gene in the regulatory
pathway. It is known that four pathways are involved in dauer larva
regulation: the cGMP, TGF-ß, insulin and steroid hormone signaling
pathways (Riddle and Albert,
1997; Gerisch et al.,
2001
; Jia et al.,
2002
). The following two sets of experiments indicate that
mrp-1 does not act in the cGMP or TGF-ß signaling pathway.
|
Second, the Daf-c phenotype of the daf-2(e1370);mrp-1(pk89) double
mutant was not suppressed by daf-d mutations in the cGMP or
TGF-ß signaling pathway. It is known that the daf-2(e1370)
mutant, which affects the insulin signaling pathway, shows a strong Daf-c
phenotype at 25°C, but not at 20°C
(Gems et al., 1998). Because
the daf-2(e1370);mrp-1(pk89) double mutant showed a strong Daf-c
phenotype at 20°C (Fig.
8A), we examined whether this phenotype was suppressed by
che-3(e1124) or daf-5(e1386), daf-d mutations in the cGMP
and TGF-ß signaling pathway, respectively. As shown in
Fig. 8B,C, the Daf-c phenotype
of daf-2;mrp-1 was suppressed neither by che-3 nor by
daf-5. It is known that che-3 and daf-5 suppress
all the daf-c mutations in the cGMP and TGF-ß signaling pathway,
respectively, but not daf-2(e1370)
(Vowels and Thomas, 1992
;
Thomas et al., 1993
). Hence,
we concluded that mrp-1 acts in neither the cGMP nor the TGF-ß
signaling pathway. As expected, the Daf-c phenotype of
daf-2(e1370);mrp-1(pk89) was suppressed by daf-16(mu86)
(Fig. 8D), a daf-d
mutation in the insulin signaling pathway.
As the mrp-1 gene acts in neither the cGMP nor the TGF-ß
signaling pathway, it may act through either the insulin or steroid hormone
signaling pathway, which interact each other
(Ohkura et al., 2003;
Matyash et al., 2004
). In
fact, the enhancement of dauer larva formation by mrp-1 mutations is
especially strong in the background of daf-2(e1370ts) at 20°C or
unc-31(e169) at 25.5°C, i.e. if the insulin signaling pathway is
partially blocked (Kimura et al.,
1997
; Ailion and Thomas,
2000
). Our attempts to prove that mrp-1 acts in the
insulin signaling pathway have failed so far. We planned to test whether
daf-16 mutations, which suppress daf-c mutations in the
insulin signaling pathway, suppress the Daf-c phenotype of
daf-1(m40);mrp-1, daf-14(m77);mrp-1 or daf-11(m47);mrp-1,
where daf-1 and daf-14 genes belong to the TGF-ß
signaling pathway and daf-11 belongs to the cGMP signaling pathway.
However, these experiments turned out to be inappropriate, because
daf-16 partially suppressed daf-1, daf-14 and
daf-11 at the temperatures at which mrp-1 mutations enhanced
the Daf-c phenotype of these mutations (T.Y., T.I. and I.K., unpublished)
(Vowels and Thomas, 1992
).
Furthermore, we could not find conditions under which mrp-1 mutants
produce the dauer-like larva, which is characteristic of mutants in the
steroid hormone signaling pathway (Gerisch
et al., 2001
; Jia et al.,
2002
; Ohkura et al.,
2003
).
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Discussion |
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ABC transporters form a large superfamily of proteins that are found in all
kingdoms (Higgins, 1992;
Dassa and Bouige, 2001
). The
function of most ABC transporters is the export of a wide variety of
substrates, such as the extrusion of noxious substances, secretion of
extracellular toxins, and targeting of membrane components, although some
prokaryotic members are involved in the import of essential nutrients.
Functional ABC transporters have four essential domains: two membrane-spanning
domains (MSDs) and two nucleotide-binding domains (NBDs), which are contained
in a single polypeptide in many cases, but in more than one polypeptide in
some cases. The MSD consists of several transmembrane
-helices
connected by polypeptides, whereas the NBD contains three consensus motifs:
Walker A motif, Walker B motif and Linker peptide
(Schneider and Hunke,
1998
).
The human ABC transporters are classified into eight subfamilies (subfamily
A to H)
(http://www.gene.ucl.ac.uk/nomenclature/genefamily/abc.html),
of which the MRPs belong to subfamily C. All MRPs have the structure of
(MSD-NBD)x2, while some of them, including MRP1, have an additional MSD
at the N terminus (MSD0) (Bakos et al.,
1996). MSD0 is not essential for the transport function, but the
intracellular linker domain (L0) connecting MSD0 and MSD1 is
required (Bakos et al.,
1998
).
The human MRP1/ABCC1 gene was first identified as a gene
similar to multidrug resistance protein 1/P-glycoprotein 1
(MDR1/PGY1/ABCB1). MDR1 and MRP1 contribute to the
multidrug resistance of various cancer cell lines. Multidrug resistance, by
which tumor cells become resistant to multiple structurally and functionally
unrelated drugs, is due to the extrusion of drug compounds from inside cells
to outside. Besides anti-cancer drugs, human MRP1 transports various organic
anions and nonanionic compounds conjugated by glutathione, glucuronide or
sulfate, and also co-transports nonanionic compounds with glutathione without
conjugation (Russel et al.,
2002). Most of these substrates are unnecessary compounds for the
organism (conjugates, xenobiotics and detoxification products) except for
Leukotriene C4 (LTC4)
(Renes et al., 2000
), a
glutathione-conjugated organic anion that acts as an inflammatory
mediator.
Structure and function of C. elegans MRP-1
Phylogenetic tree analysis showed that C. elegans MRP-1 belongs to
the same group as human MRP1, MRP2, MRP3 and MRP6 in the ABCC subfamily
(Sheps et al., 2004). It has
homology to these human MRPs throughout the amino acid sequence, including in
the MSDs, NBDs, Walker A and B motifs, and Linker peptide. The structural
similarity and conservation of the motifs suggests that the structure-function
relationship of C. elegans MRP-1 is similar to that of these human
MRPs. The results of this study are consistent with this idea.
First, NBD1 seems to be important in C. elegans MRP-1, because the
two missense mutations in this domain, ut151 and ut155,
showed phenotypes nearly as strong as the null mutation pk89. This is
similar to the case in human MRP1, in which NBD1 mutations markedly decrease
transport activity if not completely suppress it
(Gao et al., 2000). The two
NBDs of human MRP1 possess different properties: NBD1 binds ATP with high
affinity, whereas NBD2 is hydrolytically more active and binds ADP with high
affinity (Gao et al.,
2000
).
Second, our result agrees with the importance of the fourteenth
transmembrane region for substrate specificity in human and mouse MRP1
(Zhang et al., 2003). The
isoform rescue experiments showed that both the b-type and c-type, but not the
e-type, can rescue the Daf-c phenotype of unc-31(e169);mrp-1(ut153),
whereas exon 13, which is different in these isoforms, roughly corresponds to
the fourteenth and fifteenth transmembrane regions
(Fig. 2). This may be due to a
difference in substrate specificity between b/c-type and e-type, although this
remains to be proved. It is intriguing that the Drosophila CG6214
gene, an MRP1 homolog, has two variant copies of exon 4 and seven variant
copies of exon 8 (Grailles et al.,
2003
), where exon 8 partially overlaps with the sequence encoding
the fourteenth transmembrane region.
The transport activity of MRP1 is required for the regulation of dauer larva formation
We found that wild-type human MRP1 can substitute for C. elegans
MRP-1 in dauer larva regulation. Because human MRP1 had been studied
intensively, we took advantage of this and investigated the requirements for
successful substitution. The results showed that human MRP1 must retain its
export activity. First, dmL0MRP1, which is defective in the
transport of LTC4 and E217ßG, could not rescue the
Daf-c phenotype of unc-31;mrp-1. Second, PAK-104P, a competitive
inhibitor of human MRP1 (Sumizawa et al.,
1997), antagonized the function of human MRP1 but not C.
elegans MRP-1 in dauer larva regulation. The specific effect of PAK-104P
indicates that its target is human MRP1 and not other molecules inherent in
C. elegans cells. These results also strongly suggest that C.
elegans MRP-1 acts as an exporter in the regulation of dauer larva
formation.
These data indicate that there is a dauer-regulatory substance(s) among the substrates of MRP-1. The substance would have to be a dauer-inducer, if it acts inside the cell, whereas it would have to be a dauer-inhibitor, if it acts outside the cell. The identification of the regulatory substance would be a crucial next step for elucidating the mechanism of dauer larva regulation by MRP-1.
Possible involvement of stress response in dauer larva regulation by MRP-1
mrp-1 mutations strongly enhance the Daf-c phenotype of insulin
signaling mutants, when compared with that of cGMP or TGF-ß signaling
mutants. This regulation may involve a stress response, as suggested by the
following indirect evidence. (1) Insulin signaling genes are closely related
to stress response in their control of expression
(Murphy et al., 2003;
McElwee et al., 2004
) and
their mutant phenotypes (Honda and Honda,
1999
; Baryste et al., 2001). (2) RNAi of the heat shock factor
(hsf-1) gene, which is essential for stress response, suppresses the
Daf-c phenotype of insulin signaling mutants
(Walker et al., 2003
;
Morley and Morimoto, 2004
).
(3) Both high temperature and sodium arsenite, which induce stress response,
enhance the Daf-c phenotype of insulin signaling mutants efficiently, as do
mrp-1 mutations (Ailion and
Thomas, 2003
) (this study). (4) Human MRP1 transports the arsenite
ion as a complex with glutathione (Zaman
et al., 1995
), and probably the same is true for C.
elegans MRP-1, because its mutants are hypersensitive to arsenite ions
(Broeks et al., 1996
).
![]() |
ACKNOWLEDGMENTS |
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![]() |
Footnotes |
---|
Present address: Laboratory for Cell Migration, RIKEN Center for
Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 605-0074,
Japan
Present address: Department of Biology, Faculty of Sciences, Kyushu
University Graduate School, Hakozaki, Fukuoka 812-8581, Japan
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