Department of Human Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK
* Author for correspondence (e-mail: deborah.goberdhan{at}anat.ox.ac.uk)
Accepted 8 March 2005
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SUMMARY |
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Key words: Insulin signalling, Drosophila, Cancer, TOR, Diabetes, Nutrition, SLC36
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Introduction |
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Eukaryotes possess a second and more ancient nutrition-regulated system,
involving the kinase TOR (target of rapamycin)
(Jacinto and Hall, 2003),
which is responsive to local nutrient levels, particularly amino acids. In
higher eukaryotes TOR controls downstream translational regulators, including
p70-S6 kinase (S6K) and 4E-binding protein (4EBP) and is essential for normal
cell-autonomous growth (Zhang et al.,
2000
; Oldham et al.,
2000
). Studies in Drosophila have recently linked the
endocrine InR signalling system with TOR (reviewed by
Goberdhan and Wilson, 2003a
;
Aspuria and Tamanoi, 2004
). It
is proposed that Akt, a key InR-regulated kinase, phosphorylates the tumour
suppressor protein, Tuberous sclerosis complex 2 (Tsc2), dissociating the
Tsc1/Tsc2 complex. This activates the G protein Rheb, which positively
regulates TOR.
TOR activity can be affected by changes in intracellular amino acid levels
(Christie et al., 2002;
Beugnet et al., 2003
).
Experiments in cell culture indicate that this response is modulated by amino
acid transporters, but no amino acid transporter has yet been shown to
regulate growth of non-endocrine cells in vivo. We have therefore genetically
screened for such transporters. Although a number of different classes of
mammalian amino acid transporter have been identified (see
Hediger, 2004
), many still
have poorly characterised in vivo functions. In Drosophila, members
of two transporter classes have been implicated in growth: minidiscs
encodes a component of the heterodimeric family of transporters
(Martin et al., 2000
); and
slimfast (slif)
(Colombani et al., 2003
)
encodes a cationic amino acid transporter. These genes are required for normal
growth, but they both primarily act in the fat body, an amino acid-sensitive,
growth-regulatory endocrine organ with functional similarities to the
mammalian liver and white adipose tissue (Britton et al., 1998).
Here, we show that two genes encoding proteins related to a third class of
transporters of previously unknown biological function, the proton-assisted
transporter (PAT or SLC36) family
(Bermingham and Pennington,
2004), specifically modulate tissue-autonomous growth in multiple
non-endocrine tissues in vivo. They also genetically interact with TOR and
other InR signalling components, indicating a direct or indirect regulatory
link with this signalling system. One of these transporter genes,
pathetic (path), is expressed in a wide range of tissues and
is essential for both endocrine and local growth regulation. PATH has unusual
functional properties, establishing it as the founder member of a new class of
low capacity, very high-affinity PAT-related transporters, which control
growth via a novel amino acid sensing mechanism. As the functions of
path can be substituted by another Drosophila PAT-related
transporter whose properties are much more similar to mammalian PATs, we
propose that this gene family has a conserved and unique role in regulating
TOR-dependent growth and the response to insulin-like molecules in development
and disease.
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Materials and methods |
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Molecular analysis
Putative full-length path-RA (RH24992; almost all 80 available
ESTs represent the RA form) and CG1139 (LP06969) cDNAs were subcloned
into transformation vector pUAST (Brand and
Perrimon, 1993) using the restriction sites flanking the cDNA
inserts (EcoRI/XbaI and EcoRI respectively).
Transgenics were produced using standard procedures. At least four independent
lines were established and tested. In situ hybridisation was based on work by
Tautz and Pfeifle (Tautz and Pfeifle,
1989
), using a PCR-amplified path template cDNA. cRNA was
synthesised by in vitro transcription (mMessage mMachine, Ambion).
Functional analysis in Xenopus oocytes
Healthy looking Xenopus laevis oocytes (stage V and VI) were
obtained as described (Meredith et al.,
1998), and maintained at 18°C in modified Barth's medium.
Transport measurements were performed at least 72 hours after microinjection
of oocytes with 27.6 nl cRNA (1 µg/µl) with daily medium changes. Uptake
assays with [3H]-alanine (59 Ci/mmole, Amersham Biosciences) were
performed essentially as described, with five oocytes per data point
(Meredith et al., 1998
).
Membrane potential was measured using a Dagan CA-1B amplifier. Oocytes impaled
with a AgCl/3M KCl-filled glass microelectrode (resistance approximately 0.5
M
) were superfused with uptake medium containing 2 mM amino acid or
uptake medium alone. Intracellular amino acid concentrations were calculated
based on the work of Christie et al.
(Christie et al., 2002
).
Western blotting followed standard procedures. Oocytes were lysed in 3 µl/oocyte lysis buffer and loaded with 1:1 (v:v) loading buffer. PVDF membranes were probed with 1:1000 (v:v) anti-phospho-p70 S6K (Thr389) antibody (Signaling Technologies, NEB) or 1:200 (v:v) anti-total S6K (Santa Cruz), followed by 1:2000 goat anti-rabbit HRP-conjugated secondary antibody (Santa Cruz) and the membrane developed using the ECL system (Amersham).
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Results |
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Heterodimeric and cationic transporter genes produced little or no effect on growth when expressed in differentiating ommatidia using GMR-GAL4 (e.g. slif; Fig. 1B). By contrast, overexpression of GS elements upstream of CG3424 (subsequently renamed path) and CG1139, which encode proteins similar to the mammalian PATs (>30% identity; see Fig. S1A in the supplementary material), increased both ommatidial and overall eye size (Fig. 1C,D, respectively). When two GS insertions in path (Fig. 1E,L) or in CG1139 (Fig. 1F,M) were co-expressed, the resulting eyes bulged more from the head capsule and the ommatidial array was mildly disorganised, indicating a more extreme dose-dependent overgrowth phenotype. Similar growth effects were also produced by UAS-path (e.g. Fig. 2K-M) and UAS-CG1139 (Fig. 1H) transgenes, confirming that these genes caused the GS-associated phenotypes.
|
CG1139 and path also modulated growth in the developing wing, but their effects differed. Although slif had no significant effect with the MS1096-GAL4 driver (Fig. 2B), which is expressed throughout the developing adult wing, but preferentially on the dorsal surface, in animals overexpressing CG1139 GS insertions, overall wing size was increased (Fig. 2D) and excess growth on the dorsal surface caused the wing to turn down. However, the effects of CG1139 on wing growth were not simple. Surprisingly, overexpression of the UAS-CG1139 transgene, which has a more potent growth-stimulating effect in the eye than CG1139 GS inserts (Fig. 1H), reduced wing growth and produced upturned wings (Fig. 2F), suggesting that this gene might inhibit growth in some developmental contexts, if sufficiently overexpressed.
This effect was reminiscent of UAS-Tor, which, despite the
well-documented growth-promoting functions of Tor, reduces growth
when overexpressed in the wing (Fig.
2E) (Hennig and Neufeld,
2002). It has been proposed that TOR overexpression interferes
with proper formation of a TOR-containing multiprotein complex in the wing to
inhibit growth. To test whether a similar dominant-negative mechanism might
explain the growth inhibitory effects of CG1139, we overexpressed two
CG1139 GS insertions. Although both these insertions promoted growth
when expressed independently, in combination they significantly reduced growth
(Fig. 2I). In addition,
co-expression of a CG1139 GS insertion with UAS-CG1139
enhanced the growth inhibitory effect of the latter construct
(Fig. 2J).
Co-overexpression of CG1139 GS inserts with Tor greatly exaggerated the reduced growth phenotype produced by Tor alone (Fig. 2H), suggesting that these molecules might exert their dominant-negative effects on interacting complexes. Overexpression of path GS insertions or UAS-path reduced wing size in the MS1096-GAL4 assay (e.g. Fig. 2C), suggesting that path also has dominant-negative effects in the wing. In addition, like CG1139, path overexpression strongly enhanced the dominant-negative effects of Tor (Fig. 2G).
To quantify growth changes in the wing, the transporter genes were
overexpressed in the posterior wing compartment with en-GAL4. Effects
on overall wing growth mirrored those observed with the MS1096-GAL4
driver (Fig. 2K) and in most
cases total cell number was significantly reduced
(Fig. 2M). Unexpectedly,
however, path and CG1139 overexpression produced an increase
in cell size, even when overall wing area was reduced
(Fig. 2L). For each transgenic
line tested, the increase was approximately inversely related to the reduction
in cell number. Again, we tested whether these effects might be comparable
with those of TOR. It has been reported that TOR overexpression in wing
imaginal discs reduces the size of proliferating cells, as does a
dominant-negative form of TOR, TOR-TED
(Hennig and Neufeld, 2002).
However, they did not analyse the size of cells in the adult wing. In fact,
posterior wing cells were enlarged in en-GAL4 UAS-Tor adult animals,
despite the reduction in overall compartment size, mimicking the effects of
PAT-related transporters (Fig.
2L). The effects of overexpressing PATH, CG1139 and TOR on cell
growth and proliferation therefore vary in parallel, depending on
developmental context, with their growth inhibitory actions in the wing most
likely being caused by dominant-negative effects during the proliferative
stage of development.
path normally promotes growth in flies
To test whether PAT-related transporters normally promote growth in flies,
we searched for specific mutations in either CG1139 or path.
We identified a transposable element insertion,
pathKG06640, located within the first intron of the larger
path transcription unit, RA (see
Fig. 3A). The KG transposon
contains gene silencing elements, which increase the likelihood that
insertions will affect gene expression
(Roseman et al., 1995). Flies
homozygous for pathKG06640 were markedly smaller than
wild-type animals (Fig. 3B) and
40% lighter in weight (Fig.
3C). Wing size was decreased by nearly 20%
(Fig. 3D), with significant
reductions in both cell number and size (P<0.001). Mutant eyes
contained small ommatidia (compare Fig. 3E
with 3F). Like many viable mutants in which InR signalling is
disrupted (e.g. Böhni et al.,
1999
), pathKG06640 homozygotes were
developmentally delayed, eclosing roughly 3 days later than normal. Homozygous
females also laid no eggs.
|
|
path is expressed in growing tissues during development and regulates both global and local growth signals
path is highly represented in the EST database. RNA in situ
hybridisation performed on embryonic and late larval stages revealed that it
is expressed in most tissues. However, transcript levels were highly dynamic
in embryogenesis with surges of expression in many structures, including
muscle primordia, salivary glands, proventriculus (and other parts of the
gut), trachea and gonads (see Fig.
4A-C). Expression was strongly reduced in homozygous
pathKG06640 mutants
(Fig. 4D). Larval imaginal
discs also expressed path in all or most cells. Expression was
particularly strong in the pouch and hinge regions of the wing disc
(Fig. 4E) and in the
morphogenetic furrow of the eye disc (Fig.
4G). Transcript levels were reduced in
pathKG06640 homozygotes
(Fig. 4F,H), but the reduction
was less severe than in embryos, particularly in the eye.
The relatively modest reduction in path expression in mutant
imaginal discs suggested that the significant growth defects associated with
this mutant might not be explained by a cell-autonomous reduction in PATH
activity. Indeed, when the eyFLP/FRT approach
(Newsome et al., 2000) was
used to generate eyes in which many ommatidia were derived from
pathKG06640 homozygous cells, no significant growth
disadvantage was observed in these cells. Even when almost all the eye was
mutant for path, because the other clonal material produced was
homozygous for a cell lethal chromosome, the resulting eyes were
indistinguishable in size from normal eyes (compare
Fig. 4J with 4I and 4K).
However, pathKG06640 could produce tissue-autonomous effects in the eye in specific genetic backgrounds. For example, in our hands, it was difficult to produce viable animals with eyes that mainly consisted of homozygous Tsc2 clones, using eyFLP-induced recombination with a cell lethal chromosome. Less than 1% of females and no males of the appropriate genotype survived through pupal stages to adulthood. When clonal material in the eye was mutant for both Tsc2 and pathKG06640 in an otherwise heterozygous animal, the lethal effects of Tsc2 were suppressed, and about 10% of mutant animals, both males and females, eclosed. Their eyes were smaller and bulged less than Tsc2 mutant eyes (compare Fig. 4M,N), revealing a tissue-autonomous effect of path on the Tsc2 phenotype.
FOXO-induced cell death is specifically enhanced by the TOR signalling cascade and overexpression of PAT-related transporters
Our genetic data showed a role for PAT-related transporters in growth
regulation and indicated that, like other amino acid transporters, this effect
might involve TOR. However, they did not reveal whether these transporters
directly modulate TOR signalling or have an indirect effect via a parallel
growth regulatory pathway. To investigate this issue further, we developed an
in vivo genetic assay to detect changes in TOR/S6K signalling activity in a
tissue where PAT-related transporters stimulate growth. The forkhead
transcription factor FOXO is a target of InR signalling in higher eukaryotes,
including flies (Jünger et al.,
2003; Puig et al.,
2003
). FOXO normally inhibits cell proliferation and stimulates
apoptosis, but is inactivated when phosphorylated by InR-regulated Akt.
Overexpression of a GS insertion in the first intron of foxo,
foxoGS9928, with GMR-GAL4 produced distinctive
ommatidial loss particularly in the posteroventral part of the eye
(Fig. 5B)
(Jünger et al.,
2003
).
|
Radimerski et al. (Radimerski et al.,
2002a; Radimerski et al.,
2002b
) have provided biochemical and genetic evidence that TOR
signalling can negatively feed back to the InR signalling cascade, thus
inhibiting Akt1 activity. We therefore reasoned that co-overexpression of TOR
signalling components with GMR-GAL4 foxoGS9928 might
enhance the growth inhibitory effects of FOXO. Indeed, S6K, which normally
promotes modest overgrowth with GMR-GAL4
(Fig. 3M)
(Zhang et al., 2000
), strongly
enhanced the GMR-GAL4 foxoGS9928 reduced eye phenotype
(Fig. 3N), mirroring the
effects of dominant-negative Dp110 (Fig.
5E). A UAS-containing insertion in Rheb,
RhebAV4 (Patel et al.,
2003
), which by itself produces a greatly enlarged eye
(Fig. 5G,J)
(Patel et al., 2003
), enhanced
FOXO activity even more strongly in the eye, leading to loss of virtually all
ommatidia in females (Fig. 5H)
and pupal lethality in males with massive tissue degeneration in the head of
late pupae. As expected, these phenotypes were suppressed by Akt1
overexpression (Fig. 5I,L), but
not by co-expression of other UAS constructs (e.g. UAS-GFP,
UAS-lacZ). Thus, growth-stimulating TOR signalling components act
entirely differently to all other positive growth regulators in this assay,
reducing eye size when co-expressed with FOXO.
Co-expression of foxo with either CG1139 or path also enhanced the foxo reduced eye phenotype (Fig. 5P,S), an effect completely suppressed by Akt1 (Fig. 5Q,T), consistent with a role for these transporters in modulating TOR signalling. Interestingly, although TOR alone did not affect the GMR-GAL4 foxoGS9928 phenotype (Fig. 5X), it did enhance the effects of a weakly expressed UAS-path transgene (compare Fig. 5V with 5Y). Thus, TOR and the PAT-related transporters interact genetically in multiple developmental assays, and produce similar phenotypes when overexpressed in multiple tissues and in an in vivo assay that is specific for the TOR signalling pathway.
PATH defines a new type of PAT-related transporter with remarkably high substrate affinity and a novel signalling mechanism
To examine the transport properties of PATH and CG1139, we overexpressed
each in Xenopus oocytes. This heterologous system has been
extensively employed in the characterisation of many mammalian amino acid
transporters, including PATs (e.g. Boll et
al., 2002; Chen et al.,
2003b
). CG1139 behaved very similarly to mammalian PATs in
radiolabelled amino acid influx (Fig.
6A,B) and inhibition (Fig.
6C,E) assays, and in electrophysiological studies
(Fig. 6F,G). Like human PAT1
and PAT2, CG1139 specifically transported alanine, glycine and proline in an
electrogenic and proton-stimulated fashion. Not only was influx of
radiolabelled amino acids enhanced by extracellular acidification
(Fig. 6A,B), but when compared
with results obtained at pH 7.4, uptake of alanine, glycine and proline
produced a larger plasma membrane depolarisation at pH 5.5, with reversal of
membrane polarity (Fig. 6F,G).
CG1139 and PAT1 have similar affinity for alanine [Km of
1.2±0.2 mM (R2=0.99; Fig.
6E) versus an IC50 of 1.7±0.23 mM for PAT1
(Chen et al., 2003a
)], glycine
(estimated IC50=3 mM, 2.3 mM for PAT1;
Fig. 6C) and proline (estimated
IC50=8 mM, 2.0 mM for PAT1).
|
In vivo genetic studies described above show that PATH and CG1139 have
similar effects on growth and in path rescue experiments. However, in
Xenopus oocytes, PATH has a transport capacity about 400 times lower
than CG1139. It would thus be expected to increase overall intracellular
levels of alanine by less than 0.2% during a 30 minute incubation. We tested
whether despite this low transport capacity, extracellular alanine could
activate the downstream target of TOR, S6K, in PATH-expressing
Xenopus oocytes. S6K activation was detected in oocytes microinjected
with leucine or alanine using an antibody that specifically crossreacts with
the phosphorylated activated form of this molecule
(Fig. 6H; data not shown)
(Christie et al., 2002).
Extracellular alanine had no effect on control oocytes, but did stimulate S6K
signalling in oocytes expressing PATH (Fig.
6H). Thus, PATH can activate growth and a TOR target, S6K, in the
absence of significant overall change in intracellular amino acid
concentration.
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Discussion |
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path and CG1139 phenocopy the growth effects of
Tor in the eye and wing, and strongly interact with Tor in
multiple genetic assays. In addition, like positive growth regulatory
components of the TOR signalling pathway, the transporters reduce eye size in
the FOXO interaction assay. Members of other classes of transporter, including
the cationic transporter Slif, did not produce this range of effects. Our data
are therefore consistent with a model in which both TOR
(Fang et al., 2001;
Wedaman et al., 2003
) and the
PAT-related transporters act within one or more membrane-associated molecular
complexes required for TOR activation. We cannot eliminate the possibility
that PATs act in a parallel growth regulatory pathway, but our data indicate
that one function of this pathway would be to alter the sensitivity of the
cell to InR activation (Fig.
7).
The TOR signalling pathway feeds back to negatively regulate InR signalling
Under normal conditions, the net result of TOR activation is overgrowth,
but when FOXO, a direct target of Akt1 that is not part of the
Akt1/Tsc/Rheb/TOR link (Fig.
7), is overexpressed in the differentiating eye, the TOR-induced
reduction in Akt1 signalling increases cell death. Currently, the feedback
mechanism involved has not been fully elucidated, although studies in mammals,
where a similar phenomenon occurs, suggest that the phosphorylation state of
IRS-1, an adaptor molecule for the InR, is involved
(Jaeschke et al., 2002;
Um et al., 2004
;
Harrington et al., 2005
). One
consequence of Akt repression by TOR signalling is to make cells more insulin
resistant, a defining cellular and genetic defect in Type II diabetes. As
PAT-related transporters enhance the effects of FOXO, partial cell surface
inhibition of PAT activity might prove beneficial in individuals with Type II
diabetes.
PATH regulates growth via local and global signalling mechanisms
Consistent with our proposal that path functions by modulating the
response to InR/TOR signalling, the effects of reduced path on
growth, developmental timing and female fertility broadly mirror those seen in
InR signalling mutants. path is still transcribed, albeit at reduced
levels, in pathKG06640 mutant imaginal discs, and clonal
analysis reveals that at least in the eye, an altered external growth signal
is primarily responsible for the path mutant phenotype. As this
phenotype is strongly suppressed by a combination of heterozygous
Tsc1 and Tsc2 mutations, this global function of
path probably involves modulation of InR/TOR signalling. Tor
also affects endocrine as well as cell-autonomous growth regulation, at least
in part by modulating fat body signalling
(Colombani et al., 2003).
However, the endocrine effects of path are unlikely to involve the
fat body, as this organ is relatively unaffected in path mutants and
path is weakly expressed in this tissue. These differences in tissue
expression may explain why, unlike Tor mutants, ERTs and primordia of
adult structures ultimately grow at a slower rate to roughly normal size in
path larvae.
The partial suppression of a Tsc2 clonal overgrowth phenotype in the eye by the pathKG06640 mutation indicates that this allele does have tissue- and presumably cell-autonomous effects on growth in non-endocrine tissues in sensitised InR signalling backgrounds. As we have recently identified another broadly expressed PAT-related transporter with growth regulatory activity, the relatively modest effects of the path mutation in imaginal discs may also be partly explained by functional redundancy in this family.
The transport properties of PATH reveal a novel mechanism for growth regulation
Analysis of the transport characteristics of CG1139 in the heterologous
Xenopus oocyte system revealed remarkable similarities to mammalian
PATs. By contrast, PATH displayed many novel properties, despite sharing
similar levels of sequence identity with mammalian PATs (see Fig. S1A,B in the
supplementary material). These include a mechanism inhibited by extracellular
acidification, altered substrate specificity, remarkably high substrate
affinity and low transport capacity. Although these experiments were not
performed in vivo, to our knowledge there is no reported example where the
functional properties of an amino acid transporter in the Xenopus
oocyte system are not very similar to its properties in vivo, and it seems
particularly unlikely that the substrate affinity of PATH would specifically
increase by 500-fold when synthesised in the oocyte.
Taken together with our primary finding that PATH and CG1139 are important
regulators of growth in vivo, these data have at least three major
implications. First, amino acid transporters have previously been shown to
modulate TOR by changing intracellular amino acid concentrations
(Christie et al., 2002;
Beugnet et al., 2003
), although
observations in whole tissues suggest sensing of extracellular amino acid
levels may be more important in regulating protein metabolism in vivo
(Mortimore et al., 1992
;
Bohé et al., 2003
). PATH
can promote growth in vivo and in the heterologous oocyte system it can
modestly activate S6K, despite its extremely limited transport capacity,
which, during the time course of our Xenopus assays, is predicted to
change bulk intracellular amino acid concentrations by less than 0.2%. PATH
must therefore control growth via an alternative and novel mechanism, either
by modulating local amino acid concentrations in a restricted compartment near
the transporter, or through transport-independent amino acid sensing
(Fig. 7). Based on genetic and
biochemical experiments, our current hypothesis is that this mechanism
involves TOR activation and is likely to be conserved for other PAT family
members, given the ability of CG1139 to largely substitute for
path function in vivo. Interestingly, if PATs regulate growth via a
transport-independent mechanism, the recent discovery that short chain fatty
acids are also substrates for human PAT1 and PAT2
(Foltz et al., 2004
) opens up
the possibility that these molecules could act as novel growth regulators.
Second, the fact that PATH and CG1139, a transporter with 500-fold lower
affinity, can have similar effects on growth in vivo, strongly suggests that
PATH (Km 3 µM) is saturated in developing tissues. Indeed,
extracellular amino acid concentrations are estimated to be in the millimolar
range in flies (e.g. Pierce et al.,
1999
). Thus, the growth-promoting ability of PATH is effectively
determined by its pattern and levels of expression and not by the
extracellular amino acid concentration. Of course, it is possible that in some
physiological conditions or within certain tissues, the high affinity of PATH
will be crucial, but to date, rescue experiments of
pathKG06640 mutant animals with both path and
CG1139 have revealed no obvious differences between these
molecules.
If PATH couples to TOR signalling, its properties ensure that all tissues
that express this transporter will normally activate TOR at least at a basal
level. Under these conditions, endocrine signalling by insulin-like molecules
will therefore partially dominate over local nutrient signals in determining
cell growth rates, consistent with previous observations
(Britton et al., 2002)
(Fig. 7). Interestingly, other
insect genomes encode proteins with high sequence homology to PATH (XM 308237
in the mosquito Anopheles gambiae; XP 396451 in the Western honeybee
Apis mellifera; see Fig. S1A,B in the supplementary material), and
presumably employ similar mechanisms. Although there is no evidence at present
for vertebrate sequence homologues, it is noteworthy that standard transport
assays, which initially suggested PATH was an orphan transporter, have also
failed to identify substrates for PAT3 and the ubiquitously expressed
PAT4.
Finally, PAT-related transporters have been identified at the cell surface
and in lysosomes. Subcellular shuttling of transporters has a precedent in
yeast, where TOR modulates movement of at least one amino acid permease
between the cell surface and the vacuole
(Chen and Kaiser, 2003).
Interestingly, yeast Tsc1, Tsc2 and Rheb have also been implicated in the
regulation of amino acid transporters
(Urano et al., 2000
;
van Slegtenhorst et al.,
2004
). However, contrary to expectations from multicellular
organisms, where the Tsc complex inhibits TOR signalling, yeast Tsc
upregulates and Rheb downregulates one or more amino acid permeases. Perhaps
in higher eukaryotes Rheb positively regulates some transporters and
negatively regulates others (Saucedo et
al., 2003
). Alternatively, endosomal and lysosomal targeting of
PAT-related transporters upon Rheb activation could increase activity,
although in this model, activation of PATH might only occur if it acts via a
transport-independent mechanism, as its transport activity appears to be
inhibited by acidification. Resolution of this issue will require a
biochemical and cell biological analysis of the interactions between upstream
InR signalling components, TOR complexes and PAT-related transporters.
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/10/2365/DC1
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