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First published online 20 April 2005
doi: 10.1242/dev.01821

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PAT-related amino acid transporters regulate growth via a novel mechanism that does not require bulk transport of amino acids

Department of Human Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK

View larger version (45K): [in a new window]   Fig. 2. PAT-related transporters phenocopy the effects of TOR on growth and cell number in the wing. (A-H) Expressing transgenes under the control of MS1096-GAL4, which drives transcription predominantly on the dorsal surface of the wing, resulted in an overall increase in wing size for CG1139GS10666 (D), but a reduction for pathGS13857(C), Tor (E) and UAS-CG1139 (F). Overexpression of slif had no significant effect (B) relative to control wings (A). Both pathGS13857 (G) and CG1139GS10666 (H) significantly enhanced the phenotype produced by overexpressing Tor. Co-expression of two CG1139 GS insertions (CG1139GS10666 and CG1139GS7120) inhibited growth (I) and CG1139GS10666 enhanced the growth inhibitory effect of UAS-CG1139 (J). (K-M) Histograms showing measurements of posterior compartment size (ratio of a specific wing area expressing the transgene and a non-expressing region=P/A) (K), cell size (L) and cell number (M) taken from adult female wings expressing transgenes under en-GAL4 control. Expression of each PAT-related transporter (two GS insertions and a UAS-transgene), TOR or a dominant-negative form of TOR (TOR-TED), but not Slif, produced highly significant changes in wing and cell size (P<0.001). Cell number was significantly reduced upon overexpression of TOR, TOR-TED and those PAT-related transporter constructs that have the greatest effects on growth (*P<0.01; **P<0.001).

View larger version (68K): [in a new window]   Fig. 1. Overexpression of Drosophila PAT-related transporters induces overgrowth in the differentiating eye. GMR-GAL4 was used to overexpress transgenes in differentiating cells of the fly eye (B-J,L,M). Compared with controls (A,K), much greater overgrowth was induced by pathGS13857 (C,E,L), CG1139GS10666 (D,F,M) and UAS-CG1139 (H), than by slif (B). Tor had no obvious effect on growth by itself (G), but enhanced the overgrowth phenotype induced by either pathGS13857 (I) or CG1139GS10666 (J). Where the ommatidial array was not disorganised, relative ommatidial size was measured (bottom of panel: mean±s.d.; *P<0.01; **P<0.001). Eyes bulged from the head in more extreme cases of overgrowth (compare dorsal views in L and M with control in K; white bars are of equal length in these panels), so that only the central portion of the eye is in focus in lateral images (E,F,H-J).

View larger version (58K): [in a new window]   Fig. 3. path regulates growth in Drosophila. (A) Map of the path (CG3424) genomic region. Two predicted transcription units for path have been defined: RA and RC. Coding (red) and non-coding (blue) regions are indicated. roo (grey) is a naturally occurring transposable element found in path. KG06640, GS13857 and GS11111 insertions are shown in green. (B) pathKG06640 homozygotes and pathKG06640/Df(3L)AC1 transheterozygotes were reduced in size compared with wild-type controls. (C,D) Histograms show adult weights and wing sizes for different path mutant genotypes. Values for mutant flies were strongly reduced compared with wild type (P<0.001), and were significantly rescued (P<0.001; light grey bars) by both UAS-path and CG1139GS10666 expression under arm-GAL4 control and by the presence of combined heterozygous mutations in Tsc1 and Tsc2, which produce only a very modest size increase in otherwise normal flies. (E-H) Eyes from control (E), a pathKG06640 homozygote (F), a pathKG06640/Df(3L)AC1 transheterozygote (G) and a pathKG06640 homozygote expressing UAS-path constitutively (H). (I) path mutant animals (here pathKG06640/Df(3L)AC1 transheterozygotes) were reduced in size throughout larval and pupal development, and developmentally delayed. However, pathKG06640 homozygous climbing third instar larvae had wing imaginal discs of roughly normal size (K) compared with wild type (J). Genotype of Tsc1/Tsc2-rescued flies in C and D is pathKG06640 Tsc2192/pathKG06640 Tsc129.

View larger version (73K): [in a new window]   Fig. 4. path is broadly expressed during development and affects global and local growth signals. (A-C) In situ hybridisation with a path antisense probe reveals expression in almost all tissues during embryonic development, including stages 11 (A), 13 (B) and 16 (C). High transcript levels were observed in specific tissues, such as the muscle primordia (white arrowhead), midgut (white arrow), proventriculus (black arrowhead) and salivary glands (black arrow). A path sense probe gave no signal (data not shown). In pathKG06640 homozygous embryos (D), a signal was observed only after extended staining times. (E-H) In situ hybridisation of wing (E,F) and eye antennal (G,H) imaginal discs from wild-type (E,G) and pathKG06640 homozygous (F,H; overstained to detect signal) climbing third instar larvae. path is normally most strongly expressed in the pouch (arrow) and hinge (arrowhead) regions of the developing wing and at the morphogenetic furrow (arrowhead) in the eye. (I-K) Eyes that are almost entirely generated from homozygous pathKG06640 cells via the eyFLP/FRT cell lethal method (J) were very similar in size to eyes of normal flies (I), and not small like those of pathKG06640 homozygotes (K). (L-N) Dorsal views of eyes almost entirely generated from Tsc2 mutant cells (M) and Tsc2 pathKG06640 double mutant cells (N) were compared with normal controls (L). Fly genotypes are: y w eyFLP/w; pathKG06640 FRT80B/rps174 P[w+] FRT80B (J); y w eyFLP/w; Tsc2192 FRT80B/rps174 P[w+] FRT80B (M); and y w eyFLP/w; pathKG06640 Tsc2192 FRT80B/rps174 P[w+] FRT80B (N).

View larger version (68K): [in a new window]   Fig. 5. PAT-related transporters and InR/TOR signalling components enhance FOXO activity by inhibiting the InR pathway. GMR-GAL4 was used to drive expression of the following transgenes in the differentiating eye: dominant-negative Dp110D954A (D-F), RhebAV4 (G-L), UAS-S6K (M,N), CG1139GS10666 (O-Q), pathGS13857 (R-T), UAS-path (U,V,Y) and UAS-Tor (W-Y). Flies overexpressing these transgenes together with foxoGS9928 (E,H,K,N,P,S,V,X,Y), or with foxoGS9928 and UAS-Akt1 (F,I,L,Q,T) were compared with GMR-GAL4 controls expressing the transgenes alone (D,G,J,M,O,R,U,W), no transgene (A), foxoGS9928 alone (B) or a combination of foxoGS9928 and UAS-Akt1 (C). A small eye phenotype and posteroventral reduction (arrow in B) is produced by GMR-GAL4 foxoGS9928. All eyes except G-I are from adult males, which produced stronger foxoGS9928 genetic interactions.

View larger version (26K): [in a new window]   Fig. 6. PATH has very different transport properties from CG1139 and mammalian PATs. (A,B) Histograms showing pH-dependent differences (P<0.001) in [3H]-alanine uptake by Xenopus laevis oocytes expressing either CG1139 (grey) or PATH (white) in acidic (pH 5.5) versus neutral (pH 7.4) medium, using 50 µM (A) and 75 nM (B) extracellular alanine (both well below the Km of CG1139). (C,D) Histograms showing inhibition of [3H]-alanine uptake in CG1139-expressing (C; 50 µM [3H]-alanine, pH 5.5) and PATH-expressing (D; 75 nM [3H]-alanine, pH 7.4) oocytes by exogenous alanine, proline and glycine. All effects are statistically significant (P<0.03), except that of proline on PATH. (E) Self-inhibition of alanine uptake into oocytes expressing either CG1139 (50 µM [3H]-alanine, pH 5.5; black circles) or PATH (75 nM [3H]-alanine, pH 7.4; white circles). Inset shows data for PATH over low concentration range (0 to 0.25 mM). (F,G) Measurements of membrane potential from oocytes expressing CG1139 in the absence or presence of exogenous alanine, glycine and proline at pH 7.4 (F) and pH 5.5 (G). None of the other common amino acids produced a response. (H,I) Western blots carrying identical oocyte extracts probed with antibodies specific for activated S6K (H) and total S6K (I; phosphorylated and non-phosphorylated S6K are not fully separated in this gel). The blots were subsequently stained with Coomassie Blue to confirm equal loading. Oocytes incubated with external alanine (10 mM, 30 min; lane 1) produced no detectable signal, while S6K was activated by microinjection of 27 nl of 10 mM alanine (lane 2). Incubation of PATH-expressing oocytes in an alanine-containing solution (10 mM, 30 min; lane 3) also activated S6K.

View larger version (36K): [in a new window]   Fig. 7. Regulation of growth by PAT-related transporters. Model illustrating the role of PAT-related transporters in Drosophila cell growth. Although general amino acid transporters (AAT) and PATs can potentially activate TOR by increasing bulk intracellular amino acid concentrations (grey arrows), PAT-related transporters (here labelled PAT) have unique growth regulatory properties that, at least in the case of PATH, involve a local sensing mechanism (red arrows from PATH and PAT), which is modulated by InR/Tsc/Rheb. Genetic data suggest that these transporters modulate TOR signalling, but they may also affect growth via a parallel pathway that appears to affect S6K activation. The TOR signalling pathway negatively feeds back to upstream InR signalling via a mechanism that is uncharacterised in Drosophila (red bracket).