Drosophila target of rapamycin kinase functions as a multimer

Abstract

Target of rapamycin (TOR) is a conserved regulator of cell growth and metabolism that integrates energy, growth factor, and nutrient signals. The 280-kDa TOR protein functions as the catalytic component of two large multiprotein complexes and consists of an N-terminal HEAT-repeat domain and a C-terminal Ser/Thr kinase domain. Here we describe an allelic series of mutations in the Drosophila Tor gene and show that combinations of mutations in the HEAT and kinase domains of TOR display the rare genetic phenomenon of intragenic complementation, in which two or more defective proteins assemble to form a functional multimer. We present biochemical evidence that TOR self-associates in vivo and show that this multimerization is unaffected by positive or negative signals upstream of TOR. Consistent with multimerization of TOR, recessive mutations in the HEAT and kinase domains can dominantly interfere with wild-type TOR function in cells lacking TSC1 or TSC2. TOR multimerization thus partially accounts for the high apparent molecular weight of TOR complexes and offers novel therapeutic strategies for pathologies stemming from TOR hyperactivity.

Figure 1.

Graphical representation of Tor mutant alleles. (A) Mutations are shown relative to defined structural and functional domains of the TOR polypeptide and are classified according to phenotypic severity and genetic interactions (see text). (B) Alignment of the four HEAT repeats carrying class I mutations. The two α-helices of each repeat are indicated above the alignments. The seven hydrophobic core residues of each repeat (Andrade et al. 2001) are highlighted in yellow, and mutated residues are highlighted in red. HEAT-repeat numbering (in parentheses) is from Perry and Kleckner (2003). (C) Mutations in the TOR kinase domain (residues 2065–2351) map to the catalytic and activation loops (green), and to the hydrophobic region of the ATP-binding pocket (residues predicted to interact with ATP are represented by dark blue stripes). The 153 amino acid Rheb-binding domain (Long et al. 2005) is indicated. (D) Multi-species sequence alignment of the region surrounding each TOR substitution mutation. Solid boxes indicate identical residues. Tor^R248stop and Tor^P2293L are from Oldham et al. (2000), who refers to these alleles as 2L19 and 2L1, respectively.

Figure 2.

Defective growth and gonadogenesis in Tor class I homozygous escaper adults. (A) Control (top; Tor^A948V/CyO) and class I homozygous mutant (bottom; Tor^A948V/Tor^A948V) adult females. (B) Brightfield image of ovaries from control (top) and Tor^A948V homozygotes (bottom). (C and D) Ovarioles from control (C) and Tor^A948V homozygous (D) mutant females, dissected and stained unfixed with acridine orange (apoptotic cells, red) and Hoechst 33342 (DNA, blue), are shown. (E and F) Testes from control (E) and Tor^A948V homozygous (C) males, stained for tubulin (red) and DNA (blue), are shown.

Figure 3.

Genetic and physical self-interaction of TOR proteins. (A) Survival, growth and fertility parameters for control (wt), class I homoallelic (I/I), and class I/class II heteroallelic (I/II) mutants. Data shown are for Tor^A948V (class I) and Tor^G2256D (class II). Error bars indicate standard deviation from the mean. (B) Summary of complementation tests between class I, II, and III Tor mutants. Crosses of any of the four class I alleles with either of the two class II alleles result in viable, fertile progeny. (C) TOR–TOR interaction in Drosophila S2 cells. Drosophila S2 cells were transfected with FLAG–TOR (lane 1) or FLAG–TOR plus HA–TOR (lanes 2–4) and treated with rapamycin or insulin as indicated. A small fraction of the cell lysate was probed with anti-FLAG to evaluate FLAG–TOR protein level (bottom gel). The remaining lysate was immunoprecipitated (IP) with anti-HA antibody. The HA–IP product was probed with anti-FLAG (top gel) and anti-HA (middle gel). FLAG–TOR was detected in the HA–IP product in the presence (lane 2), but not in the absence (lane 1), of HA–TOR, indicating a specific interaction between FLAG–TOR and HA–TOR. This interaction was not appreciably affected by rapamycin (lane 3) or insulin treatment (lane 4).

Figure 4.

Tor point mutants dominantly suppress Tsc2 lethality. (A) Shown is the percentage of viability of Tsc2¹⁰⁹/Tsc2¹⁹³ adults heterozygous for the indicated Tor alleles. Note that Tsc2 lethality is suppressed to a greater extent by the class I and class II point mutants than by the class III null alleles. (B) Class II but not class III Tor alleles can dominantly suppress Tsc2 lethality even in the presence of two copies of wild-type Tor. One copy of a Tor genomic rescue construct (P[Tor^WT]) blocks the ability of Tor^ΔP (class III) but not Tor^P2993L (class II) heterozygotes to rescue Tsc2¹⁰⁹/Tsc2¹⁹³ animals to pupal viability. n, number of animals scored. Genotypes: (A) Tor*/+; Tsc2¹⁰⁹/Tsc2¹⁹³, (B) Tor*/+; Tsc2¹⁰⁹/Tsc2¹⁹³, and Tor*/+; Tsc2¹⁰⁹ P[Tor^WT]/Tsc2¹⁹³.