TORC1 regulators Iml1/GATOR1 and GATOR2 control meiotic entry and oocyte development in Drosophila

In single-cell eukaryotes the pathways that monitor nutrient availability are central to initiating the meiotic program and gametogenesis. In Saccharomyces cerevisiae an essential step in the transition to the meiotic cycle is the down-regulation of the nutrient-sensitive target of rapamycin complex 1 (TORC1) by the increased minichromosome loss 1/ GTPase-activating proteins toward Rags 1 (Iml1/GATOR1) complex in response to amino acid starvation. How metabolic inputs influence early meiotic progression and gametogenesis remains poorly understood in metazoans. Here we define opposing functions for the TORC1 regulatory complexes Iml1/GATOR1 and GATOR2 during Drosophila oogenesis. We demonstrate that, as is observed in yeast, the Iml1/GATOR1 complex inhibits TORC1 activity to slow cellular metabolism and drive the mitotic/meiotic transition in developing ovarian cysts. In iml1 germline depletions, ovarian cysts undergo an extra mitotic division before meiotic entry. The TORC1 inhibitor rapamycin can suppress this extra mitotic division. Thus, high TORC1 activity delays the mitotic/meiotic transition. Conversely, mutations in Tor, which encodes the catalytic subunit of the TORC1 complex, result in premature meiotic entry. Later in oogenesis, the GATOR2 components Mio and Seh1 are required to oppose Iml1/GATOR1 activity to prevent the constitutive inhibition of TORC1 and a block to oocyte growth and development. To our knowledge, these studies represent the first examination of the regulatory relationship between the Iml1/GATOR1 and GATOR2 complexes within the context of a multicellular organism. Our data imply that the central role of the Iml1/GATOR1 complex in the regulation of TORC1 activity in the early meiotic cycle has been conserved from single cell to multicellular organisms.In yeast, the inhibition of the nutrient-sensitive target of rapamycin complex 1 (TORC1) in response to amino acid limitation is essential for cells to transit from the mitotic cycle to the meiotic cycle (1, 2). In response to amino acid starvation, the Iml1 complex, comprising the Iml1, Npr2, and Npr3 proteins in yeast and the respective orthologs DEPDC5, Nprl2, and Nprl3 in mammals, inhibits TORC1 activity (3–5). The Iml1 complex, which has been renamed the “GTPase-activating proteins toward Rags 1” (GATOR1) complex in higher eukaryotes, functions as a GTPase-activating protein complex that inactivates RagsA/B or Gtr1 in mammals and yeast, respectively, thus preventing the activation of TORC1 (3, 4). In the yeast Saccharomyces cerevisiae, mutations in the Iml1 complex components Npr2 and Npr3 result in a failure to down-regulate TORC1 activity in response to amino acid starvation and block meiosis and sporulation (6–9). As is observed in yeast, in Drosophila, Nprl2 and Nprl3 mediate a critical response to amino acid starvation (10). However, their roles in meiosis and gametogenesis remain unexplored.Recent reports indicate that the Iml1, Npr2, and Npr3 proteins are components of a large multiprotein complex originally named the “Seh1-associated” (SEA) complex in budding yeast and the “GATOR” complex in higher eukaryotes (3, 11). The SEA/GATOR complex contains eight highly conserved proteins. The three proteins described above, Iml1/DEPDC5, Npr2/Nprl2, and Npr3/Nprl3, form the Iml1/GATOR1 complex and inhibit TORC1 (3, 4). The five remaining proteins in the complex, Seh1, Sec13, Sea4/Mio, Sea2/WDR24, and Sea3/WDR59, which have been designated the “GATOR2” complex in multicellular organisms, oppose the activity of Iml1/GATOR1 and thus promote TORC1 activity (3, 4, 12).Little is known about the physiological and/or developmental requirements for the GATOR2 complex in multicellular organisms. However, in Drosophila the GATOR2 components Mio and Seh1 interact physically and genetically and exhibit strikingly similar ovarian phenotypes, with null mutations in both genes resulting in female sterility (13, 14). In Drosophila females, oocyte development takes place within the context of an interconnected germline syncytium, also referred to as an “ovarian cyst” (15). Ovarian cyst formation begins at the tip of the germarium when a cystoblast, the daughter of a germline stem cell, undergoes four synchronous divisions with incomplete cytokinesis to produce 16 interconnected cells. Actin-stabilized cleavage furrows, called “ring canals,” connect cells within the cyst (16). Each 16-cell cyst develops with a single oocyte and 15 polyploid nurse cells which ultimately are encapsulated by a somatically derived layer of follicle cells to produce an egg chamber (17). Each ovary is comprised of ∼15 ovarioles that consist of a single germarium followed by a string of egg chambers in successively older stages of development. In mio- and seh1-mutant egg chambers, the oocyte enters the meiotic cycle, but as oogenesis proceeds, the oocyte fate and the meiotic cycle are not maintained stably (13, 14). Ultimately, a large fraction of mio and seh1 oocytes enter the endocycle and develop as polyploid nurse cells. A mechanistic understanding of how mio and seh1 influence meiotic progression and oocyte fate has remained elusive.Here we demonstrate that the Iml1/GATOR1 complex down-regulates TORC1 activity to promote the mitotic/meiotic transition in Drosophila ovarian cysts. We find that depleting iml1 in the female germ line delays the mitotic/meiotic transition, so that ovarian cysts undergo an extra mitotic division. Conversely, mutations in Tor result in premature meiotic entry before the completion of the four mitotic divisions. Moreover, we demonstrate that in the female germ line, the GATOR2 components Mio and Seh1 are required to oppose the TORC1 inhibitory activity of the Iml1/GATOR1 complex to prevent the constitutive down-regulation of TORC1 activity in later stages of oogenesis. To our knowledge, these studies represent the first examination of the regulatory relationship between Iml1/GATOR1 and GATOR2 components within the context of a multicellular animal. Finally, our data reveal a surprising tissue-specific requirement for the GATOR2 complex in multicellular organisms and suggest a conserved role for the SEA/GATOR complex in the regulation of TORC1 activity during gametogenesis.