Autophagy is an intracellular degradation system, by which cytoplasmic contents are degraded in lysosomes. Autophagy is dynamically induced by nutrient depletion to provide necessary amino acids within cells, thus helping them adapt to starvation. Although it has been suggested that mTOR is a major negative regulator of autophagy, how it controls autophagy has not yet been determined. Here, we report a novel mammalian autophagy factor, Atg13, which forms a stable approximately 3-MDa protein complex with ULK1 and FIP200. Atg13 localizes on the autophagic isolation membrane and is essential for autophagosome formation. In contrast to yeast counterparts, formation of the ULK1-Atg13-FIP200 complex is not altered by nutrient conditions. Importantly, mTORC1 is incorporated into the ULK1-Atg13-FIP200 complex through ULK1 in a nutrient-dependent manner and mTOR phosphorylates ULK1 and Atg13. ULK1 is dephosphorylated by rapamycin treatment or starvation. These data suggest that mTORC1 suppresses autophagy through direct regulation of the approximately 3-MDa ULK1-Atg13-FIP200 complex.

mTOR controls cell growth, in part by regulating p70 S6 kinase alpha (p70alpha) and eukaryotic initiation factor 4E binding protein 1 (4EBP1). Raptor is a 150 kDa mTOR binding protein that also binds 4EBP1 and p70alpha. The binding of raptor to mTOR is necessary for the mTOR-catalyzed phosphorylation of 4EBP1 in vitro, and it strongly enhances the mTOR kinase activity toward p70alpha. Rapamycin or amino acid withdrawal increases, whereas insulin strongly inhibits, the recovery of 4EBP1 and raptor on 7-methyl-GTP Sepharose. Partial inhibition of raptor expression by RNA interference (RNAi) reduces mTOR-catalyzed 4EBP1 phosphorylation in vitro. RNAi of C. elegans raptor yields an array of phenotypes that closely resemble those produced by inactivation of Ce-TOR. Thus, raptor is an essential scaffold for the mTOR-catalyzed phosphorylation of 4EBP1 and mediates TOR action in vivo.

TOR is a serine-threonine kinase that was originally identified as a target of rapamycin in Saccharomyces cerevisiae and then found to be highly conserved among eukaryotes. In Drosophila melanogaster, inactivation of TOR or its substrate, S6 kinase, results in reduced cell size and embryonic lethality, indicating a critical role for the TOR pathway in cell growth control. However, the in vivo functions of mammalian TOR (mTOR) remain unclear. In this study, we disrupted the kinase domain of mouse mTOR by homologous recombination. While heterozygous mutant mice were normal and fertile, homozygous mutant embryos died shortly after implantation due to impaired cell proliferation in both embryonic and extraembryonic compartments. Homozygous blastocysts looked normal, but their inner cell mass and trophoblast failed to proliferate in vitro. Deletion of the C-terminal six amino acids of mTOR, which are essential for kinase activity, resulted in reduced cell size and proliferation arrest in embryonic stem cells. These data show that mTOR controls both cell size and proliferation in early mouse embryos and embryonic stem cells. TOR (target of rapamycin) was originally identified in two mutantSaccharomyces cerevisiae strains, TOR1-1 and TOR2-1, that are resistant to the growth-inhibiting effect of the immunophilin-immunosuppressant complex FKBP (FK506 binding protein) and rapamycin (17). TOR1 and TOR2 are large proteins (Ϸ280 kDa) and are Ϸ70% identical (26,28 (21, 48). mTOR and other members of this family, including ATM, ATR/FPR, and DNA-PKcs, contain C-terminal regions with high similarity to the catalytic domains of phosphoinositide (PI)-3 kinase and PI-4 kinase (26, 28). However, PIKK members are not lipid kinases but rather function as serine-threonine kinases (4, 20). The PIKK proteins contain a short segment at the extreme C terminus that is essential for protein kinase activity and is not present in PI-3 and PI-4 kinases (51).Cell culture studies have demonstrated that mTOR controls protein synthesis, in part by phosphorylating downstream substrates, including p70 s6 kinase (p70 S6K1 ) (3, 5, 20) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) (4, 5, 13, 15). p70 S6K phosphorylates the 40S ribosomal protein S6 and is proposed to play a crucial role in the translation of 5Ј-terminal oligopyrimidine tract mRNAs, which primarily encode ribosomal proteins and components of the translation apparatus (22, 23). Phosphorylation of 4E-BP1 disrupts its binding to eIF4E, a protein that binds the 5Ј cap structure of mRNA. Released eIF4E then forms a functional translation initiation complex with eIF4G, eIF4A, and eIF3 ribosomes, enhancing translation (29, 45). Inactivation of 4E-BP1 and family proteins has a profound effect on translation of mRNAs with complex 5Ј untranslated regions, which often encode regulatory proteins such as protooncogenes (45). The recent discoveries of a 150-kDa binding partner of mTOR, named raptor (regulatory-associated protein of mTOR) (14,27), and its Saccharomyces cerevisi...

The mammalian target of rapamycin (mTOR) controls multiple cellular functions in response to amino acids and growth factors, in part by regulating the phosphorylation of p70 S6 kinase (p70S6k) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Raptor (regulatory associated protein of mTOR) is a recently identified mTOR binding partner that also binds p70S6k and 4E-BP1 and is essential for TOR signaling in vivo. Herein we demonstrate that raptor binds to p70S6k and 4E-BP1 through their respective TOS (conserved TOR signaling) motifs to be required for amino acid-and mTOR-dependent regulation of these mTOR substrates in vivo. A point mutation of the TOS motif also eliminates all in vitro mTOR-catalyzed 4E-BP1 phosphorylation and abolishes the raptor-dependent component of mTOR-catalyzed p70S6k phosphorylation in vitro. Raptor appears to serve as an mTOR scaffold protein, the binding of which to the TOS motif of mTOR substrates is necessary for effective mTOR-catalyzed phosphorylation in vivo and perhaps for conferring their sensitivity to rapamycin and amino acid sufficiency.The target of rapamycin (TOR) 1 proteins are protein kinases that were first identified in Saccharomyces cerevisiae through mutants that confer resistance to growth inhibition induced by the immunosuppressive macrolide rapamycin (1). In mammalian cells, rapamycin blocks phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) (2, 3) and p70 S6 kinase (p70S6k) (4,5) by interfering with the function of mTOR (6, 7) (also known as FRAP, RAFT1, or RAPT). Although mTOR can phosphorylate both these targets directly in vitro (8 -10), the mechanism of mTOR regulation of these phosphorylations in vivo remains incompletely understood (11).The p70S6k is activated through a sequential multisite phosphorylation in response to insulin or mitogens in vivo (11). In addition, nutrients, especially amino acids, have been shown to regulate the phosphorylation of p70S6k and 4E-BP1 and to be necessary for insulin or mitogen regulation (12-17). The activity of p70S6k␣1 in vivo is most closely related to the phosphorylation at Thr-412, situated in a hydrophobic motif C-terminal to the canonical catalytic domain (18,19). The identity of the kinase(s) acting on this site in vivo is uncertain; however, this site can be phosphorylated directly by mTOR in vitro (9, 10). Recently, site-specific mutagenesis was employed to define a five-amino acid sequence called the TOS (TOR signaling) motif as the minimal functionally important region within this p70S6k noncatalytic N-terminal segment (21). As with N-terminal deletion, mutation of a single Phe within the TOS motif to Ala causes marked inhibition of activity of full-length p70S6k and a loss of sensitivity to rapamycin and amino acid withdrawal in the p70S6k-⌬CT104, lacking C-terminal noncatalytic tail, background. In addition, a TOS motif was identified in the 4E-BPs, wherein mutation of 4E-BP1 Phe-114 to Ala inhibits amino acid-and serum-induced 4E-BP1 phosphorylation.Raptor is a recently...