Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Jul 22;111(29):10586-91.
doi: 10.1073/pnas.1406305111. Epub 2014 Jul 7.

TOR complex 2-Ypk1 signaling is an essential positive regulator of the general amino acid control response and autophagy

Ariadne Vlahakis  1 , Martin Graef  1 , Jodi Nunnari  1 , Ted Powers  2
Affiliations

TOR complex 2-Ypk1 signaling is an essential positive regulator of the general amino acid control response and autophagy

Ariadne Vlahakis et al. Proc Natl Acad Sci U S A. .

Abstract

The highly conserved Target of Rapamycin (TOR) kinase is a central regulator of cell growth and metabolism in response to nutrient availability. TOR functions in two structurally and functionally distinct complexes, TOR Complex 1 (TORC1) and TOR Complex 2 (TORC2). Through TORC1, TOR negatively regulates autophagy, a conserved process that functions in quality control and cellular homeostasis and, in this capacity, is part of an adaptive nutrient deprivation response. Here we demonstrate that during amino acid starvation TOR also operates independently as a positive regulator of autophagy through the conserved TORC2 and its downstream target protein kinase, Ypk1. Under these conditions, TORC2-Ypk1 signaling negatively regulates the Ca(2+)/calmodulin-dependent phosphatase, calcineurin, to enable the activation of the amino acid-sensing eIF2α kinase, Gcn2, and to promote autophagy. Our work reveals that the TORC2 pathway regulates autophagy in an opposing manner to TORC1 to provide a tunable response to cellular metabolic status.

Keywords: Atg8; Gcn4.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
TORC2 is required for amino acid starvation induced autophagy. Wild-type (WT) and torc2-ts (PLY1141) cells expressing pRS416 prATG8-GFP-ATG8 were grown at 25 °C to log phase and then transferred to (A) nitrogen starvation media, (B) amino acid starvation media, (C) or treated with 200 nM rapamycin in SCD growth media for 6 h at both 25 °C (permissive) and 30 °C (nonpermissive). Cells were analyzed at indicated time points by whole-cell protein extraction and Western blot analysis. Membranes were probed with α-GFP and α-G6PDH (Zwf1) primary antibodies. Quantification of autophagy flux is shown as a ratio of free GFP to total GFP (GFP-Atg8 and free GFP) after 6 h of starvation (Materials and Methods).
Fig. 2.
Fig. 2.
Ypk1 is required for amino acid starvation induced autophagy. WT, atg7Δ , ypk1Δ, and ypk2Δ cells expressing pRS416 prATG8-GFP-ATG8 [and, when indicated, plasmids expressing wild-type Ypk1 (pPL250) or Ypk1S644A/T662A (pPL491)] were grown at 30 °C to log phase and then transferred to (A) amino acid starvation media, (B) nitrogen starvation media, (C) or treated with 200 nM rapamycin in SCD growth media for 6 h at 30 °C. Analysis of GFP-Atg8 and quantification of autophagy flux were performed as described in Fig. 1. (D) WT and ypk1Δ cells expressing either pRS416 prATG8-GFP-ATG8 or pRS426- prATG8-GFP were grown at 30 °C to log phase and harvested, and Western blot analysis was performed as described in Fig. 1. GFP or GFP-Atg8 protein bands were normalized to G6PDH (Zwf1), and fold increase was calculated relative to WT.
Fig. 3.
Fig. 3.
Calcineurin inhibits autophagy downstream of TORC2/Ypk1. (A) WT, cnb1Δ, ypk1Δ, and ypk1Δ cnb1Δ cells containing plasmid pAMS363 expressing a 2xCDRE:lacZ fusion were grown at 30 °C to log phase and harvested. β-Galactosidase activity was measured (Materials and Methods) and is given in units of nanomoles of ONPG converted per minute per milligram of protein. (B) WT, cnb1Δ, ypk1Δ, and ypk1Δ cnb1Δ cells expressing pRS416 prATG8-GFP-ATG8 were grown at 30 °C to log phase and then transferred to amino acid starvation media for 6 h at 30 °C. Analysis of GFP-Atg8 and quantification of autophagy flux were preformed as described in Fig. 1. (C) WT, cnb1Δ, ypk1Δ, and ypk1cnb1Δ cells expressing endogenously tagged prATG8-2xyEGFP-ATG8 were grown as in B and transferred to amino acid starvation media supplemented with adenine. GFP-Atg8 localization was analyzed at 6 h of starvation using fluorescence microscopy (n > 200 cells for each strain). (Scale bar, 5 μm.) (D) WT, crz1Δ, ypk1Δ, and ypk1Δ crz1Δ and (E) WT, cmd1-6, ypk1Δ, and ypk1Δ cmd1-6 cells expressing pRS416 prATG8-GFP-ATG8 were grown and subjected to amino acid starvation, and autophagy flux was analyzed as in B.
Fig. 4.
Fig. 4.
TORC2/Ypk1 are required for the GAAC response during amino acid starvation. (A) WT, gcn2Δ, ypk1Δ, and ypk1Δ cnb1Δ cells were grown at 30 °C to log phase and then transferred to amino acid starvation media for 6 h at 30 °C. Gcn2-dependent phosphorylation of eIF2α at Ser-51 was determined by Western blot using α-phospho eIF2α Ser-51, α-Sui2 (eIF2α), and α-G6PDH antibodies. Quantification represents the ratio of phospho eIF2α Ser-51 and total eIF2α (Sui2) signal with fold change relative to WT at growing conditions (t = 0 h). (B) WT, ypk1Δ, and ypk1Δ cnb1Δ cells expressing the GCN4 derepression lacZ reporter plasmid (p180) were grown and starved as described in A. β-Galactosidase activity was measured (Materials and Methods) and is given in units of nanomoles of ONPG converted per minute per milligram of protein. (C and D) gcn2Δ and ypk1Δ gcn2Δ cells harboring pRS415 prATG8-GFP-ATG8, and, when indicated, plasmids expressing either wild-type GCN2 or GCN2C S577A, were grown and starved as described in A. (C) Analysis of GFP-Atg8 and quantification of autophagy flux were preformed as described in Fig. 1. (D) GFP-Atg8 localization was analyzed using fluorescence microscopy as described in Fig. 3C (n > 200 cells for each strain). (Scale bar, 5 μm.)
Fig. 5.
Fig. 5.
A model for TORC2 regulation of autophagy and the GAAC response. TORC2 and Ypk1 promote autophagy upon amino acid starvation in a pathway distinct from TORC1. Specifically, TORC2 and Ypk1 promote amino acid starvation-induced autophagy by negatively regulating Calmodulin (CaM)/Calcineurin, whose activity inhibits the GAAC response (Gcn2-Gcn4). Dashed line depicts potential autophagy-mediated degradation of Ypk1 protein upon nutrient limitation (see Discussion for details).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

  • Calcium channel regulator Mid1 links TORC2-mediated changes in mitochondrial respiration to autophagy.
    Vlahakis A, Lopez Muniozguren N, Powers T. Vlahakis A, et al. J Cell Biol. 2016 Dec 19;215(6):779-788. doi: 10.1083/jcb.201605030. Epub 2016 Nov 29. J Cell Biol. 2016. PMID: 27899413 Free PMC article.
  • Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition).
    Klionsky DJ, Abdelmohsen K, Abe A, Abedin MJ, Abeliovich H, Acevedo Arozena A, Adachi H, Adams CM, Adams PD, Adeli K, Adhihetty PJ, Adler SG, Agam G, Agarwal R, Aghi MK, Agnello M, Agostinis P, Aguilar PV, Aguirre-Ghiso J, Airoldi EM, Ait-Si-Ali S, Akematsu T, Akporiaye ET, Al-Rubeai M, Albaiceta GM, Albanese C, Albani D, Albert ML, Aldudo J, Algül H, Alirezaei M, Alloza I, Almasan A, Almonte-Beceril M, Alnemri ES, Alonso C, Altan-Bonnet N, Altieri DC, Alvarez S, Alvarez-Erviti L, Alves S, Amadoro G, Amano A, Amantini C, Ambrosio S, Amelio I, Amer AO, Amessou M, Amon A, An Z, Anania FA, Andersen SU, Andley UP, Andreadi CK, Andrieu-Abadie N, Anel A, Ann DK, Anoopkumar-Dukie S, Antonioli M, Aoki H, Apostolova N, Aquila S, Aquilano K, Araki K, Arama E, Aranda A, Araya J, Arcaro A, Arias E, Arimoto H, Ariosa AR, Armstrong JL, Arnould T, Arsov I, Asanuma K, Askanas V, Asselin E, Atarashi R, Atherton SS, Atkin JD, Attardi LD, Auberger P, Auburger G, Aurelian L, Autelli R, Avagliano L, Avantaggiati ML, Avrahami L, Awale S, Azad N, Bachetti T, Backer JM, Bae DH, Bae JS, Bae ON, Bae SH, Baehrecke EH, Baek SH, Baghdiguian S, Bagniewska-Zadworna A, Bai H, Bai J, Bai XY, Bailly Y, Balaji KN, … See abstract for full author list ➔ Klionsky DJ, et al. Autophagy. 2016;12(1):1-222. doi: 10.1080/15548627.2015.1100356. Autophagy. 2016. PMID: 26799652 Free PMC article. No abstract available.
  • Autophagy modulation: a potential therapeutic approach in cardiac hypertrophy.
    Wang X, Cui T. Wang X, et al. Am J Physiol Heart Circ Physiol. 2017 Aug 1;313(2):H304-H319. doi: 10.1152/ajpheart.00145.2017. Epub 2017 Jun 2. Am J Physiol Heart Circ Physiol. 2017. PMID: 28576834 Free PMC article. Review.
  • The mTORC1/4E-BP pathway coordinates hemoglobin production with L-leucine availability.
    Chung J, Bauer DE, Ghamari A, Nizzi CP, Deck KM, Kingsley PD, Yien YY, Huston NC, Chen C, Schultz IJ, Dalton AJ, Wittig JG, Palis J, Orkin SH, Lodish HF, Eisenstein RS, Cantor AB, Paw BH. Chung J, et al. Sci Signal. 2015 Apr 14;8(372):ra34. doi: 10.1126/scisignal.aaa5903. Sci Signal. 2015. PMID: 25872869 Free PMC article.
  • A Signaling Lipid Associated with Alzheimer's Disease Promotes Mitochondrial Dysfunction.
    Kennedy MA, Moffat TC, Gable K, Ganesan S, Niewola-Staszkowska K, Johnston A, Nislow C, Giaever G, Harris LJ, Loewith R, Zaremberg V, Harper ME, Dunn T, Bennett SAL, Baetz K. Kennedy MA, et al. Sci Rep. 2016 Jan 13;6:19332. doi: 10.1038/srep19332. Sci Rep. 2016. PMID: 26757638 Free PMC article.
See all "Cited by" articles

References

    1. Takeshige K, Baba M, Tsuboi S, Noda T, Ohsumi Y. Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction. J Cell Biol. 1992;119(2):301–311. - PMC - PubMed
    1. Tsukada M, Ohsumi Y. Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae. FEBS Lett. 1993;333(1-2):169–174. - PubMed
    1. Onodera J, Ohsumi Y. Autophagy is required for maintenance of amino acid levels and protein synthesis under nitrogen starvation. J Biol Chem. 2005;280(36):31582–31586. - PubMed
    1. Madeo F, Tavernarakis N, Kroemer G. Can autophagy promote longevity? Nat Cell Biol. 2010;12(9):842–846. - PubMed
    1. Ryter SW, Cloonan SM, Choi AM. Autophagy: A critical regulator of cellular metabolism and homeostasis. Mol Cells. 2013;36(1):7–16. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources