GATOR1 regulates nitrogenic cataplerotic reactions of the mitochondrial TCA cycle. Jun Chen, Benjamin M. Sutter, Lei Shi, Benjamin P.

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1 Supplementary Information GATOR1 regulates nitrogenic cataplerotic reactions of the mitochondrial TCA cycle Jun Chen, Benjamin M. Sutter, Lei Shi, Benjamin P. Tu* Department of Biochemistry University of Texas Southwestern Medical Center 52 Harry Hines Boulevard Dallas, TX , USA *Correspondence: Nature Chemical Biology: doi:10.108/nchembio.2478

2 Supplementary Results Supplementary Table 1 Yeast strains used in this study. Strain Background Genotype Reference MAT α 1 MAT α nprδ::kanmx MAT α npr2δ::kanmx 2 MAT α iml1δ::kanmx 2 MAT α npr2δ::loxp this study MAT α Rtg1-GFP::HYG Nic96-mCherry::KanMX npr2δ::loxp this study MAT α Rtg1-GFP::HYG Nic96-mCherry::KanMX this study MAT α Rtg1-GFP-NLS::HYG npr2δ::loxp this study MAT α Rtg1-GFP-NLS::HYG this study MAT α Cit1-XHA::HYG this study MAT α Cit1-XHA::HYG npr2δ::kanmx this study MAT α Aco1-XHA::HYG this study MAT α Aco1-XHA::HYG npr2δ::kanmx this study MAT α Lsc1-XHA::HYG this study MAT α Lsc1-XHA::HYG npr2δ::kanmx this study MAT α Mdh1-XHA::HYG this study MAT α Mdh1-XHA::HYG npr2δ::kanmx this study MAT α Sdh1-XHA::HYG this study MAT α Sdh1-XHA::HYG npr2δ::kanmx this study MAT α Fum1-XHA::HYG this study MAT α Fum1-XHA::HYG npr2δ::kanmx this study MAT α Idh1-XHA::HYG this study MAT α Idh1-XHA::HYG npr2δ::kanmx this study MAT α Kgd1-XHA::HYG this study MAT α Kgd1-XHA::HYG npr2δ::kanmx this study MAT α Cit1-XHA::HYG Cit2-XFlag::natNT this study MAT α Cit1-XHA::HYG Cit2-Xflag::natNT npr2δ::kanmx this study MAT a Gtr1 Q65L-XFlag::HYG this study MAT a Gtr1 Q65L-XFlag::HYG npr2δ::loxp this study MAT α Pyc1-XHA::HYG this study MAT α Pyc1-XHA::HYG npr2δ::kanmx this study MAT α Pyc2-XHA::HYG this study MAT α Pyc2-XHA::HYG npr2δ::kanmx this study MAT α Aat1-Xflag::KanMX this study MAT α Aat1-XHA::KanMX npr2 ::loxp this study MAT α Aat2-Xflag::KanMX this study MAT α Aat2-XHA::KanMX npr2 ::loxp this study MAT α Pda1-Xflag::HYG this study MAT α Pda1-XHA::HYG npr2 ::loxp this study MAT α Mpc-Xflag::HYG this study 2 Nature Chemical Biology: doi:10.108/nchembio.2478

3 MAT α Mpc-XHA::HYG npr2 ::loxp this study MAT α Npr1-XHA::KanMX this study MAT α Npr1-XHA::KanMX npr2 ::HYG this study MAT a Sch9-xHA::natNT MAT a Sch9-xHA::natNT npr2δ::kanmx MAT a Gln-xFlag::natNT MAT a Gln-xFlag::natNT npr2δ::kanmx MAT a Gdh1-xFlag::natNT MAT a Gdh1-xFlag::natNT npr2δ::kanmx MAT a Gdh-xFlag::natNT MAT a Gdh-xFlag::natNT npr2δ::kanmx MAT a Gln1-xFlag::natNT MAT a Gln1-xFlag::natNT npr2δ::kanmx MAT a Glt1-xFlag::natNT MAT a Glt1-xFlag::natNT npr2δ::kanmx Nature Chemical Biology: doi:10.108/nchembio.2478

4 Supplementary Table 2 List of labeled TCA metabolites, glutamine and aspartate from glucose- 1 C 6 tracing experiments by LC-MS/MS, related to Fig C Pyruvate -> 1 C 2 Acetyl-CoA Name MW 1 C MW Q1/Q m/z (neg. mode) 1 C 2 -Isocitrate /75 1 C 2 -Succinate /75 1 C 2 -Glutamine /129 1 C 2 -Aspartate /117 2nd round metabolites 1 C 4 -α-ketoglutarate /104 1 C -Malate /118 1 C -Glutamine /112 1 C Pyruvate -> 1 C Oxaloacetate 1 C 5 -Citrate /111 1 C -Succinate /75 1 C -Fumarate /7 1 C -Aspartate /118 1 C 4 -Glutamine /11 Nature Chemical Biology: doi:10.108/nchembio.2478

5 Nature Chemical Biology: doi:10.108/nchembio.2478

6 Supplementary Fig.1 Effect of amino acids on growth of npr2 and related strains involved in the negative regulation of TORC1, related to Fig. 1. (A) Serial dilution of cells of the indicated genotypes (WT, npr2δ, nprδ and iml1δ) were spotted onto plates (10,000, 1,000, 100, 10 cells for each spot) and incubated at 0⁰C for 2 days. Growth media were YPD (left), SD (middle) or SD with 5 mm aspartate (right). (B) Serial dilution of cells (WT, npr2δ, gtr1δ, GTR1 Q65L-flag and npr2δgtr1δ) were spotted onto plates as in (A). Growth media were YPD (left) or SD (right). GTR1 Q65L-Flag is a GTP-locked mutant of Gtr1p 4. (C) Growth curves of WT, npr2 and npr2, Npr2-flag (npr2 with reintroduction of Npr2-flag) in SD. Data were collected from 2 independent experiments. (D) Western blots depicting Glnp, Sch9p, and Npr1p proteoforms in WT and npr2δ cells (top) and phospho-s6 in WT, npr2δ, GTR1-Q65L and GTR1-Q65L, npr2δ cells (bottom) switched from starter cultures grown in SD medium to fresh SD medium for the indicated times. 200 ng/ml rapamycin or 25 μg/ml cycloheximide was added as controls for inhibition or stimulation of TORC1 signaling. p-s6 amounts were quantified against G6PDH by ImageJ and normalized against the earliest WT sample. Full gel images are shown in Supplementary Fig. 6. (E) Upper panel: growth curves of WT, npr and iml1 in SD supplemented with 2 mm aspartate or 2 nm rapamycin. Data were collected from 2 independent experiments. Lower panel: Growth of WT or npr2δ cells in SD medium or on agar plate with indicated amount of rapamycin. Data were collected from 2 different independent experiments. (F) Growth curves of WT and npr2 in either SD or SD + CSM (complete supplement mixture). Data were collected from 2 independent experiments. (G) Doubling time of WT and npr2δ cells in SD medium supplemented with 2 mm of the indicated amino acids (tyrosine was added to 1 mm due to low solubility). Data were mean ± s.d. from independent experiments. ***p < by two-tailed Student s t-test. Nature Chemical Biology: doi:10.108/nchembio.2478

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8 Supplementary Fig. 2 Amounts of TCA cycle metabolites and enzymes in WT vs. npr2 cells, related to Fig. 2. (A) Dissolved oxygen levels as a function of growth following inoculation into SD medium. WT, npr2, npr, iml1, and GTR1 Q65L strains were grown in parallel bioreactor vessels, and the npr2 strains were also supplemented with 2 mm aspartate or glutamine (left). On the right panel, the OD 600 readings of corresponding time points were labeled on the dissolved oxygen traces for WT (black), npr2 (red), and npr2 supplemented with 5 mm aspartate (blue). Representative data were selected from 2 independent experiments. (B) Mitochondrial DNA content in WT and npr2δ cells following switch from YPD to SD for a period of 1,, 6 h. Data were mean ± s.d. from one set of triplicates, 2 independent experiments were performed. (C) Acetate and glucose amounts in the extracellular medium over time in SD. Data were collected from two independent experiments. (D) TCA cycle metabolite levels were quantitated by LC-MS/MS. Cells were collected after switch from YPD to SD for 6 h. Data were mean ± s.d. from independent experiments. **p < 0.01 by two-tailed Student s t test. (E) α-ketoglutarate levels were quantitated by LC-MS/MS. Cells were collected after switch from YPD to SD or SD supplemented with 2 mm dimethyl 2-oxoglutarate (membrane permeable α-kg, labeled as α-kg) for 6 h. Data were mean ± s.d. from one set of 4 repeats, 2 independent experiments were performed. **p < 0.01 by two-tailed Student s t test. (F) Western blots indicating amounts of Gdh1p (NADP + -dependent glutamate dehydrogenase), Gdhp (Gdh1p paralog), Glt1p (NAD + - dependent glutamate synthase) and Gln1p (glutamine synthetase) in WT and npr2δ cells collected after switch from YPD to SD for 6 h. Full gels are shown in Supplementary Fig. 6. (G) Western blots indicating amounts of Aat1p/Aat2p (aspartate aminotransferase), Pyc1p/Pyc2p (pyruvate carboxylase), Pda1p (E1α subunit of pyruvate dehydrogenase complex), and Mpcp (mitochondrial pyruvate carrier) in WT and npr2δ cells collected following switch from YPD to SD for 1,, 6 h. The Pda1 samples were run on phostag gels with 25 μm of Phos-Tag TM AAL-107 (NARD institute, Ltd.). Full gels are shown in Supplementary Fig. 6. (H) mrna expression amounts for AAT1 and AAT2 across 16 time points in the yeast metabolic cycle (YMC). Data are from a previous published RNA-Seq dataset 5. Note that both AAT1 and AAT2 have expression peaks in either RC or RB phase which are correlated with stress response or mitochondria, as opposed to growth (OX). Nature Chemical Biology: doi:10.108/nchembio.2478

9 Supplementary Fig. Altered retrograde response in npr2 cells, related to Fig. 2 and Fig.. (A) Intracellular NAD + /NADH ratio in YPD or after switch to SD for or 6 h. Data were mean ± s.d. from one set of triplicates, 2 independent experiments were performed. *p < 0.05 by two-tailed Student s t test. (B) mrna amounts of transcriptional targets of the retrograde response (RTG) pathway, ACO1, CIT1 and CIT2 in WT and npr2δ cells collected following growth in SD for 6 h. Data were mean ± s.d. from two independent experiments. *p < 0.05, ***p < 0.001, by two-tailed Student s t-test. (C) Images of cells expressing Rtg1-NLS-GFP showing the forced nuclear localization of Rtg1p in cells growing in YPD medium. Scale bar 5 µm. Nature Chemical Biology: doi:10.108/nchembio.2478

10 Supplementary Fig. 4 Nucleoside or nucleobase supplementation is not sufficient to rescue growth of npr2 cells and metabolomes of WT and npr2 cells with or without glutamine or aspartate supplementation, related to Fig. 4. (A) Heat map depicting abundances of the indicated metabolites obtained by LC-MS/MS analyses of WT or npr2δ or Rtg1-NLS-GFP cells switched from YPD to SD with or without 2 mm glutamine for 6 h. Data were mean ± s.d. from one set of triplicates, 2 independent experiments were performed. (B) Heat maps depicting abundances of the indicated metabolites obtained by LC-MS/MS analyses of WT or npr2δ cells grown to OD = 1, in SD or SD supplemented with 2 mm aspartate. (C) Growth of WT and npr2δ cells in SD supplemented with different combinations of nucleosides at 20 mg/l. A (adenosine), C (cytidine), G (guanosine), T (thymidine), U (uridine), AG (adenosine and guanosine, all purine nucleosides), UTC (uridine, thymidine, cytidine, all pyrimidine nucleosides), AUTCG (all nucleosides). Data were mean ± s.d. from one set of triplicates, 2 independent experiments were performed. (D) Growth of WT and npr2δ cells in SD supplemented with different combinations of nucleobases at 20 mg/l, with 5 mm of ribose. A (adenine), C (cytosine), T (thymine), U (uracil), UTC (uracil, thymine, cytosine, all pyrimidine nucleobases), AUTC (all nucleobases). Guanine is poorly soluble in water and not supplemented in the experiments. Data were mean ± s.d. from one set of triplicates, 2 independent experiments were performed. Nature Chemical Biology: doi:10.108/nchembio.2478

11 Supplementary Fig. 5 Full gel images of Western blots for Fig. 1b (A), Fig. 2b (B), Fig. b (C). Nature Chemical Biology: doi:10.108/nchembio.2478

12 Supplementary Fig. 6 Full gel images of Western blots for Supplementary Fig. 1D (A), Supplementary Fig. 2F (B), Supplementary Fig. 2G (C). Nature Chemical Biology: doi:10.108/nchembio.2478

13 Supplementary Information References 1. van Dijken, J.P. et al. An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains. Enzyme Microb Technol 26, (2000). 2. Wu, X. & Tu, B.P. Selective regulation of autophagy by the Iml1-Npr2-Npr complex in the absence of nitrogen starvation. Mol Biol Cell 22, (2011).. Laxman, S., Sutter, B.M., Shi, L. & Tu, B.P. Npr2 inhibits TORC1 to prevent inappropriate utilization of glutamine for biosynthesis of nitrogen-containing metabolites. Sci Signal 7, ra120 (2014). 4. Nakashima, N., Noguchi, E. & Nishimoto, T. Saccharomyces cerevisiae putative G protein, Gtr1p, which forms complexes with itself and a novel protein designated as Gtr2p, negatively regulates the Ran/Gsp1p G protein cycle through Gtr2p. Genetics 152, (1999). 5. Kuang, Z. et al. High-temporal-resolution view of transcription and chromatin states across distinct metabolic states in budding yeast. Nature structural & molecular biology 21, (2014). Nature Chemical Biology: doi:10.108/nchembio.2478