Abstract
Glutaminase (GLS), which deaminates glutamine to form glutamate, is a mitochondrial tetrameric protein complex. Although inorganic phosphate (Pi) is known to promote GLS filamentation and activation, the molecular basis of this mechanism is unknown. Here we aimed to determine the molecular mechanism of Pi-induced mouse GLS filamentation and its impact on mitochondrial physiology. Single-particle cryogenic electron microscopy revealed an allosteric mechanism in which Pi binding at the tetramer interface and the activation loop is coupled to direct nucleophile activation at the active site. The active conformation is prone to enzyme filamentation. Notably, human GLS filaments form inside tubulated mitochondria following glutamine withdrawal, as shown by in situ cryo-electron tomography of cells thinned by cryo-focused ion beam milling. Mitochondria with GLS filaments exhibit increased protection from mitophagy. We reveal roles of filamentous GLS in mitochondrial morphology and recycling.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$189.00 per year
only $15.75 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
The fGLS PDB and EMDB accession codes are 8EC6 and EMD-28013, respectively. The datasets and unique biological materials generated and analyzed during the current study are available from the corresponding authors upon reasonable request. Source data are provided with this paper.
Code availability
The code generated for fluorescence microscopy analysis is available on GitHub (https://github.com/douglasadamoski/mitoMorphology).
References
Shijie, J. et al. Blockade of glutamate release from microglia attenuates experimental autoimmune encephalomyelitis in mice. Tohoku J. Exp. Med. 217, 87–92 (2009).
Erdmann, N. et al. Glutamate production by HIV-1 infected human macrophage is blocked by the inhibition of glutaminase. J. Neurochem. 102, 539–549 (2007).
Rambold, A. S., Kostelecky, B., Elia, N. & Lippincott-Schwartz, J. Tubular network formation protects mitochondria from autophagosomal degradation during nutrient starvation. Proc. Natl Acad. Sci. USA 108, 10190–10195 (2011).
Cai, W.-F. et al. Glutaminase GLS1 senses glutamine availability in a non-enzymatic manner triggering mitochondrial fusion. Cell Res. 28, 865–867 (2018).
Gomes, L. C., Benedetto Di, G. & Scorrano, L. During autophagy mitochondria elongate, are spared from degradation and sustain cell viability. Nat. Cell Biol. 13, 589–598 (2011).
Palikaras, K., Lionaki, E. & Tavernarakis, N. Mechanisms of mitophagy in cellular homeostasis, physiology and pathology. Nat. Cell Biol. 20, 1013–1022 (2018).
Kvamme, E., Tveit, B. & Svenneby, G. Glutaminase from pig renal cortex: I. Purification and general properties. J. Biol. Chem. 245, 1871–1877 (1970).
Olsen, B. R. & Svenneby, G. Formation and ultrastructure of enzymically active polymers of pig renal glutaminase. J. Mol. Biol. 52, 239–245 (1970).
Cassago, A. et al. Mitochondrial localization and structure-based phosphate activation mechanism of glutaminase C with implications for cancer metabolism. Proc. Natl Acad. Sci. USA 109, 1092–1097 (2012).
Ferreira, A. P. S. et al. Active glutaminase C self-assembles into a supratetrameric oligomer that can be disrupted by an allosteric inhibitor. J. Biol. Chem. 288, 28009–28020 (2013).
Rumping, L. et al. GLS hyperactivity causes glutamate excess, infantile cataract and profound developmental delay. Hum. Mol. Genet. 28, 96–104 (2019).
Jiang, B. et al. Filamentous GLS1 promotes ROS-induced apoptosis upon glutamine deprivation via insufficient asparagine synthesis. Mol. Cell 82, 1821–1835.e6 (2022).
Huang, Q. et al. Characterization of the interactions of potent allosteric inhibitors with glutaminase C, a key enzyme in cancer cell glutamine metabolism. J. Biol. Chem. 293, 3535–3545 (2018).
Brown, G. et al. Functional and structural characterization of four glutaminases from Escherichia coli and Bacillus subtilis. Biochemistry 47, 5724–5735 (2008).
Jones, S. & Thornton, J. M. Principles of protein-protein interactions. Proc. Natl Acad. Sci. USA 93, 13–20 (1996).
Li, Y. et al. Mechanistic basis of glutaminase activation: a key enzyme that promotes glutamine metabolism in cancer cells. J. Biol. Chem. 291, 20900–20910 (2016).
Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).
Acuner Ozbabacan, S. E., Engin, H. B., Gursoy, A. & Keskin, O. Transient protein–protein interactions. Protein Eng. Des. Sel. 24 635–648 (2011).
Pasquali, C. C. et al. The origin and evolution of human glutaminases and their atypical C-terminal ankyrin repeats. J. Biol. Chem. 292, 11572–11585 (2017).
Ferreira, I. M. et al. Structure and activation mechanism of the human liver-type glutaminase GLS2. Biochimie 185, 96–104 (2021).
Boysen, G. et al. Glutaminase inhibitor CB-839 increases radiation sensitivity of lung tumor cells and human lung tumor xenografts in mice. Int. J. Radiat. Biol. 95, 436–442 (2019).
Robinson, M. M. et al. Novel mechanism of inhibition of rat kidney-type glutaminase by bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES). Biochem. J. 406, 407–414 (2007).
Ramachandran, S. et al. Structural basis for exploring the allosteric inhibition of human kidney type glutaminase. Oncotarget 7, 57943–57954 (2016).
Liesa, M. & Shirihai, O. S. Mitochondrial dynamics in the regulation of nutrient utilization and energy expenditure. Cell Metab. 17, 491–506 (2013).
Kawajiri, S. et al. PINK1 is recruited to mitochondria with parkin and associates with LC3 in mitophagy. FEBS Lett. 584, 1073–1079 (2010).
Soth, M. J. et al. Discovery of IPN60090, a clinical stage selective glutaminase-1 (GLS-1) inhibitor with excellent pharmacokinetic and physicochemical properties. J. Med. Chem. 63, 12957–12977 (2020).
Laake, J. H. et al. Postembedding immunogold labelling reveals subcellular localization and pathway-specific enrichment of phosphate activated glutaminase in rat cerebellum. Neuroscience 88, 1137–1151 (1999).
Zacharias, N. M. et al. Metabolic differences in glutamine utilization lead to metabolic vulnerabilities in prostate cancer. Sci. Rep. 7, 16159 (2017).
Ma, Y., Wang, L. & Jia, R. The role of mitochondrial dynamics in human cancers. Am. J. Cancer Res. 10, 1278–1293 (2020).
Valera-Alberni, M. et al. Crosstalk between Drp1 phosphorylation sites during mitochondrial remodeling and their impact on metabolic adaptation. Cell Rep. 36, 109565 (2021).
Ding, W.-X. & Yin, X.-M. Mitophagy: mechanisms, pathophysiological roles, and analysis. Biol. Chem. 393, 547–564 (2012).
Cruzat, V., Macedo Rogero, M., Noel Keane, K., Curi, R. & Newsholme, P. Glutamine: metabolism and immune function, supplementation and clinical translation. Nutrients 10, 1564 (2018).
Chin, R. M., Panavas, T., Brown, J. M. & Johnson, K. K. Optimized mitochondrial targeting of proteins encoded by modified mRNAs rescues cells harboring mutations in mtATP6. Cell Rep. 22, 2818–2826 (2018).
Strack, R. L. et al. A rapidly maturing far-red derivative of DsRed-Express2 for whole-cell labeling. Biochemistry 48, 8279–8281 (2009).
de Guzzi Cassago, C. A. et al. Glutaminase affects the transcriptional activity of peroxisome proliferator-activated receptor γ (PPARγ) via direct interaction. Biochemistry 57, 6293–6307 (2018).
Wang, W. & Malcolm, B. A. Two-stage PCR protocol allowing introduction of multiple mutations, deletions and insertions using QuikChange site-directed mutagenesis. Biotechniques 26, 680–682 (1999).
Tian, S. & Das, R. Primerize-2D: automated primer design for RNA multidimensional chemical mapping. Bioinformatics 33, 1405–1406 (2017).
Winter, D. J. rentrez: an R package for the NCBI eUtils API. R Journal 9, 520–526 (2017).
Bodenhofer, U., Bonatesta, E., Horejš-Kainrath, C. & Hochreiter, S. msa: an R package for multiple sequence alignment. Bioinformatics 31, 3997–3999 (2015).
Katoh, K., Misawa, K., Kuma, K.-I. & Miyata, T. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 30, 3059–3066 (2002).
Piñeiro, C., Abuín, J. M. & Pichel, J. C. Very Fast Tree: speeding up the estimation of phylogenies for large alignments through parallelization and vectorization strategies. Bioinformatics 36, 4658–4659 (2020).
Wang, L.-G. et al. Treeio: an R package for phylogenetic tree input and output with richly annotated and associated data. Mol. Biol. Evol. 37, 599–603 (2020).
Kass, I. & Horovitz, A. Mapping pathways of allosteric communication in GroEL by analysis of correlated mutations. Proteins 48, 611–617 (2002).
Jacob, A. I. et al. The regulatory domain of protein kinase C delta positively regulates insulin receptor signaling. J. Mol. Endocrinol. 44, 155–169 (2010).
Afanasyev, P. et al. A posteriori correction of camera characteristics from large image data sets. Sci. Rep. 5, 10317 (2015).
van Heel, M. et al. Single-particle electron cryo-microscopy: towards atomic resolution. Q. Rev. Biophys. 33, 307–369 (2000).
Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).
Yamashita, K., Palmer, C. M., Burnley, T. & Murshudov, G. N. Cryo-EM single-particle structure refinement and map calculation using Servalcat. Acta Crystallogr. D 77, 1282–1291 (2021).
Toro-Nahuelpan, M. et al. Tailoring cryo-electron microscopy grids by photo-micropatterning for in-cell structural studies. Nat. Methods 17, 50–54 (2020).
Strale, P.-O. et al. Multiprotein printing by light-induced molecular adsorption. Adv. Mater. 28, 2024–2029 (2016).
Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).
Wolff, G. et al. Mind the gap: micro-expansion joints drastically decrease the bending of FIB-milled cryo-lamellae. J. Struct. Biol. 208, 107389 (2019).
Klumpe, S. et al. A modular platform for automated cryo-FIB workflows. eLife 10, e70506 (2021).
Kremer, J. R., Mastronarde, D. N. & McIntosh, J. R. Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116, 71–76 (1996).
Lennon, F. E. et al. Unique fractal evaluation and therapeutic implications of mitochondrial morphology in malignant mesothelioma. Sci. Rep. 6, 24578 (2016).
Acknowledgements
We acknowledge the access and services provided by the Imaging Centre at the European Molecular Biology Laboratory (EMBL), generously supported by the Boehringer Ingelheim Foundation. The Leica SP8 Cryo-CLEM system was developed in collaboration with Leica Microsystems. This research also used facilities of LNNano and the Brazilian Biosciences National Laboratory (LNBio), both part of the Brazilian Centre for Research in Energy and Materials, a private nonprofit organization under the supervision of the Brazilian Ministry for Science, Technology, and Innovations. We acknowledge the cryo-microscopy staff for their assistance during the experiments at LNNano under internal proposal no. TEM26134, and Â. Saito from LNBio for generously donating the MEF cells used in this work. This work was supported by São Paulo Research Foundation (FAPESP) grants 2019/16351-3 (to S.M.G.D.), 2014/20673-2 and 2017/11766-5 (to A.L.B.A.), 2021/03933-4 (to J.E.N.Q.), 2021/13736-1 (to B.N.d.S), 2016/09077-4 (to R.K.E.C.), 2014/12663-7 (to Z.I.), 2021/01504-9 (to S.R.C.), 2017/15340-2 (to R.V.P.) and 2020/06062-1 (to M.G.v.H.). We acknowledge CIBFar (FAPESP RDIC 2013/07600-3) for full support and thank A. Girasole for administrative support.
Author information
Authors and Affiliations
Contributions
S.M.G.D. and A.L.B.A. designed the study and wrote the manuscript. D.A., M.M.D., J.E.N.Q. and Z.Y. performed experiments and data analysis with the support of I.Z., A.M.S., C.T.R., A.C.d.S.B., B.N.d.S., R.K.E.C., F.M.O.d.A., Z.I., A.C., M.G.v.H., S.R.C., S.M., J.M. and R.V.P. All authors read and approved the final manuscript.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
Peer review
Peer review information
Nature Structural & Molecular Biology thanks Tom Laughlin and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editors: Carolina Perdigoto and Katarzyna Ciazynska, in collaboration with the Nature Structural & Molecular Biology team.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data
Extended Data Fig. 1 Workflow for cryo-EM structure determination of fGLSm.
(a) Depiction of reference numbering and domain composition of the two kidney-type GLS splicing variants from mouse (Uniprot accession codes: D3Z7P3-1 for KGA and D3Z7P3-2 for GAC), featuring a Glutaminase domain (green) that is both well-conserved and structurally analogous to bacterial glutaminases14. In KGA, the Glutaminase domain is flanked by an extended N-terminal domain folded in an EF-hand-like four-helix bundle (EH-like) and a C-terminal domain with three ANK repeats19. GAC, in contrast, has a shorter, unstructured C-terminus lacking any recognizable motif or domain19. (b) An image of a typical raw micrograph obtained by negative staining of fGLSm, revealing a high concentration of long filaments. (c) A representative raw cryo-EM micrograph, captured using a Volta Phase plate to enhance particle visibility, illustrating significant filament dissociation upon vitrification. Insets showcase short helical filament segments, extending down to a half-turn. (d) An example of the single-particle picking approach utilized in the study. (e) Cryo-EM data processing was entirely carried out in CryoSPARC47 according to the schematic workflow. The panel includes representative 2D class averages of filament, accompanied by Gold-standard Fourier Shell Correlations, and viewing direction distributions for helical and local refinement with mask tightening with D2 symmetry-expanded particles. The orthogonal views of the fGLSm density map are colored by local resolution. (f) A wall-eyed stereoscopic depiction of the Fo-Fc omit map, aiding in the visualization of phosphate and neighboring densities, contoured at 13σ (where σ represents the standard deviation within the mask). This difference map was computed using Servalcat48 software, which is based on Bayesian statistics derived from half-map variances, after omitting the phosphate ions and mutating the side chains of interacting residues (those in contact with the phosphate ion) to alanine.
Extended Data Fig. 2 The overall structural similarity between inactive and active GLS and key-to-filamentation differences in main chain tracing.
(a) Cartoon representation of the superposition of the glutaminase domains, according to α-carbons, of the protein′s three states: (i) the active state, in green, is observed in the structure of filamentous mouse GLS (fGLSm) bound to Pi; (ii) the inhibited state, in magenta, is found in the structure of human GAC (GACh) bound to the allosteric inhibitor CB-839; (iii) in a ligand-free state of mouse GAC (GACm), in orange (dashed orange lines depicts the unstructured activation loop). (b) The black inset highlights the variations in the conformational state of the activation loop. (c) The red inset displays the stacking between F323m (activation loop) and Y471m (active site), which is stabilized by a hydrogen bond between the main chains of L321m and D472m in the fGLSm but not in the GACh:CB-839 and GACm ligand-free (the latter superpose identically in the region of D472m). Consequently, in fGLSm, the α-helix that starts with D472m is significantly shifted and contributes to the extended dimer interface near the activation loop. The yellow arrow indicates the relative position of this hydrogen bonding in panels (a) and (c).
Extended Data Fig. 3 The relationship between the filamentation and the activation of GLS.
(a) Dynamic light scattering curves, expressing the size distribution as a function of hydrodynamic radii by volume, for mouse GLS wild-type and point mutants, in the absence (empty peak tracing) and presence (filled peak tracing) of 20 mM phosphate. The expected size for the tetramer is indicated by a vertical gray dashed line and the region corresponding to heterogeneous filaments colored in light yellow. (b) Michaelis-Menten curves for all the constructs. We employed a streamlined enzymatic reaction that couples the glutaminase and glutamate dehydrogenase activities with a stoichiometric balance of 1:1; for each glutamate molecule produced by glutaminase in the first reaction, one NAD+ is reduced and can be tracked at 340 nm. (c) The direct relationship between the enzyme efficiency and particle size confirms that the R387mD mutant does not form filament (Rh ~5 nm) and is not activated by Pi. Abrogating the F323m:Y471m stacking (F232mS mutant) also blocks activity and filamentation, while K325mA mutant filament is constitutive, as previously shown by us10. Quantification of n = 5 or more light scattering replicates (a) is shown as smoothed distribution curves, each represented point in the Michaelis-Menten curves (b) is a mean with SD derived from a minimum of n = 6 replicates, the correlation between enzymatic efficiency and hydrodynamic radius (c) is reported using mean with SD for a minimum of n = 6 replicates.
Extended Data Fig. 4 Activation loop movement between inactive and active states requires a flip between phenylalanine and leucine to occupy the same hydrophobic pocket at the tetramer interface.
(a) Overall view of the cartoon representation of the fGLSm glutaminase domain activated state. (b) Inset of the tetramer interface, highlighting that L326m is locked in a symmetric hydrophobic groove (view on the right, yellow arrow) at the tetramer interface, with a significant flip of F327m (yellow arrow) toward the interior of the tetramer groove. We have previously shown that F327m occupies this contact in the inactive state, while L326m is disordered, along with the activation loop9.
Extended Data Fig. 5 GLS fibrillar-like staining upon QD is seen in different cell types.
(a) Immunofluorescence (IF) staining of endogenous GLS (green) of PC3 (prostate cancer), Hs578T (breast cancer), A549 (lung cancer), or HT-29 (colon cancer) cells in SQ (panel on the left) or QD (panels on the right). Cells were incubated with standard glutamine concentration medium (SQ) or glutamine-deprived medium (QD) for 6 hs. Nuclei were stained with DAPI (blue). Scale bar 1 µm. (b) Time course of filament formation in PC3 cells expressing GLS-EGFP after QD up to 24 hs; the filamentation of GLS is reversible upon the addition of dimethyl-α -ketoglutarate, glutamine (Gln). (c) Removing glutamine (QD) but not glucose (Glc) leads to the formation of fibrils. Scale bar for b-c are 10 µm. Electron micrographs of OsO4-stained PC-3 cells expressing GLS-APEX2 (d) or GLS-V5 after incubation with anti-V5 coupled to a secondary antibody linked to horseradish peroxidase (HRP) (e). Scale bars are indicated in the panels.
Extended Data Fig. 6 GLS-enriched mitochondria increase tubulation upon glutamine withdrawal.
PC-3 cells were transduced to express mitochondria signaling peptide Mitotag33-E2-Crimson34, GLS.WT/GLS.K320hA/GLS.R382hD-mKO2 (when indicated) and DM-Drp1 (when indicated). Drp1 and GLS expression levels are shown in Extended Data Fig. 8a. PC-3 cells were incubated with standard glutamine concentration medium (SQ) or glutamine-deprived medium (QD) for 6 hs, followed by DMSO or CB-839 incubation (1 µM) for another 6 hours. Mitochondria fluorescence (red), IF staining of endogenous GLS (green) (a), and the fluorescence of GLS.WT-mKO2 (b), GLS.K320A-mKO2 (c), or GLS.R382D-mKO2 (d) mutants (green) and merge (GLS+mito) are shown in representative figures on the left (inset of each gated area is shown at the right-down corner). Under QD, endogenous GLS-enriched mitochondria had increased tubulation (from 25 ± 2% in SQ to 35 ± 2%), which resisted DM-Drp1 treatment but was reduced by combining CB-839 and DM-Drp1 (to 29 ± 2%). GLS.WT-enriched mitochondria′s tubulation dropped only with CB-839, revealing GAC′s role in influencing tubulation. Interestingly, tubulation rose in GAC-depleted mitochondria under QD, hinting at GAC.WT′s broad effect. GLS.K320hA consistently elevated tubulation levels, unaffected by CB-839 and DM-Drp1. However, introducing GLS.R382hD negated QD-induced tubulation. Scale bar 30 µm; inset represents 3 times magnification of the image. Image quantifications are shown on the right (above, GLS-enriched mitochondria; below, GLS-depleted mitochondria). Quantification for n = 3 independent wells; at least 291 cells (a), 794 cells (b), 412 cells (c), 760 cells (d), were scored per condition per replicate is shown as the mean and SEM of the percentage of cells classified as having fragmented, intermediate, and tubulated mitochondria using ImageJ software and an in-house developed script. Pairwise two-tailed t tests (a) or one-way ANOVA followed by Tukey′s test (b-d) were applied. The significance of the statistical tests for the tubulated mitochondria is displayed at the most right (significance of the statistical tests for the fragmented and intermediated mitochondria are presented in the Supplementary Fig. 5b).
Extended Data Fig. 7 Cryo-CLEM workflow for the identification of GLS filaments inside cells.
(a) The fluorescent signal of pcDNA3.1_hGAC_EGFP transfected PC-3 cells, under glutamine deprivation (QD), showing filament formation. (i) Maximum intensity projection of the cryo confocal fluorescence signal overlayed onto the scanning electron microscopy image to help identify the region of interest (red square); scale bar 50 µm. (ii) A lamella (blue square) was produced based on the fluorescent signal in the focused ion beam-scanning electron microscope; scale bar 10 µm. (iii) Transmission electron microscopy montage of the lamella in (ii); scale bar 5 µm. (iv) An area on the montage (cyan square) showed an elongated mitochondrion with filamentous structures. (v) A slice of a weighted back projection reconstructed tomogram of the tilt series collected on the region in (iv); scale bar 100 nm. Inset: zoom in view of the white square shows the filament morphology. (b) A Fourier Shell Correlation (FSC) curve for the subtomogram averaging experiment estimated a resolution of 28 Å using the 0.143 threshold, and 32 Å at the 0.5 threshold.
Extended Data Fig. 8 DM-Drp1 and GLS.mKO2 expression in both PC-3 and MEF cells.
(a) Cells were transduced to express DM-Drp1; Drp1 was detected by IF with an antibody anti-Drp1 (green). Nuclei were stained with DAPI (blue) (cell panels on the left). The frequency of cells expressing the construct was determined considering the endogenous level as a cut-off value, over which all the measured signal was related to the DM-Drp1 construct (graphic on the above-right); Fluorescence intensity (sum) violin and boxplot per cell is also shown (graphic on the bottom-right). The same experimental conditions were applied in the mitochondria morphology quantification assays. (b). Fluorescence intensity (sum) violin and boxplot per cell is shown for endogenous GLS (IF) and GLS.WT/K320hA/R382hD-mKO2 in MEF (on the left) and PC3 (on the right). Quantification for n = 2 independent wells; at least 1254 cells (a), or n = 3 independent wells; at least of 160 cells (b, MEF), 602 cells (b, PC3), were scored per condition per replicate is shown as the mean and SEM for barplots as a violin plot and boxplot (centerline, median; box limits, upper and lower quartiles). One-way ANOVA followed by Tukey’s test was applied. Heatmaps of the statistical significance are shown at the right of each graph.
Extended Data Fig. 9 Tubulated mitochondria size correlates positively to fiber-formation.
PC-3 (a) and MEF (b) cells were transduced to express Mitotag23-E2-Crimson33 GLS.WT/GLS.K320hA/GLS.R382hD-mKO2 and DM-Drp1 (when indicated). Cells were incubated with standard glutamine concentration medium (SQ) or glutamine-deprived medium (QD) for 6 hs, followed by DMSO or CB-839 incubation (1 µM) for another 6 hours. Tubulated mitochondria size (as defined by fractal D55; the higher the number, the bigger the mitochondria) correlated positively to GLS.WT fiber formation as defined by intensity Kurtosis (the higher the number the bigger the fiber) when glutamine was removed. CB-839, but not DM-Drp1, was effective in decreasing the positive correlation for GAC.WT. Positive correlation was the highest in cells expressing GLS.K320hA and the lowest in cells expressing GLS.R382hD.
Extended Data Fig. 10 GLS-enriched mitochondria have decreased mitophagy upon glutamine withdrawal.
PC-3 cells were transduced to express Mitotag33-E2-Crimson34, either Parkin-mTurquoise2 or LC3- mTurquoise2 and GLS.WT/GLS.K320hA/GLS.R382hD-mKO2 (when indicated). GLS expression levels are shown in Extended Data Fig. 8b. PC-3 and MEF cells were incubated with standard glutamine concentration medium (SQ) or glutamine-deprived medium (QD) for 6 hs, followed by DMSO (Crtl) or CB-839 incubation (1 µM) for another 6 hours. Parkin (panels on the left) or LC3 (panels on the right) (blue) associated with mitochondria fluorescence (red + blue, called Parkin or LC3-positive mitochondria) or with IF staining of endogenous GLS (in MEF, a; in PC-3, b), the fluorescence of GLS.WT-mKO2 (c), GLS.K320A-mKO2 (d), or GLS.R382D-mKO2 (e) mutants (green + blue), as well as the combined Parkin/LC3+Mito+GLS, are shown in representative figures on the left (inset of each gated area is shown at the right-down corner; white arrow indicate PARKIN or LC3/GLS/mito co-localization signal, while pink arrow indicates Parkin or LC3/mito co-localization signal). In PC-3 cells, glutamine depletion amplified Parkin-positive mitochondria with endogenous GLS and GLS-WT (median values for endogenous GLS were 24% in SQ to 50% in QD; for GLS.WT, it was 15% to 28%). LC3-positivity on GLS-WT-enriched mitochondria declined under QD (from 11% to 7%), indicating less autophagosome recruitment. CB-839 reduced Parkin and led to LC3 accumulation in QD. GLS K320hA-enriched mitochondria displayed higher Parkin but lower LC3 than their depleted counterparts, whereas GLS R382hD showed the inverse pattern. The scale bar size is 30 µm; the inset represents 3 times the magnification of the image. Image quantifications are shown on the right (above, PARKIN-signal; below, PC-3-signal). Quantification (farthest right) was performed using ImageJ software and an in-house developed script and is shown as a violin plot and boxplot (centerline, median; box limits, upper and lower quartiles) of the percentage of Parkin/LC3-positive mitochondria per cell for n = 3 independent wells; at least 820 cells (a), 1032 cells (b), 742 cells (c), 829 cells (d), 1290 cells (e), and were scored per condition per replicate (Kruskal‒Wallis test followed by pairwise Wilcoxon test, FDR-adjusted). Significances of the statistical tests are displayed on the right.
Supplementary information
Supplementary Information
Supplementary results and Figs. 1–9.
Supplementary Video 1
Simulated trajectory morphing between the X-ray crystal structure of GACm inhibited by CB-839 (I-state, 5HL1) and the single-particle cryo-EM structure bound to Pi (A-state, 8EC6) highlighting the rotational movement of individual monomers that allows for reorganization of the activation loop and filament extension.
Supplementary Video 2
GLS forms filaments upon glutamine withdrawal. PC-3 cells were transduced to express Mitotag-E2-Crimson and GLS.WTh-mKO2 and plated in 200-nm-bottom-thickness multiwell plates. Cells were incubated with HEPES-buffered QD medium and imaged for 1 h 45 min using an HC PL APO CS2 ×63/1.20 water objective in a Leica SP8 confocal system with temperature control. The red channel represents the mitochondria and the green channel the GLS.WT-mKO2 signal.
Supplementary Data File
Supplementary data sources for Supplementary Figs. 4 and 6–9.
Source data
Source Data Fig. 4
Statistical source data.
Source Data Fig. 6
Statistical source data.
Source Data Extended Data Fig. 3
Statistical source data.
Source Data Extended Data Fig. 6
Statistical source data.
Source Data Extended Data Fig. 8
Statistical source data.
Source Data Extended Data Fig. 9
Statistical source data.
Source Data Extended Data Fig. 10
Statistical source data.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Adamoski, D., Dias, M.M., Quesñay, J.E.N. et al. Molecular mechanism of glutaminase activation through filamentation and the role of filaments in mitophagy protection. Nat Struct Mol Biol 30, 1902–1912 (2023). https://doi.org/10.1038/s41594-023-01118-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41594-023-01118-0
This article is cited by
-
Looking back at 30 years of Nature Structural & Molecular Biology
Nature Structural & Molecular Biology (2024)
