Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Jul 25;20(4):895-908.
doi: 10.1016/j.celrep.2017.06.082.

Glycolytic Enzymes Coalesce in G Bodies under Hypoxic Stress

Meiyan Jin  1 , Gregory G Fuller  2 , Ting Han  3 , Yao Yao  4 , Amelia F Alessi  5 , Mallory A Freeberg  6 , Nathan P Roach  2 , James J Moresco  7 , Alla Karnovsky  8 , Misuzu Baba  9 , John R Yates 3rd  7 , Aaron D Gitler  10 , Ken Inoki  11 , Daniel J Klionsky  12 , John K Kim  13
Affiliations

Glycolytic Enzymes Coalesce in G Bodies under Hypoxic Stress

Meiyan Jin et al. Cell Rep. .

Abstract

Glycolysis is upregulated under conditions such as hypoxia and high energy demand to promote cell proliferation, although the mechanism remains poorly understood. We find that hypoxia in Saccharomyces cerevisiae induces concentration of glycolytic enzymes, including the Pfk2p subunit of the rate-limiting phosphofructokinase, into a single, non-membrane-bound granule termed the "glycolytic body" or "G body." A yeast kinome screen identifies the yeast ortholog of AMP-activated protein kinase, Snf1p, as necessary for G-body formation. Many G-body components identified by proteomics are required for G-body integrity. Cells incapable of forming G bodies in hypoxia display abnormal cell division and produce inviable daughter cells. Conversely, cells with G bodies show increased glucose consumption and decreased levels of glycolytic intermediates. Importantly, G bodies form in human hepatocarcinoma cells in hypoxia. Together, our results suggest that G body formation is a conserved, adaptive response to increase glycolytic output during hypoxia or tumorigenesis.

Keywords: RNA binding protein; RNA granule; glycolysis; hypoxia; intrinsically disordered region; phase transitions.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Glycolytic enzymes form G bodies in response to hypoxia
(A) Glycolytic enzymes form granules upon hypoxia treatment. Yeast cells with the indicated GFP-tagged proteins were cultured with oxygen (+) or in a hypoxic chamber (−) for 24 h. (B) Pfk2p does not form granules in glucose (-Glu) or nitrogen (-N) starved yeast or in yeast grown on a non-fermentable carbon source (EtOH+Gly). (C) Pfk2p colocalizes with Pfk1p, Fba1p, and Cdc19p in yeast grown in hypoxia for 24 h. GFP, green; Azurite, magenta. (D) Pfk2p-GFP forms non-membrane-bound structures after 24 h hypoxic treatment as measured by immunogold (anti-GFP) staining and TEM. (E) Yeast strains co-expressing GFP-tagged Pab1p or Edc3p with Pfk2p-Azurite were subjected to glucose or oxygen deprivation, respectively. Cells were cultured in SMD (5%) to the mid-log phase, and shifted to SM medium for 30 min to induce stress granule formation, or cultured in the hypoxic chamber for 24 h to induce Pfk2 granule formation. Images are representative fluorescence images taken with FITC (GFP) and DAPI (Azurite) channels. (F) Pfk2p granule formation adjacent to a P-body (Edc3p-GFP) was observed occasionally in -O2 treated cells in (E). Scale bar 5 μm (A–E). [See also Figures S1–S2.]
Figure 2
Figure 2. Identification of the G body proteome
(A) Schematic of partial purification of G bodies. Cells were lysed, then G body fractions were concentrated and immunopurified using a monoclonal mouse anti-GFP antibody on Dynabeads. The eluted fraction was then subjected to proteomic mass spectrometry. (B) Cartoon and image of Pfk2p-GFP labeled G bodies bound to Dynabeads. (C) Cellular localization of candidate G body-interacting proteins. Left: Representative confocal maximum intensity projections of Pfk2p-Azurite colocalization in hypoxia with the indicated GFP-tagged protein candidate. Right: Quantitation of the percent of cells with either overlapping or adjacent foci in hypoxia. Top 14 proteins showing colocalizing are shown with Pfk1p-GFP control. (D) Mutant analysis to determine if mass spectrometry candidates are required for Pfk2p-GFP foci/G body formation. Left: Representative maximum intensity projections of Pfk2p-GFP localization in hypoxia in deletions of proteome candidates. Right: Quantitation of Pfk2-GFP G body formation. All scale bars 10 μm. [See also Figures S3–S5]
Figure 3
Figure 3. G Body formation is Snf1-dependent
(A) Pfk2p intrinsically disordered domain (IDR) as predicted by IUPred in wild-type and 140–165Δ Pfk2p. (B) Cells expressing GFP-tagged 140–165Δ Pfk2p and subjected to 16 h hypoxic treatment contain multiple fragmented granules (indicated by arrows) (p<0.05 versus wild-type by student’s t-test). Average intensity of granules measured by ImageJ (p<0.01 versus wild-type). Error bars represent SEM for at least three independent experiments. (C) A majority of snf1Δ cells display no or fragmented Pfk2p-GFP granules after 16 h hypoxia. Error bars represent SEM for more than three independent experiments. At 8 h, t-test comparing snf1Δ to wild-type, p<0.05 for cells without granules and cells with more than one granule and p<10−4 for cells with one granule. At 16 h, p<0.05 for cells without granules, p<0.01 for cells with more than one granule and cells with one granule. (D–F) Phosphorylation of Pfk2p-GFP is compromised in snf1Δ cells and Pfk2p is phosphorylated at its IDR. Cells expressing GFP-tagged glycolytic enzymes or Pfk2pΔ140–165 were collected after specified durations of anaerobic treatment. Protein extracts were separated by Mn2+-Phos-tag SDS-PAGE and subjected to immunoblotting using a GFP antibody. The images in D and F are from the same blot with the same wild-type control. All scale bars 5 μm. [See also figure S6]
Figure 4
Figure 4. Snf1 regulates glycolytic activity under hypoxic conditions
(A) Hypoxia and snf1 deletion result in remodeling of metabolic pathways. Cross-validated partial least squares discriminant analysis (PLS-DA) score plot (R2Y=0.936; Q2=0.454) shows separation of wild-type and snf1Δ samples at time 0 and after 16 h hypoxic treatment. Shaded ovals represent 95% confidence intervals. Solid circles represent individual replicates. (B) Four key metabolites in the glycolysis pathway contribute to the metabolic differences observed in (A). Boxplot colors represent samples as in (A). (C–E) Phloxine B staining indicates high bud cell death in pfk2Δ cells after 24 h hypoxia. Dead cells were defined as those with fluorescence signal in the FITC channel. Quantification in (D, E) is based on N>300 cells per strain. Cartoon is of mother cell and daughters in the dotted box, green indicates Phloxine B. (F) Pfk1p-GFP and Cdc19p-GFP form granules in pfk2Δ cells. C, F scale bar 5 μm. Cartoon is of mother cell and daughters in the dotted box, green indicates GFP-tagged protein. (G) Cells with G bodies have significantly less glucose than cells lacking G bodies by t-test at t = 3 h (p=0.0035) and t = 4.5 h (p=0.035). Error bars represent SEM for three biological replicates in each condition. [See also Figure S7]
Figure 5
Figure 5. G bodies containing PFKL form in hypoxic mammalian cells
(A) Pfk2p homolog, PFKL, forms granules in HepG2 cells maintained in 1% oxygen for 24 h. (B) Time course of PFKL granule formation under hypoxic conditions. HepG2 cells were kept in 1% oxygen for indicated times (1, 3, 8, and 24 h). As a control, cells were cultured under normoxic conditions. (C) Formation of PFKL bodies is dependent on RNA. RNaseA treatment of cells maintained in 1% oxygen for 24 h decreases PFKL bodies compared to vehicle. (A–C) All cells were fixed and stained with anti-PFKL antibody to examine granule formation. All scale bars 10 μm. [See also Figure S8]
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. An S, Kumar R, Sheets ED, Benkovic SJ. Reversible compartmentalization of de novo purine biosynthetic complexes in living cells. Science. 2008;320:103–106. - PubMed
    1. Anderson P, Kedersha N. RNA granules: post-transcriptional and epigenetic modulators of gene expression. Nature Reviews Molecular Cell Biology. 2009;10:430–436. - PubMed
    1. Atsumi T, Chesney J, Metz C, Leng L, Donnelly S, Makita Z, Mitchell R, Bucala R. High expression of inducible 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (iPFK-2; PFKFB3) in human cancers. Cancer Research. 2002;62:5881–5887. - PubMed
    1. Bah A, Forman-Kay JD. Modulation of Intrinsically Disordered Protein Function by Post-translational Modifications. Journal of Biological Chemistry. 2016;291(13):6696–6705. - PMC - PubMed
    1. Breslow DK, Cameron DM, Collins SR, Schuldiner M, Stewart-Ornstein J, Newman HW, Braun S, Madhani HD, Krogan NJ, Weissman JS. A comprehensive strategy enabling high-resolution functional analysis of the yeast genome. Nat Methods. 2008;5:711–718. - PMC - PubMed

Publication types