Research ArticleCell Biology

Stress granules sense metabolic stress at the plasma membrane and potentiate recovery by storing active Pkc1

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Science Signaling  17 Mar 2020:
Vol. 13, Issue 623, eaaz6339
DOI: 10.1126/scisignal.aaz6339
  • Fig. 1 SGs associate with eisosomes during starvation.

    (A) Strategy for BioID proteomics analysis of yeast proteins adjacent to Pab1 and Pub1 after starvation but not Pab1- or Pub1-adjacent without starvation. LC-MS/MS, liquid chromatography–tandem mass spectrometry. (B and C) Gene ontology (GO) analysis of SG proteins significantly enriched in Pab1 and Pub1 starvation interactomes hierarchically arranged (B) and eisosome components identified under starvation conditions (C). n = 3 biological replicates. ATP, adenosine triphosphate. (D) Confocal microscopy of SGs and eisosomes in yeast expressing endogenously tagged Pil1-mCH and Pab1-GFP that were starved for 30 min. Arrowheads indicate SGs. Images and kymograph are representative of four independent experiments. Quantification of the ratio of SGs proximal to eisosomes is noted in the merged images and represent the means ± SEM of 100 cells pooled from all experiments (see also fig. S1I). (E) BiFC analysis of Pab1 and Pil1 proximity in cells expressing endogenously tagged Pil1-mCH-VenusC (Vc) and inducible VenusN-Pab1-IFP (Vn) that were starved for 1 hour. Images are representative of three independent experiments. Fold change of fluorescence intensity are means ± SEM of 30 cells per strain pooled from all experiments, *P < 0.05 by Student’s t test. (F) Live-cell SIM of SG and eisosomes during starvation. Images are representative of four independent experiments. (G) Distances between eisosomes and SGs as measured by SIM are indicated as means ± SEM of 30 pairs pooled from all experiments, *P < 0.05 by Student’s t test. au, arbitrary units. (H) SG formation in wild-type (WT) and pil1lsp1∆ strains under starvation conditions. Images are representative of three independent experiments. (I) SG formation in pil1lsp1∆ (control) and pil1lsp1∆ complemented with Pil1-mCH strains under starvation conditions. Images are representative of three independent experiments. (J) Western blot for endogenous Pab1 tagged with green fluorescent protein (GFP) in lysates of WT and pil1lsp1∆ yeast. The numbers below the blot represent combined densitometry results from three independent experiments normalized to WT control, P = 0.17 by Kruskal-Wallis test. Scale bars, 1 μm (D, E, F, H, and I) and 200 nm [inset in (F)].

  • Fig. 2 Pil1 undergoes Pkc1-dependent rearrangements during starvation.

    (A) Confocal and TIRF/SIM microscopy of eisosomes in live yeast expressing endogenously tagged Pil1-GFP and starved for 1 hour. Images are representative of three independent experiments. Imaging plane is indicated above the frames. (B) Quantification of the distance between eisosomes on a radial cell section is shown as a mean ± SEM, *P < 0.05. Student’s t test, n = 100 cells per condition in three independent experiments. (C) Western blot for endogenous GFP-tagged Pil1 in lysates of WT yeast under control and 1-hour starvation conditions. The numbers below the blot represent densitometry combined from three independent experiments, P = 0.61 by Mann-Whitney test. (D) Schematic of Pil1 and Pab1 showing protein domains, low-complexity regions (LCR), and intrinsically disordered regions (IDR), RNA recognition motif (RRM), and polyadenylate-binding protein domain (PBP). Phosphosites in Pil1 identified during starvation conditions (Ser163 and Ser230) are indicated. (E) Confocal microscopy in live pil1lsp1∆ yeast expressing endogenously tagged Pab1-GFP and induced to express mCH-tagged WT, S230A, S230D, S163A, S163D, or truncated (1 to 276) Pil1 before the onset of starvation. Images are representative of three independent experiments. (F) Quantification of the ratio of cells in the population with SGs and the distance between the eisosomes. Data represent means ± SEM of 100 cells per strain pooled from all experiments for distance quantification and 1000 cells per strain pooled from all experiments for ratio quantification, N = 20. Pearson correlation between SG ratio and the distance between eisosomes = −0.74. Asterisk (*) indicates significant differences in the distance or ratio of cells with SGs comparing to WT, *P < 0.05 with one-way analysis of variance (ANOVA). (G) Experimental scheme for assessing eisosome formation in yeast expressing endogenously tagged Pkc1-AID, an integrated copy of the auxin receptor OsTIR (50), and endogenously tagged Pil1-GFP. Yeast were grown to mid-log phase, and then, Pkc1 depletion was initiated by addition of a synthetic auxin analog before cells were starved and visualized. (H) Confocal imaging and quantification of the distance between eisosomes in cells treated as in (G). Data are means ± SEM of 30 cells per group per condition pooled from three independent experiments. Scale bars, 1 μm [A, E (top row), and H), 500 nm [E (bottom row)], and 100 nm (insets in (A)].

  • Fig. 3 Pkc1 localizes to SGs and is required for SG formation.

    (A) Schematic of Pkc1 showing protein domains, LCR, and IDRs. The R398P mutation renders Pkc1 constitutively active (31). The T983A mutation blocks Pkc1 phosphorylation by Pkh2. (B) Live confocal microscopy of yeast expressing endogenously tagged Pab1-GFP and either inducible WT or constitutively active (Pkc1-R398P) Pkc1-mCH grown under control or starvation conditions. Quantified portion of SGs with detectable Pkc1 are noted in the merged images. Fluorescence intensity of Pkc1-mCH in SGs was quantified and presented as means ± SEM from 30 cells per genotype per condition pooled from three independent experiments. **P < 0.01. (C) Quantified ratios of dead cells in the population of WT and pil1lsp1∆ yeast overexpressing an integrated copy of Pkc1-mCH or Pkc1-R398P-mCH stained with propidium iodide. Data represent means ± SEM of 100 cells per genotype per condition pooled from three independent experiments. *P < 0.05. (D) Confocal microscopy of SGs in pil1lsp1∆ yeast overexpressing Pkc1 or Pkc1-R398P before the starvation and quantification of the ratios of cells in the population with SGs. Data represent means ± SEM of 30 images containing at least 20 cells of each type from three independent experiments. (E) Confocal microscopy of yeast expressing endogenously tagged Pil1-GFP and induced to express PKC1(T983A)-mCH 1 hour before starvation. Images are representative of three independent experiments. (F) Schematic of FRET sensor for Pkc1 activity and control. Ruby2 (red, acceptor) and Clover (green, donor) are fused to opposite ends of Pkc1. The control is a head-to-tail fusion of Ruby2 and Clover. Activation of Pkc1 is thought to involve a change from a closed to an open conformation (35), thus reducing the FRET signal. (G) Confocal microscopy showing Pkc1 activity as assessed by FRET in WT yeast grown under control or starvation conditions or in the presence of the Pkc1-activating compound capsofungin. (H) Quantification of donor (Clover)/acceptor(Ruby2) fluorescence intensities inside cells during control and starvation conditions. Graphs are representative of 10 independent experiments. Data represent means ± SEM of 30 cells per condition pooled from three independent experiments, *P < 0.05, with one-way ANOVA. (I) Quantification of FRET activity in (H). (J) Model of eisosome rearrangement during starvation. Pkc1-dependent phosphorylation of eisosomes during starvation results in their clustering, which primes formation of SGs that recruit active Pkc1. Scale bars, 1 μm.

  • Fig. 4 SGs store Pkc1 and enable rapid recovery from stress.

    (A) Experimental scheme and confocal microscopy of middle-log phase yeast induced to express Pkc1-mCH for 2 hours and then starved for 24 hours before being placed in fresh glucose-containing media for 2 hours. (B) Quantification of whole-cell fluorescence intensities from (A). Data represent means ± SEM of 30 cells per genotype per condition pooled from three independent experiments. *P < 0.05. (C) Growth curves of WT and pil1lsp1∆ yeast that were grown under control conditions to middle-log phase or for 24 hours in stationary phase and then diluted in fresh glucose-containing media. Graphs are representative of three independent experiments. Data represent means ± SEM. OD600, optical density at 600 nm. (D) Model of WT and pil1lsp1∆ recovery after long-term starvation, showing that the absence of SGs results in delayed resumption of division. Scale bars, 1 μm.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/13/623/eaaz6339/DC1

    Fig. S1. SGs associate with eisosomes during starvation.

    Fig. S2. Eisosomes undergo Pkc1-dependent clustering during starvation.

    Fig. S3. Pkc1 localizes to SGs during starvation.

    Table S1. S. cerevisiae strains used in this study.

    Table S2. Plasmids used in this study.

    Table S3. Pab1 and Pub1 interactomes identified by BioID and Pil1 phosphorylation during control and starvation.

  • The PDF file includes:

    • Fig. S1. SGs associate with eisosomes during starvation.
    • Fig. S2. Eisosomes undergo Pkc1-dependent clustering during starvation.
    • Fig. S3. Pkc1 localizes to SGs during starvation.
    • Table S1. S. cerevisiae strains used in this study.
    • Table S2. Plasmids used in this study.
    • Legend for table S3

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S3 (Microsoft Excel format). Pab1 and Pub1 interactomes identified by BioID and Pil1 phosphorylation during control and starvation.

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