Research ArticleCELL BIOLOGY

GADD34 is a modulator of autophagy during starvation

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Science Advances  25 Sep 2020:
Vol. 6, no. 39, eabb0205
DOI: 10.1126/sciadv.abb0205
  • Fig. 1 Identification of TFEB direct targets following TFEB overexpression.

    (A) Characterization of inducible expression system in human embryonic kidney (HEK) 293 cells with genomic integration of the expression cassette in fig. S2. Exogenous TFEB-FLAG and tTA transactivator levels measured at the indicated time points by quantitative real-time polymerase chain reaction (qRT-PCR). (B) Quantification by densitometry of (C) immunoblotting assay (WB) of TFEB-FLAG in nuclear and cytoplasmatic fractions at the indicated time points expressed as fold change relative to time 0. (D) Distribution of TFEB binding sites detected by ChIP-seq relative to the transcription start site (TSS). (E) Selection of 557 bona fide TFEB direct targets and (F) de novo motif finding in their proximal promoter revealing the CLEAR binding site. (G) Expression levels of the 557 TFEB direct targets at increasing TFEB-FLAG levels at the indicated time points. Genes are ordered according to their correlation with TFEB-FLAG expression. (H) Scatter plot of GADD34 versus TFEB-FLAG expression levels. (I) qRT-PCR of the indicated gene during amino acid deprivation in untreated cells (control, CTRL) or following small interfering RNA–mediated knockdown (KD) of TFEB and TFE3 (TFEB/TFE3 KD); P value refers to two-way analysis of variance (ANOVA) after post hoc correction.

  • Fig. 2 GADD34 activity is required for sustained autophagic flux in starved cells.

    (A) Cells lacking GADD34 have an impaired autophagy flux. WT cells or knocked out for GADD34 (GADD34 KO) in growth medium or under amino acid starvation [Hanks’ balanced salt solution (HBSS)] with or without bafilomycin A1 treatment (BafA1, 100 nM) and immunostained for LC3. Scale bar, 10 μm. (B) LC3 puncta for untreated WT and GADD34 KO cells; means ± SD. n = 150 cells per condition from three experiments; GADD34 KO cells have more LC3 puncta than WT cells (P = 1.96 × 10−12, two-way ANOVA after post hoc correction). (C and D) LC3 puncta for WT and GADD34 KO cells with BafA1 in (C), or with 50 μM salubrinal in (D), or BafA1 + salubrinal in fig. S5. P values refer to t tests between salubrinal-treated and untreated cells, or between salubrinal + Baf1–treated cells and Baf1-treated cells. (E) Western blot (WB) of Chinese hamster ovary (CHO)–WT cells following amino acid deprivation alone (CTRL), or with 100 nM BafA1, or with 50 μM salubrinal, or with BafA1 + salubrinal. (F) Densitometry of p62 and LC3-II. Means ± SD. n = 2 independent experiments; P values refer to two-way ANOVA after post hoc correction. (G) WB of GADD34 KO cells treated as in (E). (H) Densitometry of WBs. Means ± SD. n = 2 independent experiments. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. The BafA1-dependent and salubrinal-dependent increase are highlighted in orange and red, respectively.

  • Fig. 3 GADD34 activity is required for lysosomal biogenesis following starvation.

    (A) Lysosomal biogenesis assay in a model of Fabry disease, i.e., HeLa cells knocked out for α-galactosidase (GLA) gene. Storage accumulation [globotriaosylceramide (Gb3)] discriminates small newly formed storage-free lysosomes from older and larger storage-filled lysosomes. (B) Lysosomes and Gb3 labeled with anti-LAMP1 antibody (green) and CY3-conjugated Shiga Toxin Subunit B (CY3-ShTxB) in GLA-KO cells in growth medium or starved (HBSS) for 24 hours in the absence or presence of salubrinal (50 μM). (C) Total number of lysosomes in GLA-KO cells across conditions in (B); means ± SD. n = 60 cells per condition from three experiments; P values from t tests. (D) Distribution of lysosome size and storage (Gb3) content across the indicated conditions as a percentage of their total number; n = 60 cells per condition from three experiments. Lysosomes were binned into three classes according to their area [small, S = area < 0.1 μm2; medium, M = area in (0.1 μm2, 1 μm2); large, L = area > 1 μm2] (P = 7.06 × 10−16 from Fisher’s exact test). (E) Lysosomes were labeled with anti-LAMP1 antibody in CHO-WT and GADD34 KO cells in growth medium or starved (HBSS) for 24 hours. (F) Total number of lysosomes in WT and GADD34 KO cells across conditions in (E); means ± SD. n = 50 cells per condition from three experiments; P value from t tests. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

  • Fig. 4 GADD34 activity is required for the starvation-induced transcriptional program to be implemented at the protein level.

    (A) Quantification of neosynthesized proteins by LC-MS following bio-orthogonal amino acid tagging with AHA for 6 hours in the following conditions: full medium (growth) and 6-hour amino acid starvation (HBSS) with or without salubrinal (Sal). Each protein is shown as a dot whose coordinates are the relative (fold) change following starvation alone (HBSS/growth – x axis) or starvation in the presence of 100 μM salubrinal (HBSS + Sal/HBSS – y axis). For each condition, the average fold change was computed from five independent samples. The trend line is shown as a solid black line. The dashed vertical line separates proteins increasing following starvation (right) from those decreasing (left). The red horizontal line separates proteins for which Sal treatment in starvation resulted in increased protein levels with respect to starvation alone (top) from those which resulted in decreased protein levels (bottom). Of 3667 proteins detected in all the samples (gray dots), HBSS treatment alone significantly changed 968 proteins (black dots). (B) Quantification of changes in protein levels measured as described in (A) for selected TFEB targets following starvation in the absence (black) or presence (gray) of salubrinal (P < 0.1). (C) Model of TFEB-mediated regulation of protein translation during starvation.

Supplementary Materials

  • Supplementary Materials

    GADD34 is a modulator of autophagy during starvation

    Gennaro Gambardella, Leopoldo Staiano, Maria Nicoletta Moretti, Rossella De Cegli, Luca Fagnocchi, Giuseppe Di Tullio, Sara Polletti, Clarissa Braccia, Andrea Armirotti, Alessio Zippo, Andrea Ballabio, Maria Antonietta De Matteis, Diego di Bernardo

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