eIF5A is required for autophagy by mediating ATG3 translation

Abstract

Autophagy is an essential catabolic process responsible for recycling of intracellular material and preserving cellular fidelity. Key to the autophagy pathway is the ubiquitin-like conjugation system mediating lipidation of Atg8 proteins and their anchoring to autophagosomal membranes. While regulation of autophagy has been characterized at the level of transcription, protein interactions and post-translational modifications, its translational regulation remains elusive. Here we describe a role for the conserved eukaryotic translation initiation factor 5A (eIF5A) in autophagy. Identified from a high-throughput screen, we find that eIF5A is required for lipidation of LC3B and its paralogs and promotes autophagosome formation. This feature is evolutionarily conserved and results from the translation of the E2-like ATG3 protein. Mechanistically, we identify an amino acid motif in ATG3 causing eIF5A dependency for its efficient translation. Our study identifies eIF5A as a key requirement for autophagosome formation and demonstrates the importance of translation in mediating efficient autophagy.

Keywords: ATG3; autophagy; eIF5A; translation.

Figure 1. High‐throughput RNAi screen identifies eIF5A as an autophagy regulator

1. Schematic overview of high‐throughput siRNA screening procedure performed in MCF‐7 cells stably expressing GFP‐LC3B.

2. Venn diagrams showing distribution and overlap of significantly scoring RBP candidates identified from basal and Torin‐1‐treated screens based on statistical filtering by RSA analysis (see Materials and Methods for details and Dataset EV1 for full data sets). Dotted lines indicate selection of candidates based on LogP values, degree of puncta deregulation, number of scoring siRNAs and manual curation based on RNA‐binding potential and subcellular localization. Left: RBP candidates for which knockdown upregulated GFP‐LC3B puncta (orange). Right: RBP candidates for which knockdown downregulated GFP‐LC3B puncta (blue). Candidates are ordered alphabetically.

3. Secondary validation screen (basal autophagy) for 23 hits from (B). Data shown are the percentage of GFP‐LC3B puncta‐positive cells relative to the scramble siRNA control (indicated by dashed line) and represent the mean + SEM from three biological replicates. The two best of three siRNAs from the primary screen were used and indicated as “siRNA A” and “siRNA B”. With the exception of ASS1, KPNB1, TROVE2 and FXR2, all candidates scored significantly (P < 0.05, Student's t‐test) in the expected direction relative to scramble. CCT3, ATXN2L, CARHSP1, MYO18A, NCOA5 and PUM1 scored with 1 siRNA, while EIF4A3, UBC, VCP, CISD2, eIF5A, HDAC2, LARP1, LRPPRC, MEX3D, MKRN1, R3HDM1, RC3H1, SAMD4B scored with both siRNAs.

Figure EV1. Secondary validation screen upon Torin‐1 induction of autophagy

Secondary validation screen performed as in Fig 1C, but treated for 2 h with Torin‐1 prior to fixation. Data shown are the percentage of GFP‐LC3B puncta‐positive cells relative to the scramble siRNA control (indicated by dashed line) and represent the mean + SD from one representative experiment (n = 2).

Figure 2. eIF5A regulates lipidation of ATG8 homologs and autophagosome formation

1. Representative images of GFP‐LC3B puncta in MCF‐7 cells 72 h after transfection with cntrl and eIF5A siRNAs. For the last 2 h prior to fixation, cells were either left untreated or treated with Torin‐1 or Bafilomycin A1. Scale bars 10 μm.

2. Quantification of GFP‐LC3B puncta in MCF‐7 cells 72 h after transfection. Cells were treated 2 h prior to fixation as in (A). Data are mean + standard deviation (SD) from one representative of three independent experiments. Each datapoint represents a technical replicate with values obtained from the quantification of > 1,500 cells. **P < 0.01, ***P < 0.001. Student's t‐test.

3. Western blotting analysis of LC3B, GABARAP and eIF5A in MCF‐7 GFP‐LC3B cells transfected for 72 h with indicated siRNAs. A representative experiment is shown (n = 3). Vinculin, histone H3 and GAPDH were used as loading markers. Dashed lines indicate separate gels of the same samples.

4. Transmission electron microscopy (TEM) images from MCF‐7 GFP‐LC3 cells after 72‐h transfection. Arrowheads indicate mature autophagosomes. Images (right) are enlargements of boxed area in overview images (left). Scale bars 5 μm (left), 1 μm (right).

5. Quantification of autophagosomes per cytoplasmic area from TEM images. Data are mean + standard error of the mean (SEM) (n = 2) (30–35 cells/sample, counting a total of 90 autophagosomes in cntrl si and 54 autophagosomes in eIF5A si samples).

6. Representative images from body‐wall muscle, terminal pharyngeal bulb and intestinal cells (individual cells are outlined) of Caenorhabditis elegans expressing GFP::LGG‐1 subjected to CTRL (empty vector) or iff‐2 RNAi. Arrowheads denote individual GFP::LGG‐1 puncta. Scale bars 10 μm.

7. Quantification of GFP::LGG‐1 puncta in intestinal cells (n = 91), terminal pharyngeal bulbs (n = 43) and body‐wall muscle cell areas (n = 61–78) of animals subjected to CTRL (empty vector) or iff‐2 RNAi. Data are mean + SEM from four independent experiments. Student's t‐test: ns P > 0.05, *P < 0.05, ***P < 0.001.

Source data are available online for this figure.

Figure EV2. eIF5A depletion effects on specific autophagy markers

1. Western blotting analysis of p‐mTOR (Ser 2448) in MCF‐7 GFP‐LC3B cells after indicated treatments (2 h) and siRNA transfections (72 h). A representative experiment is shown (n = 2).

2. Western blot of MCF‐7 GFP‐LC3B cells after 72‐h transfection with indicated siRNAs. A representative experiment is shown (n = 3).

3. qRT–PCR of LC3B, GABARAP and GATE‐16 mRNA levels in MCF‐7 GFP‐LC3B cells of indicated transcripts after 72‐h transfection with indicated siRNAs. Data represent the mean + SD (n = 3).

4. Western blots of LC3B in indicated cells lines and siRNA transfections (72 h). A representative experiment is shown (n = 3).

5. Western blot in ATG5 WT or ATG5 KO HeLa cells with indicated siRNAs transfections for 72 h. A representative experiment is shown (n = 3).

6. Western blot of MCF‐7 cells with indicated siRNAs for 72 h. Bafilomycin treatment for the last 2 h prior to harvest. A representative experiment is shown (n = 3).

7. Mature autophagosomes identified from TEM images were scored as having diameter of > 300 or < 300 nm, and their percentage‐wise distribution in cntrl or eIF5A siRNA‐treated cells is shown (n = 2).

8. MCF‐7 cells were transfected for 72 h as indicated, fixed and immunostained for ATG16L1. Cells containing more that 5 puncta were defined as ATG16L1 puncta‐positive cells. Data represent the mean + SD (n = 3). Student's t‐test: **P < 0.01.

Source data are available online for this figure.

Figure 3. eIF5A hypusination is required for autophagy regulation

1. Western blotting analysis of eIF5A levels in cell lines expressing WT and K50A eIF5A at indicated time points after doxycycline treatment. A representative experiment is shown (n = 3).

2. Quantification of GFP‐LC3B puncta in MCF‐7 eIF5A WT or K50A cell lines at indicated time points of doxycycline treatment. For GC7 samples, treatment was given 24 h prior to fixation. Data are mean + standard deviation (SD) from one representative of three independent experiments. Each datapoint represents a technical replicate with values obtained from the quantification of > 1,500 cells. Statistical significance for each (no dox, 24 h dox and 48 h dox) was tested relative to corresponding eIF5A WT samples. **P < 0.01, ***P < 0.001. Student's t‐test.

3. Quantification of GFP‐LC3B puncta in MCF‐7 eIF5A WT or K50A cell lines after indicated siRNA transfections (72 h) and doxycycline treatments (48 h). Data are mean + standard deviation (SD) from one representative of three independent experiments. Each datapoint represents a technical replicate with values obtained from the quantification of > 1,500 cells. Student's t‐test: ns P > 0.05, **P < 0.01.

4. Western blot for LC3B in MCF‐7 eIF5A WT and K50A cell lines after 72‐h transfection with indicated siRNAs. Doxycycline was added for last 48 h of transfection. A representative experiment is shown (n = 3). Dashed lines indicate separate gels of the same samples.

5. Western blotting analysis of eIF5A levels in pelleted ribosomes. Ribosomes were isolated through a 10% sucrose cushion from cells after indicated treatments (2 h prior to cell lysis) and analysed together with corresponding input samples. A representative experiment is shown (n = 3). Quantification of eIF5A levels relative to RPL23A in pellet is indicated below the figure (ImageJ software).

6. Analysis of eIF5A interaction with the ribosome by Western blotting analysis. Inputs and eluates obtained after purification of GFP‐eIF5A were analysed with RPS6, RPL10A, RPL23A and GFP antibodies as indicated. A cell line expressing GFP‐3xFLAG was used as a negative control. A representative experiment is shown (n = 4).

Source data are available online for this figure.

Figure EV3. eIF5A hypusination, localization and ribosome binding

1. Western blotting analysis of eIF5A and hypusine levels in cell lines expressing WT and K50A eIF5A at indicated time points after doxycycline treatment. A representative experiment is shown (n = 2).

2. Nucleus/cytoplasmic fractionations were performed in MCF‐7 cells and subsequently analysed by Western blotting for eIF5A. Lamin A1 and GAPDH were used as nuclear and cytoplasmatic markers, respectively. A representative experiment is shown (n = 3).

3. Quantification of GFP‐LC3B puncta after indicated treatments (2 h) in MCF‐7 GFP‐LC3B eIF5A WT inducible cells (used for ribosome purifications in Fig 3E). Data shown are mean + SD (n = 2).

4. RNA inputs and eluates obtained from co‐purification of GFP‐eIF5A were analysed by qRT–PCR for 18S and 28S rRNA. A cell line expressing GFP‐3xFLAG was used as a negative control. Data are shown as eluates (RNA IP) relative to input and represent the mean + SEM (n = 4). Student's t‐test comparing treatments to untreated: **P < 0.01, ***P < 0.001.

Source data are available online for this figure.

Figure EV4. eIF5A depletion from OPP assay

Quantification of eIF5A knockdown in MCF‐7 cells from Fig 4A by immunostaining using Alexa 594‐coupled secondary antibody. Data are mean + SEM from four independent experiments.

Figure 4. eIF5A regulates translation of ATG3

1. Global translation measurement upon eIF5A depletion by pulse labelling of newly translated proteins with Alexa Fluor 488‐tagged O‐propargyl‐puromycin (OPP) followed by imaging and fluorescent quantification. MCF‐7 cells were transfected for 72 h, and a positive control was treated with cycloheximide (CHX) for 5 min. Representative images (left) show OPP (green) and Hoechst signals (blue). Quantification (right) shows mean values of fluorescent signal per well from four wells of three combined experiments. Error bars represent SD. Scale bars 100 μm. Student's t‐test: ns P > 0.05, ***P < 0.001.

2. Schematic overview of labelling and mass spectrometry‐based analysis of newly translated proteins. Cells were transfected as indicated for 72 h and incubated 2 h with the methionine analogue L‐azidohomoalanine (+AHA) or left untreated (−AHA/mock). The Click‐iT chemistry‐based kit was used for covalent capture of newly synthesized proteins, which were subsequently subjected to LC‐MS analysis. The experiment was performed in biological triplicates.

3. Flow diagram illustrating data analysis stages of newly synthesized protein LC‐MS described in (B). A set of newly synthesized proteins was defined using a fivefold enrichment threshold above mock (unlabelled −AHA) sample (2,308 proteins). Further filtering by applying P‐value and fold change thresholds (see Materials and Methods) identified 350 newly synthesized proteins deregulated by eIF5A of which 278 proteins were downregulated upon eIF5A depletion. Comparison to total RNA‐seq data and exclusion of proteins for which mRNA transcripts were deregulated by eIF5A narrowed down this selection to 211 candidates. Merging these with a set of 468 autophagy‐associated genes (geneontology.org) resulted in an overlap of nine autophagy‐related proteins.

Figure EV5. eIF5A affects autophagy via ATG3

1. Western blotting analysis of LC3B and ATG3 protein levels after 72‐h transfection with indicated siRNAs. Representative experiment is shown (n = 3).

2. Western blotting analysis of effects of p62 depletion on LC3B lipidation in MCF‐7 cells after 72‐h transfection with indicated siRNAs. Representative experiment is shown (n = 2).

3. Analysis of eIF5A effect on single or combined mutations of ATG3‐mCherry constructs assessed by imaging and automated quantification of mCherry fluorescent signal. HEK 293 cells were transfected with indicated siRNAs for 72 h. For the last 48 h, cells were transfected with indicated ATG3‐mCherry constructs. Data show quantification of mean mCherry fluorescence + SD from one representative experiment (n = 2).

Source data are available online for this figure.

Figure 5. Regulation of autophagy by eIF5A is mediated via ATG3 translation

1. Western blotting of ATG3 protein levels in HEK 293 and MCF‐7 cells (left) and qRT–PCR of ATG3 mRNA levels from MCF‐7 cells (right) after 72‐h transfection with cntrl and eIF5A siRNAs. For the Western blot, a representative experiment is shown (n = 3). Quantifications of ATG3 intensity relative to Vinculin are shown below the Western blot (ImageJ software). For the qRT–PCR, data shown are mean + SD (n = 3).

2. Analysis of newly synthesized ATG3 levels upon depletion of eIF5A. Pulse‐labelling and coupling of newly synthesized proteins to biotin followed by streptavidin pull‐down and Western blotting; 1 and 2 indicate replicate samples from one experiment. A representative experiment is shown (n = 3). The asterisk indicates a nonspecific band.

3. Western blot of HEK 293 cells transfected with pLVX‐empty or pLVX‐ATG3 constructs together with indicated siRNAs for 72 h. All samples were treated with doxycycline for 72 h to induce pLVX expression. A representative experiment is shown (n = 3). The asterisk indicates a nonspecific band.

4. Schematic of ATG3‐mCherry WT and mutant constructs.

5. Western blot of HEK 293 cells transfected with indicated siRNAs for 72 h. For the last 48 h, cells were transfected with indicated ATG3‐mCherry constructs from (D). ATG3‐mCherry is detected with anti‐mCherry Ab. A representative experiment is shown (n = 3), and the full‐length ATG3‐mCherry band intensity (lower band) is quantified relative to Vinculin and shown below the figure (ImageJ software).

6. HEK 293 cells transfected as described in (E), fixed and imaged for automated quantification of cytoplasmic mCherry fluorescence. Percentage of mCherry positive cells was defined based on arbitrary fluorescent intensity cut‐offs. Data show the mean fluorescent signal + SEM relative to control from three independent experiments. Student's t‐test: ns P > 0.05, ***P < 0.001.

Source data are available online for this figure.

Figure 6. Model for eIF5A‐mediated regulation of autophagy

During autophagosome formation, cytosolic LC3B‐I is conjugated to the lipid phosphatidylethanolamine (PE), forming LC3B‐II which is anchored to the phagophore. This is mediated via the sequential action of the E1‐like ATG7, E2‐like ATG3 and E3‐like ATG5–ATG12–ATG16L1 complex. eIF5A, via its hypusine residue, assists the ribosome in translating the ATG3 protein at its DDG motif. This increases the efficiency of ATG3 production and facilitates LC3B lipidation and autophagosome formation. eIF5A association with ribosomes is enhanced upon autophagy induction.