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, 18 (5), 765-780

Conserved Atg8 Recognition Sites Mediate Atg4 Association With Autophagosomal Membranes and Atg8 Deconjugation

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Conserved Atg8 Recognition Sites Mediate Atg4 Association With Autophagosomal Membranes and Atg8 Deconjugation

Susana Abreu et al. EMBO Rep.

Abstract

Deconjugation of the Atg8/LC3 protein family members from phosphatidylethanolamine (PE) by Atg4 proteases is essential for autophagy progression, but how this event is regulated remains to be understood. Here, we show that yeast Atg4 is recruited onto autophagosomal membranes by direct binding to Atg8 via two evolutionarily conserved Atg8 recognition sites, a classical LC3-interacting region (LIR) at the C-terminus of the protein and a novel motif at the N-terminus. Although both sites are important for Atg4-Atg8 interaction in vivo, only the new N-terminal motif, close to the catalytic center, plays a key role in Atg4 recruitment to autophagosomal membranes and specific Atg8 deconjugation. We thus propose a model where Atg4 activity on autophagosomal membranes depends on the cooperative action of at least two sites within Atg4, in which one functions as a constitutive Atg8 binding module, while the other has a preference toward PE-bound Atg8.

Keywords: LC3; autophagosome; autophagy; deconjugation; phagophore assembly site.

Figures

Figure 1
Figure 1. Atg4 localizes to the PAS during autophagy

Subcellular distribution of Atg4‐GFP under autophagy‐inducing conditions in WT and atg1∆ strains expressing the PAS marker proteins RFP‐Ape1 (SAY071 and SAY020) and mCherry‐Atg8 (MNY006 and SAY010 transformed with pCumCherryV5Atg8). DIC, differential interference contrast. Scale bars, 5 μm.

Percentage of cells in which Atg4‐GFP is observed in a punctate structure in the experiments depicted in panel (A). Data represent the average of three independent experiments ± standard deviation (SD).

Percentage of Atg4 puncta co‐localizing with the PAS marker proteins Atg8 and Ape1 in the experiments shown in panel (A). Data represent the average of three independent experiments ± SD.

Localization of Atg4‐GFP in WT (MNY006), atg1∆ (SAY10), atg7∆ (SAY014), atg8∆ (SAY015), atg10∆ (SAY059), atg12∆ (SAY029), and atg16∆ (SAY064) mutant strains, under starvation. Cells were analyzed by fluorescence microscopy as in panel (A). DIC, differential interference contrast. Scale bars, 5 μm.

Figure EV1
Figure EV1. Analysis of Atg4 association with the PAS by time‐lapse live‐cell imaging

Cells expressing Atg4‐GFP and mCherry‐V5‐Atg8 (RGY287) were starved in SD‐N medium for 30 min and incubated with CMAC for 10 min, before being examined by time‐lapse fluorescence microscopy. PAS were considered mCherry‐Atg8‐positive puncta adjacent to the vacuole, stained with CMAC. Images of the same cells were collected every 1 min for 15 min. For the complete movie, see the supplemental data (Movie EV1). Scale bar, 2 μm.

Experiments as in panel (A) were quantified by normalizing the autophagosome cycle, defined as the interval of time form the appearance until disappearance of mCherry‐Atg8 puncta 49, to 1 and integrating Atg4‐GFP recruitment to the PAS overtime. Data are from four independent experiments where the PAS remained in the imaged focal planes over the course of the entire filming.

Figure EV2
Figure EV2. Atg4 association with the PAS does not require components of the Atg1 complex, Atg9 cycling system and PI3K complex

Fluorescence microscopy images showing the subcellular localization of Atg4‐GFP in atg13∆ (SAY030), atg2∆ (SAY109), atg9∆ (SAY016), atg18∆ (SAY017), atg6∆ (SAY013), and atg14∆ (SAY110) strains analyzed as in Fig 1. White arrows highlight Atg4‐GFP puncta. DIC, differential interference contrast. Scale bars, 5 μm.

Percentage of cells, in the experiments shown in panel (A), that display an Atg4‐GFP punctate structure. Data represent the average of three independent experiments ± SD.

Subcellular localization of Atg4‐GFP in double knockout cells lacking Atg1 and components of the conjugation systems leading to the formation of Atg8‐PE: atg7∆ (SAY032), atg3∆ (SAY031), atg8∆ (SAY033), atg10∆ (SAY035), atg12∆ (SAY134), and atg16∆ (SAY135). Cells were grown and imaged as in Fig 1. DIC, differential interference contrast. Scale bars, 5 μm.

Figure 2
Figure 2. Atg4 LIR motif at amino acid position F102 to I105 is essential for autophagy

Atg4‐TAP atg8∆ (yMS69) or atg8∆ (yCK765) strains carrying an empty plasmid (pRS416) or one expressing GFP‐Atg8 (pCK15) were grown to a log phase and exposed to 220 nM rapamycin for 1 h before preparing cell extracts. Atg4‐TAP was subsequently immunoprecipitated using IgG magnetic beads. Finally, immunoprecipitates were analyzed by Western blot for GFP and protein A.

The atg4Δ (SAY084) or the atg4Δ (JAY151) strains carrying the integrative GFP‐ATG8ΔR plasmid were transformed with the centromeric plasmids expressing either Atg4‐13xmyc, Atg4pLIR1‐13xmyc, Atg4pLIR2‐13xmyc, Atg4pLIR3‐13xmyc, or Atg4pLIR4‐13xmyc. The strains were exponentially grown before being nitrogen starved in SD‐N medium for 1 h. Cell lysates were then subjected to pull‐down experiments using GFP‐trap agarose beads. Isolated proteins, 1% of cell lysate (input) or 50% of the pull‐down material (IP: GFP), were resolved by SDS–PAGE and analyzed by Western blot using either anti‐myc or anti‐GFP antibodies. A schematic view of the distribution of the putative LIR motif (blue), that is, LIR1 (L1), LIR2 (L2), LIR3 (L3), and LIR4 (L4), and the catalytic site (red) over within Atg4 is presented on the bottom of the panel.

Quantification of the experiments shown in panel (B). Values are relative to WT Atg4 and represent the average of three independent experiments ± SD. Significant differences (P < 0.05) between cells expressing WT Atg4 were calculated using the paired two‐tailed Student's t‐test, and they are indicated with the # symbol.

The atg4Δ (SAY084) mutant was transformed with integrative vectors expressing 13xmyc‐tagged Atg4 (SAY173) or its mutant versions (Atg4PD, SAY174; Atg4pLIR1, SAY175; Atg4pLIR2, SAY176; Atg4pLIR3, SAY177; and Atg4pLIR4, SAY178). The resulting strains were grown to a log phase in SMD medium before being nitrogen starved in SD‐N medium for 3 h. Proteins were precipitated with 10% trichloroacetic acid (TCA) and analyzed by Western blot using the anti‐myc, anti‐Ape1, and anti‐Pgk1 antibodies (loading control).

The percentages of prApe1 and mApe1 in the experiment shown in panel (D) were quantified, and values were plotted. Data represent the average of five independent experiments ± SD.

The experiment described in panel (D) was repeated with the SAY130 strain (Pho8∆60 pho13∆ atg4∆) carrying an empty pRS416 vector (atg4∆) or plasmids expressing Atg4, Atg4PD, Atg4pLIR1, Atg4pLIR2, Atg4pLIR3, and Atg4pLIR4. Pho8∆60 activity was subsequently measured before (SMD) or after (SD‐N) the nitrogen starvation and expressed in arbitrary units (a.u.). Data represent the average of three independent experiments ± SD. Significant differences (P < 0.05) between cells expressing WT Atg4 were calculated using the paired two‐tailed Student's t‐test, and they are indicated with the # symbol.

Figure EV3
Figure EV3. Putative LIR (pLIR) motifs in S. cerevisiae Atg4 and their conservation among eukaryotes

Saccharomyces cerevisiae Atg4 amino acid sequence. The catalytic site (C147, D322, and H324) is highlighted in red, while the putative LIR motifs are indicated in blue.

The amino acid sequence of the regions flanking the two conserved pLIR motifs of S. cerevisiae Atg4 (in blue), that is, F102 to I105 (pLIR2) and Y424 to I427 (pLIR4), were aligned with that of homologous proteins from different species using the Kalign alignment tool (http://www.ebi.ac.uk/Tools/msa/kalign/). UniprotKB accession numbers are C. albicans Atg4 (Q59UG3), A. nidulans Atg4 (Q5B7L0), S. cerevisiae Atg4 (P53867), K. lactis Atg4 (Q6CQ60), C. elegans Atg4.1 (Q9NA30), C. elegans Atg4.2 (Q9U1N6), D. melanogaster Atg4 (M9PBM3), D. rerio Atg4B (Q6DG88), M. musculus ATG4A (Q8C9S8), M. musculus ATG4B (Q8BGE6), M. musculus ATG4C (Q811C2), M. musculus ATG4D (Q8BGV9), H. sapiens ATG4A (Q8WYN0), H. sapiens ATG4B (Q9Y4P1), H. sapiens ATG4C (Q96DT6), and H. sapiens ATG4D (Q86TL0). The asterisk indicates conservation of the residue while two dots designate similarity.

Figure EV4
Figure EV4. Atg4 LIR2 motif is essential for the Cvt pathway

The strains described in Fig 2C were grown to an exponential log phase before proteins were precipitated with TCA and analyzed by Western blotting with anti‐myc, anti‐Ape1, and anti‐Pgk1 antibodies.

The percentage of prApe1 and mApe1 in the experiment shown in panel (A) were quantified, and values were plotted. Data represent the average of five independent experiments ± SD.

Figure 3
Figure 3. Atg4 pLIR2 motif is essential for Atg8‐PE deconjugation

The atg4Δ Atg8‐GFP mutant (SAY113) was transformed with integration vectors expressing 13xmyc‐tagged Atg4 (SAY173) or its mutant variants (Atg4PD, SAY174; Atg4pLIR1, SAY175; Atg4pLIR2, SAY176; Atg4pLIR3, SAY177; and Atg4pLIR4, SAY178). Proteins were TCA‐precipitated and analyzed by Western blot using the anti‐GFP antibody.

The percentages of Atg8‐GFP processed in the experiment shown in panel (A) were quantified and values were plotted. Data represent the average of three independent experiments ± SD.

The atg4Δ strain carrying the integration plasmid pCuGFPAtg8ΔR(305) (JSY151) and an empty vector (atg4Δ) or plasmids expressing 13xmyc‐tagged Atg4 variants (Atg4, Atg4PD, Atg4pLIR1, Atg4pLIR2, Atg4pLIR3, and Atg4pLIR4) were grown in SMD, labeled with the vacuole‐specific dye CMAC, nitrogen starved in SD‐N for 3 h, and imaged. DIC, differential interference contrast. Scale bars, 5 μm.

Quantification of GFP‐Atg8 distribution in cells imaged in panel (C): vacuole lumen, vacuole rim, or both localizations (lumen + rim). Data represent the average of three independent experiments ± SD.

The atg4∆ (SAY084, top) and atg4∆ atg8∆ ATG8∆R (RHY012, bottom) mutants were transformed with an empty vector or plasmids expressing the 13xmyc‐tagged Atg4 variants (Atg4, Atg4PD, Atg4pLIR1, Atg4pLIR2, Atg4pLIR3, and Atg4pLIR4), and the resulting strains were grown to a log phase in SMD medium before being nitrogen starved in SD‐N medium for 3 h. Proteins were precipitated with TCA and analyzed by Western blot using the anti‐Atg8 and anti‐Pgk1 antibodies.

Figure 4
Figure 4. Atg4p LIR 2 mutant recruitment to the PAS is reduced

The atg1∆ cells expressing integrated RFP‐Ape1 and Atg4‐GFP (SAY136) Atg4PD‐GFP (SAY137), Atg4pLIR1‐GFP (RHY016), Atg4pLIR2‐GFP (SAY139), Atg4pLIR3‐GFP (RHY017), or Atg4pLIR4‐GFP (RHY018) were grown in YPD to an early log phase and then starved for 3 h in SD‐N medium before imaging. Scale bars, 5 μm.

Percentage of cells in which Atg4‐GFP is observed in a punctate structure in the experiments depicted in panel (A). Data represent the average of three independent experiments ± SD. Significant differences (P < 0.05) between the Atg4 mutants and the WT were calculated using the paired two‐tailed Student's t‐test, and they are indicated with the symbol #.

Percentage of Atg4 puncta co‐localizing with the PAS marker proteins Ape1 in panel (A). Data represent the average of three independent experiments ± SD.

Figure 5
Figure 5. The pLIR2 sequence is new motif involved in the specific recognition of Atg8‐PE

The atg4Δ pep4Δ strain transformed with integration plasmids expressing Atg4 (SAY144), Atg4PD (SAY145), Atg4pLIR1 (SAY146), Atg4pLIR2 (SAY147), Atg4pLIR3 (RHY009), and Atg4pLIR4 (RHY010) or an empty plasmid (JSY163) was grown and starved as in Fig 1D before being processed for EM. Autophagic bodies (AB) are highlighted in the EM micrographs with asterisks. CW, cell wall; LD, lipid droplet; M, mitochondria; N, nucleus; PM, plasma membrane; V, vacuole. Scale bars, 1 μm.

Quantification of the autophagic bodies. Average number of autophagic bodies (AB) per 50 vacuole sections ± SD. Significant differences (P < 0.05) between the Atg4 mutants and the WT were calculated using the paired two‐tailed Student's t‐test, and they are indicated with the symbol #.

Quantification of the diameter of the AB. Average diameter of the AB in 50 vacuole sections ± SD. Significant differences (P < 0.05) between the various Atg4 mutants and the WT were calculated using the paired two‐tailed Student's t‐test, and they are indicated with the symbol #.

The atg4Δ atg3Δ (FKY428) or the atg4Δ atg3Δ (FKY437) strains carrying the integrative GFP‐ATG8ΔR plasmid were transformed with the centromeric plasmids expressing either Atg4‐13xmyc, Atg4pLIR1‐13xmyc, Atg4pLIR2‐13xmyc, Atg4pLIR3‐13xmyc, or and Atg4pLIR4‐13xmyc. Strains were processed for pull‐down experiments as in Fig 2B. Quantification is shown in Fig 6A.

Atg4, Atg4pLIR2, and Atg4pLIR4 were translated and radiolabeled in vitro as described in the Materials and Methods section before pull‐down (PD) with GST or GST‐Atg8 immobilized on glutathione‐beads. Beads were successively washed, and the eluted material was resolved by SDS–PAGE. Atg4 was visualized by autoradiographs, while GST and GST‐Atg8 amounts were assessed by Coomassie brilliant blue staining of the SDS–PAGE gel. The graph is the quantification of three independent experiments ± SD, where the binding of Atg4 is set as 100%.

Figure 6
Figure 6. The double Atg4APEAR , cLIR mutant leads to enhanced in vivo defects compared to the single APEAR and cLIR mutants

Quantification of the experiments shown in Fig 5D. Values are relative to WT Atg4 and represent the average of three independent experiments ± SD. Significant differences (P < 0.05) between cells expressing WT Atg4 were calculated using the paired two‐tailed Student's t‐test, and they are indicated with the # symbol.

The atg4Δ (JSY151) strains carrying the integrative GFP‐ATG8ΔR plasmid were transformed with the centromeric plasmids expressing either Atg4‐13xmyc, Atg4APEAR‐13xmyc, Atg4cLIR‐13xmyc, or Atg4APEAR,cLIR‐13xmyc. Pull‐down experiments were carried out as in Fig 2B. 2% of cell lysate (input) or 50% of the pull‐down material (IP: GFP) were resolved by SDS–PAGE and analyzed by Western blot using either anti‐myc or anti‐GFP antibodies.

The atg4Δ (SAY084) mutant was transformed with plasmids expressing 13xmyc‐tagged Atg4, Atg4PD, and Atg4APEAR,cLIR before being grown to a log phase in SMD medium. Proteins were precipitated with TCA and subsequently analyzed by Western blot using the anti‐Ape1 and anti‐Pgk1 antibodies (loading control).

The SAY130 strain (Pho8∆60 pho13∆ atg4∆) carrying plasmids expressing Atg4, Atg4PD, Atg4APEAR, Atg4cLIR, and Atg4APEAR,cLIR was analyzed as in Fig 2E. Data represent the average of three independent experiments ± SD. Significant differences (P < 0.05) between cells expressing WT Atg4 were calculated using the paired two‐tailed Student's t‐test, and they are indicated with the # symbol.

Figure 7
Figure 7. Atg4APEAR , cLIR displays similar Atg8‐PE deconjugation defect as Atg4APEAR

The atg4Δ Atg8‐GFP mutant (SAY113) was transformed with an empty vector or plasmids expressing 13xmyc‐tagged Atg4, Atg4LIR2, Atg4LIR4, and Atg4APEAR,cLIR. Proteins from exponentially growing cells were TCA‐precipitated and analyzed by Western blot using the anti‐GFP antibody.

The atg4Δ strain carrying the integration plasmid pCuGFPAtg8ΔR(305) (JSY151) and a plasmid expressing 13xmyc‐tagged Atg4, Atg4PD, or Atg4APEAR,cLIR was processed and analyzed as in Fig 3C. DIC, differential interference contrast. Scale bars, 5 μm.

Quantification of GFP‐Atg8 distribution in cells imaged in (B): vacuole lumen, vacuole rim, or both localizations (lumen + rim). Error bars represent the SD of three independent experiments.

The atg4∆ (SAY084) and atg4∆ ATG8∆R (RHY012) mutants were transformed with plasmids expressing 13xmyc‐tagged Atg4, Atg4PD, and Atg4APEAR,cLIR, and the resulting strains were processed as in Fig 3E.

Recombinant GST‐tagged Atg4, Atg4APEAR, Atg4cLIR, and Atg4APEAR,cLIR were added to Atg8‐PE‐containing liposomes, and their deconjugation activity was assessed over time as described in the Materials and Methods section.

Figure 8
Figure 8. Models for the structure of yeast Atg4 catalytic site and for Atg4 deconjugation activity on autophagosomal membranes

Three‐dimensional model predicting yeast Atg4 structure generated using the RaptorX online program (http://raptorx.uchicago.edu/StructurePrediction/predict/) 55, which covered Atg4 sequence from aa 1–403 leaving C‐terminal region with 91 aa unpredicted. The catalytic site (C147) of Atg4 is highlighted in red while APEAR is colored in purple. The last eight C‐terminal amino acids of LC3B, in white, were positioned into the catalytic site as shown for ATG4B 37.

After proteolytic priming, the C‐terminal glycine of Atg8 gets activated via the ubiquitin‐like conjugation system and linked through an amide bond to the PE present on autophagosomal membranes. Atg8‐PE is involved in autophagosome biogenesis at the PAS (dotted arrow). At this location, Atg8‐PE also associates with various proteins (gray clouds), such as cargo receptors, by binding their LIR motifs through a defined structural pocket (yellow). Atg4 binding to Atg8‐PE has to be controlled to avoid premature deconjugation. Occupation of the LIR motif‐binding pocket by other factors and/or other regulatory mechanisms such as post‐translational modifications (depicted with a question mark) could inhibit Atg4 action on autophagosomal membranes. As soon as the autophagosome is completed, the Atg machinery, including the shielding factors, is released allowing Atg4 access to autophagosomal membranes. This latter event involves both Atg4 binding to Atg8 via cLIR (blue circle) and association of the APEAR motif (purple circle) possibly with the C‐terminal region of Atg8‐PE, which allows the correct positioning of the C‐terminus into the catalytic site (red triangle) of Atg4.

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