Cargo binding to Atg19 unmasks additional Atg8 binding sites to mediate membrane–cargo apposition during selective autophagy

Abstract

Autophagy protects cells from harmful substances such as protein aggregates, damaged mitochondria and intracellular pathogens, and has been implicated in a variety of diseases. Selectivity of autophagic processes is mediated by cargo receptors that link cargo to Atg8 family proteins on the developing autophagosomal membrane. To avoid collateral degradation during constitutive autophagic pathways, the autophagic machinery must not only select cargo but also exclude non-cargo material. Here we show that cargo directly activates the cargo receptor Atg19 by exposing multiple Atg8 binding sites. Furthermore, Atg19 mediates tight apposition of the cargo and Atg8-coated membranes in a fully reconstituted system. These properties are essential for the function of Atg19 during selective autophagy in vivo. Our results suggest that cargo receptors contribute to tight membrane bending of the isolation membrane around the cargo.

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Figure 1: Atg19 and Atg34 interaction with Atg8.
Figure 2: The C-terminus of Atg19 interacts in a cargo-dependent manner with Atg8.
Figure 3: The C-terminus of Atg19 contains multiple Atg8 binding sites.
Figure 4: Reconstitution of Atg8-dependent Atg19 recruitment to the membrane.
Figure 5: Reconstitution of Atg8- and Atg19-dependent cargo recruitment to the membrane.
Figure 6: Requirements for the Atg19–Atg8 interaction for cargo delivery during selective and bulk autophagy.
Figure 7: Model for the differential actions of cargo receptors during selective and bulk autophagy.

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Acknowledgements

We thank C. Kraft and T. Brach for providing reagents and G. Warren for critical comments on the manuscript. Funding by the University of Vienna is gratefully acknowledged. The research leading to these results has received funding from the European Research Council under the European Community’s Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement number 260304, from the FWF Austrian Science Fund (grant number P25546-B20) and by the EMBO Young Investigator Program to S.M. We also acknowledge funding by the VIPS Program of the Austrian Federal Ministry of Science and Research and the City of Vienna to J.S-M. and by the Austrian FFG to C.A.

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J.S-M., C.A, J.R., B.Z., I.I. and S.M. carried out experiments, J.S-M., C.A. and S.M. planned experiments and S.M. wrote the manuscript.

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Correspondence to Sascha Martens.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Interaction of Atg19 with Atg8 is weak but stimulated by cargo and depends on multiple interaction sites.

(a) Profile of an isothermal titration calorimetry experiment with Atg8 as ligand injected into the cell containing Atg19. The calculated KD is 35 μM. (b) Anti-Atg8 western blot of a pull down experiment with the indicated proteins. (c) Alignment of the C-termini of S. cerevisiae Atg19 and Atg34. The position of the Atg11 binding sites and the LIR motifs is indicated. (d) Coomassie stained gel showing the amounts of the indicated proteins used for the pull down experiment shown in Fig. 2d. (e) Coomassie stained gel showing the amounts of the indicated proteins used for the pull down experiment shown in Fig. 3a. (f) Coomassie stained gel showing the amounts of the indicated proteins used for the pull down experiment shown in Fig. 3b. (g) Anti-Atg8 western blot (upper part) using the indicated GST fusion proteins shown on the left as bait to pull down Atg8. The lower part shows the amount of Atg8 in the inputs on a Coomassie stained gel. Abbreviations: In: input, B: bound. The experiments shown in (a, b, g) have been conducted 2 times.

Supplementary Figure 2 The C-terminus of Atg19 interacts in a cargo-dependent manner with Atg8 via multiple sites.

(a, b) Anti-Atg8 western blots with the indicated GST fusion proteins as bait to pull down Atg8 are shown on the left. On the right the Coomassie-stained gels of the respective inputs are shown. (c) Coomassie stained gel showing the amounts of the indicated proteins used for the pull down experiment shown in Fig. 3f. Abbreviations: In: input, B: bound. The experiments shown in (a, b) have been conducted two times.

Supplementary Figure 3 Recruitment of Atg19 and Atg34 to Atg8 coated GUV and reconstitution of membrane bending during selective autophagy.

(a, b) GUVs containing 5% Nickel lipids were incubated with Atg8–His or GFP–His. Subsequently, different concentrations of mCherry–Atg19 or mCherry–Atg34 were added to the GUVs. For better visibility the mCherry signal is shown in pseudo-colour. Scale bars: 20 μm. (c) Atg19-dependent envelopment of prApe1-coated beads with Atg8 positive membranes Scale bars: 5M. The experimental setup for the experiment shown in (c) is shown in (d). Abbreviations: 3: Atg3; 5: Atg5; 7: Atg7; 8: Atg8; 12: Atg12; 16: Atg16; 19: Atg19. The experiments shown in (a, b) have been conducted two times; the experiment in (c) two times.

Supplementary Figure 4 Efficient pull down of Atg19 by GST–Atg8.

(a) Coomassie stained gel of a pull down experiment using GST–Atg8 as bait to pull down the indicated Atg19 proteins. (b) Coomassie stained gel of a pull down experiment using GST–Atg8 as bait to pull down the indicated Atg19 proteins. The quantification above the gel is based 3 independent experiments (N = 3), one of which is shown below the graph. Shown are the averages and standard deviations. The experiment shown in (a) has been conducted two times, the experiment in (b) three times.

Supplementary Figure 5 The Atg19–34 chimera localises to prApe1 oligomers.

(a, b) Representative electron micrographs of Ypt7, Atg19 yeast cells grown under Cvt conditions expressing the indicated proteins labelled with an anti-Myc primary antibody and a secondary anti-mouse antibody coupled to 10 nm gold particles. The boxes indicate the cropped regions shown in Fig. 6b. (c, d) Electron micrographs of Ypt7, Atg19 yeast cells grown under Cvt conditions expressing the indicated proteins co-labelled with a mouse anti-Myc antibody and a rabbit anti-prApe1 antiserum followed by incubation with secondary anti-mouse antibody coupled to 10 nm gold particles (black arrow) and anti-rabbit antibody coupled to 5 nm gold particles (white arrow). Scale bars: 200 nm. All experiments have conducted three times.

Supplementary Figure 6 Expression of Atg19 variants in yeast cells and function of Atg19 mutants during selective and bulk autophagy.

(a) Anti-Myc and anti-Pgk1 western blots of the same lysates shown in Fig. 6d. (b) Quantification of the percent of processed prApe1 compared to total prApe1. The graphs are based on three independent experiments (N = 3), one of which is shown in Fig. 6d. Shown are averages and standard deviations. (c) Anti-Ape1, anti-Myc and anti-Pgk1 western blots of atg17atg19 cells transformed with the indicated expression constructs. The lower mApe1 band indicates prApe1 processing and thus its delivery into the vacuole. The experiment in (a) has been conducted 2 times, the experiment in (c) two times.

Supplementary Figure 7 Uncropped gels and western blots. The red boxes indicate the cropped regions.

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Sawa-Makarska, J., Abert, C., Romanov, J. et al. Cargo binding to Atg19 unmasks additional Atg8 binding sites to mediate membrane–cargo apposition during selective autophagy. Nat Cell Biol 16, 425–433 (2014). https://doi.org/10.1038/ncb2935

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