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.

Figure 1

Atg19 and Atg34 interaction with Atg8 (a-f) Atg8 was co-incubated with Atg19 or Atg34 (wild type and mutants) at concentrations of 40μM and 800μM, respectively and run on a size exclusion column. Aliquots of individual fractions were run on SDS-PAGE gels and stained by Coomassie. The white box indicates the N-terminal domain, the light grey box the coiled-coil domain, the dark grey box the Ams1-binding domain and the purple box indicates the C-terminal domain. (g-f) Atg8 was co-incubated with the indicated Atg19 domains at 40 μM and run on a size exclusion column. Individual elution profiles are shown above; Coomassie stained SDS-PAGE gels below. Columns: (a-f, i: S200 3.2/30; g, h: S75 3.2/30). M: size marker. All experiments shown have been done two times.

Figure 2

The C-terminus of Atg19 interacts in a cargo-dependent manner with Atg8 (a) Anti-Atg8 western blot of the input and pellet fractions of a pull down experiment using GST-Atg19 as bait and Atg8 as prey. The black vertical line indicates that unrelated samples were run on the same gel but in between the two samples on the left and the one on the right (see Supplementary Fig. 7). (b) Coomassie stained gels of pull down experiments using the GST-prApe1 propeptide as bait to pull down Atg19, the Atg19-Atg34 chimera and Atg34, respectively. The Atg19-Atg34 chimera contained residues 1-361 of Atg19 and residues 348-412 of Atg34. (c) Yeast-two-hybrid assay testing for the interaction of Atg11 with Atg19, the Atg19-Atg34 chimera and Atg34, respectively. (d) Anti-Atg8 western blot using the indicated GST fusion proteins as bait to pull down Atg8. The pull downs were conducted in the presence or absence of the prApe1 propeptide. See Supplementary Fig. 1 for input gel. All experiments shown have been done two times. Images of uncropped western blots and gels can be found in Supplementary Figure 7.

Figure 3

The C-terminus of Atg19 contains multiple Atg8 binding sites (a, b) Anti-Atg8 western blots using the indicated GST fusion proteins shown on the left as bait to pull down Atg8. See Supplementary Fig. 1 for input gels. (c) Alignment of the Atg19 C-terminus lacking the LIR motif with the region around the LIR motif of human p62 (NP_003891.1). The amino acids shown in magenta and green indicate the residues mutated in the experiments shown in (d-f). (d) Anti-Atg8 western blots with the indicated GST-fusion proteins as bait to pull down Atg8. Quantification of the amount of Atg8 pulled down by the indicated GST fusion protein. The graph is based on 3 independent experiments (N = 3), one of which is shown below the graph. Shown are the averages and standard deviations. (e) GFP-Trap pull down experiment using yeast cells with integrated GFP-Atg8 transformed with the indicated Myc-Atg19 constructs. (f) Pull down experiment using the GST-prApe1 propeptide to pull down Atg8 via the indicated Atg19 proteins. The graph is based on 3 experiments (N = 3), one of which is shown below the graph. Shown are the averages and s.d. See Supplementary Fig. 2 for input gel. (g) Representative profile of a size exclusion chromatography run using a 10/300 S200 column. Atg8 was run at 400μM and the Atg19 C-terminus at 40μM. For the complex run both proteins were pre-incubated at the concentrations mentioned. The average number of Atg8 molecules shifted into the complex peak per 1 Atg19 C-terminus was 4.14 (average of 3 experiments). The experiments shown in (a, b, d, f, g) have been conducted three times; the experiment shown in (e) has been conducted two times. Images of uncropped western blots and gels can be found in Supplementary Figure 7.

Figure 4

Reconstitution of Atg8-dependent Atg19 recruitment to the membrane (a) GUVs incubated with mCherry-Atg19. The membrane was marked by incorporation of an Oregon green labelled lipid. (b) GUV coated with GFP-Atg8 incubated with mCherry-Atg19. (c) Scheme showing the proteins and reaction resulting in GFP-Atg8 conjugation to the GUV shown in (b). (d) GUVs containing nickel lipids incubated with GFP-Atg8-6xHis and wild type mCherry-Atg19. (e) Experimental setup of the experiment shown in (d). (f) Quantification of membrane binding by the indicated mCherry-Atg19 proteins. The experimental setup is shown in (e). The graph is based on 3 experiments. Shown are the averages and s.d. of these 3 experiments (N = 3, based on 318 GUVs for Atg19, 171 GUVs for Atg19 W412A, 166 GUVs for Atg19 F376A, F379A, W412A, 188 GUVs for the eGFP control). Scale bars: 5μm. Abbreviations: 3: Atg3; 5: Atg5; 7: Atg7; 8: Atg8; 12: Atg12; 16: Atg16; 19: Atg19. Experiments shown in (a, b) have been conducted two times; the experiment in (d) three times.

Figure 5

Reconstitution of Atg8 and Atg19 dependent cargo recruitment to the membrane (a) prApe1 propeptide recruitment to Atg8 harbouring GUVs in the presence and absence of Atg19. The experimental setup is shown on the right. (b) Atg19 dependent envelopment of prApe1-coated beads with Atg8 positive membranes. The experimental setup is shown on the right. The membrane was labelled by incorporation of a rhodamine labelled lipid. (c) Quantification of the percentage of GUV-associated eGFP-Ape1 coated beads showing detectable membrane bending. (N = 3; total GUVs per condition = 40). The experimental setup is shown in (b). The density of Atg8-6xHis on the membrane was titrated by different amounts of Ni-lipids in the GUV membrane. The graph is based on at least 3 independent experiments. Shown are the averages and standard deviations. (d) Quantification of the percentage of GUV-associated eGFP-Ape1 coated beads showing detectable membrane bending. The quantification is based on 3 independent experiments for the mutant Atg19 versions and 16 independent experiments for wild type Atg19 and the no Atg19 control (Atg19: N = 16, total GUVs = 382; Atg19 W412A: N = 3, total GUVs = 55; Atg19 F376A, F379A: N = 3, total 66 GUVs; Atg19 F376A, F379A, W412A: N = 3, total GUVs = 84; Atg19 F376A, F379A, P385A, E386E, W412A: N = 3, total GUVs = 60; Atg19-34: N = 4, total GUVs = 110; no Atg19: N = 16, total GUVs = 382). Shown are the averages and standard deviations. The experimental setup is as shown in (b) the p values in (c) and (d) were calculated using a 2-tailed Student’s T-test. (e) Visualization of Atg19 during the envelopment of prApe1-coated beads with Atg8 containing membranes. Note that Atg19 directly binds the prApe1-coated beads (arrows). The experimental setup is shown on the right. Scale bars: 5μM. Abbreviations: 8: Atg8; 19: Atg19; DIC: Differential interference contrast. All experiments have been conducted at least three times.

Figure 6

Requirements for the Atg19-Atg8 interaction for cargo delivery during selective and bulk autophagy. (a) Anti-Ape1 western blot of atg19Δ cells transformed with the indicated expression constructs. The lower Ape1 band indicates prApe1 processing and thus its delivery into the vacuole. (b) Representative electron micrographs of ypt7Δ, atg19Δ yeast cells grown under Cvt conditions expressing the indicated proteins labelled with an anti-Myc antibody. The white arrowheads indicate the isolation membrane. The dashed line indicates the circumference of the prApe1 oligomer. Gold particles: 10nm. Scale bars: 200nm. See Supplementary Fig. 5 for full images. (c) Quantification of electron micrographs of yeast cells expressing either Myc-Atg19 or Myc-Atg19-Atg34 co-labelled with anti-Myc (10nm gold) and anti-prApe1 (5nm gold). See also Supplementary Fig. 5. (3 independent experiments, Myc-Atg19: N = 48, Myc-Atg19-34: N = 24). (d) Anti-Ape1 western blot of atg19Δ cells transformed with the indicated expression constructs. The lower Ape1 band indicates prApe1 processing and thus its delivery into the vacuole. Supplementary Fig. 6 shows the expression of the Myc-tagged proteins and a quantification of the assay. (e) Yeast-two-hybrid assay testing for the interaction of Atg11 with Atg19 and the indicated Atg19 mutants. (f) Atg19Δ yeast cells expressing the indicated Atg19 proteins and prApe1-RFP were labelled with the vacuolar membrane dye MDY-64. Scale bars: 2μM. The experiments in (a-d, f) have been conducted 3 times; the experiment in (e) two times. Images of uncropped western blots and gels can be found in Supplementary Figure 7.

Figure 7

Model for the differential actions of cargo receptors during selective and bulk autophagy. In order to promote exclusion of non-cargo material cargo receptors interact with Atg8 via multiple sites to wrap the membrane around the cargo material.