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. 2011 Jul 22;146(2):290-302.
doi: 10.1016/j.cell.2011.06.022.

SNARE proteins are required for macroautophagy

Usha Nair  1 , Anjali Jotwani, Jiefei Geng, Noor Gammoh, Diana Richerson, Wei-Lien Yen, Janice Griffith, Shanta Nag, Ke Wang, Tyler Moss, Misuzu Baba, James A McNew, Xuejun Jiang, Fulvio Reggiori, Thomas J Melia, Daniel J Klionsky
Affiliations

SNARE proteins are required for macroautophagy

Usha Nair et al. Cell. .

Abstract

Macroautophagy mediates the degradation of long-lived proteins and organelles via the de novo formation of double-membrane autophagosomes that sequester cytoplasm and deliver it to the vacuole/lysosome; however, relatively little is known about autophagosome biogenesis. Atg8, a phosphatidylethanolamine-conjugated protein, was previously proposed to function in autophagosome membrane expansion, based on the observation that it mediates liposome tethering and hemifusion in vitro. We show here that with physiological concentrations of phosphatidylethanolamine, Atg8 does not act as a fusogen. Rather, we provide evidence for the involvement of exocytic Q/t-SNAREs in autophagosome formation, acting in the recruitment of key autophagy components to the site of autophagosome formation, and in regulating the organization of Atg9 into tubulovesicular clusters. Additionally, we found that the endosomal Q/t-SNARE Tlg2 and the R/v-SNAREs Sec22 and Ykt6 interact with Sso1-Sec9, and are required for normal Atg9 transport. Thus, multiple SNARE-mediated fusion events are likely to be involved in autophagosome biogenesis.

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Figures

Fig. 1
Fig. 1. Physiological Levels of Atg8–PE Are not Sufficient to Drive Membrane Hemifusion
(A) Atg8ΔR conjugation reactions (1 μM Atg7, 1 μM Atg3, 3 μM Atg8ΔR, 1 mM ATP, 30°C for 90 min) were run with liposomes (2.4 mM lipid) comprised of varying PE surface densities (indicated) as described in the Supplement. (B) Tethering is PE concentration dependent and closely parallels lipidation. Tethering (red) is measured as the absorbance at 450 nm of aggregating liposomes (as in Figure S1A; n = 7). Lipidation (blue) is the fraction of total Atg8 in lipidated form determined by densitometry of gels as in (A) (n = 5). (C) Lipid-mixing assays. To follow fusion, donor liposomes (carrying small amounts of NBD and rhodamine-conjugated lipids) were mixed 1:4 with acceptor liposomes (carrying no fluorophore-associated lipids). Fusion results in dilution of the fluorophores and a decrease in rhodamine-dependent quenching of NBD. The increase in NBD fluorescence is plotted as a percentage of the total NBD fluorescence achieved after detergent solubilization of the liposomes. Atg8 coupling reactions are run as in (A) but with 0.5 μM Atg7, 0.5 μM Atg3, 1 mM total lipid and the indicated concentrations of Atg8ΔR. Lower panels are negative controls missing Atg7. The liposomes have the indicated amounts of PE. Panels labeled “high [enzyme]” were run with 7.4 μM Atg7 and 5.6 μM Atg3. (D) Fusion remains highly PE-concentration dependent even when Atg8 lipidation is independent of Atg7/Atg3 activity. Atg8 with a C-terminal cysteine (Atg8G116C) was conjugated to maleimide-PE as described in the Supplement, so that conjugation was independent of PE concentration. Fusion reactions were initiated by the addition of varying concentrations of Atg8G116C as indicated. Lower panels: the maleimide reaction was blocked with 1 mM β-mercaptoethanol. See also Figure S1.
Fig. 2
Fig. 2. Both Autophagy and the Cvt Pathway Are Defective in sec9-4 and sso1Δ/2ts Mutants
(A) The sec9-4 and sso1Δ/2ts (H603) strains and the corresponding wild type (BY4742 and W303) were analyzed with the GFP-Atg8 processing assay at the indicated temperature by nitrogen starvation for 2 h. R, recovery; cells were returned to 24°C after the 34°C incubation for an additional 2 h. (B) The sec9-4 (JGY236), and sso1Δ/2ts (JGY247) strains and the corresponding wild-type (YTS158 and ZFY202) cells were analyzed for Pho8Δ60 activity before and after starvation for 2 h. The atg1Δ (TYY127) strain was used as a negative control. Error bar, SD from three independent experiments. (C) The strains from (A) were examined by pulse-chase at NPT and immunoprecipitated with anti-Ape1 antiserum. (D) Wild-type (pep4Δ vps4Δ; UNY148) or sso1Δ/2ts pep4Δ vps4Δ (UNY142) cells were analyzed by electron microscopy after 1.5 h starvation. Scale bar, 500 nm. (E) Cultures of vam3ts (UNY162), atg1Δ (TYY164), sec9-4 and sso1Δ/2ts (H603) cells were pre-incubated at 34°C for 30 min, shifted to starvation conditions for 1 h at the NPT and analyzed for sensitivity to proteinase K (PK) with or without 0.2% Triton X-100 (TX) as described in the Supplement. See also Figure S2.
Fig. 3
Fig. 3. Atg9 Anterograde Movement and Atg8 Localization Are Impaired in the sso1Δ/2ts Mutant
Wild-type (WT, UNY145) or sso1Δ/2ts (UNY138) cells expressing Atg9-3xGFP and RFP-Ape1 were grown in rich medium to mid-log phase and shifted to NPT for 0.5 h. Incubation was continued for 0.5 h in nitrogen-starvation medium. The cells were fixed, and examined by fluorescence microscopy. For each picture, sixteen Z-section images were captured and projected. Arrowheads mark the position of RFP-Ape1 at the PAS, and double arrowheads mark overlaps of Atg9-3xGFP and RFP-Ape1. (A) Representative projected images. (B) Quantification of colocalization. Error bars, standard error of the mean (SEM) from three independent experiments; n = 218 for the wild type, and 447 for the mutant. (C) Representative projected images showing the transport of Atg9 after knocking out ATG1 in wild-type (UNY149) or sso1Δ/2ts (UNY140) cells. (D) Quantification of colocalization. Error bars are SEM from three independent experiments; n = 150 for the wild type and the mutant. Scale bar, 2.5 μm. (E) WT (UNY146) or sso1Δ/2ts (UNY147) cells expressing RFP-Ape1 and transformed with a plasmid encoding GFP-Atg8, were grown in SMD-Ura medium, shifted to NPT for 30 min, and incubated in nitrogen-starvation medium at the NPT for 1 h. Cells were fixed and fluorescence microscopy was performed. (F) Quantification of colocalization. The error bars indicate the SEM; n = 256 for wild type, and 310 for the mutant. Scale bar, 2.5 μm. DIC, differential interference contrast. See also Figure S3.
Figure 4
Figure 4. Ultrastructural Analysis of the Atg9-Containing Membranes and of the prApe1 Oligomer
Wild-type (WT, UNY151, panel E) and sso1Δ/2ts (UNY141, panels A-D and F-H) cells were grown to exponential phase at PT (panels A and B) before being shifted to NPT (panels C-H) in either rich (YPD, panels A-C, F and G) or nitrogen starvation medium (SD-N, panels D, E and H) for 1.5 h. Cells were processed for immuno-EM as described in the Supplement before and after the temperature and the medium changes. Cryo-sections shown in panels A to D were immuno-labelled for GFP to detect Atg9-GFP, while those presented in panels E to H were immuno-labelled for Ape1. (A) A tubulovesicular cluster detected infrequently in the mutant strain. (B) Overview of a cluster of secretory vesicles that also contain Atg9-positive carriers. (C, D) The Atg9-containing vesicles are more dispersed throughout the cytoplasm in the sso1Δ/2ts mutant shifted to NPT. (E) Autophagic bodies accumulated in the vacuole of nitrogen-deprived wild-type cells, and some of them contain the electron-dense prApe1 oligomer. (F and G) The prApe1 oligomer is associated with vesicular structures in the sso1Δ/2ts mutant shifted to NPT in rich medium. (H) In the sso1Δ/2ts cells nitrogen starved at NPT, the prApe1 oligomer is associated with 1-3 large vesicles. The asterisks mark the circular electron-dense structures that correspond to prApe1 oligomers, often observed in proximity to Atg9-positive membranes. Scale bar, 200 nm. AB, autophagic body; CW, cell wall; ER, endoplasmic reticulum; PM, plasma membrane; V, vacuole. See also Figure S4.
Fig. 5
Fig. 5. The Overexpression of SNAREs Involved in Secretion Cannot Suppress the Autophagy Defect in the sso1Δ/2ts or sec9-4 Mutants
(A and C) sso1Δ/2ts (H603) or sec9-4 cells expressing GFP-Atg8 were transformed with a plasmid expressing the indicated protein. The cells were streaked on two plates; one was incubated at 24°C and the other at 34°C, and growth was monitored after 72 h. (B and D) The sso1Δ/2ts (H603) and sec9-4 strains above were cultured in rich medium at 24°C to mid-log phase. For each strain, half of the culture was shifted to the restrictive temperature (34°C) for 30 min, whereas the rest remained at permissive temperature (24°C). Cells were starved for 2 h at the same temperatures, and samples were collected before and after starvation. Autophagy activity was determined by examining GFP-Atg8 processing. (E) Representative image showing the colocalization between a cytosolic RFP-Sso1 punctum and Atg9-3xGFP. Wild-type (UNY108) cells were transformed with a plasmid expressing CUP1 promoter-driven RFP-Sso1. Overnight cultures of cells grown in SMD medium at 30°C were diluted to an OD600 = 0.2 in the same medium and grown until OD600 = 0.6. The cells were then observed by fluorescence microscopy. The arrowheads mark examples of overlapping puncta. DIC, differential interference contrast. Scale bar, 5 μm. See also Figure S5.
Fig. 6
Fig. 6. Tlg2 Determines the Magnitude of Autophagy, Affects Atg9 Anterograde Transport, and Interacts with Sso1 and Sec9
(A) Wild-type (WT; BY4742), tlg2Δ (KWY76) or atg1Δ (UNY5) cells were grown in rich medium to mid-log phase, then shifted to nitrogen-starvation medium for the indicated time points, and Pho8Δ60 activity was determined. The activity measured from wild-type cells was set to 100%, and the other values were normalized. Error bars are SD from three independent experiments. (B) Wild-type (JGY191) or tlg2Δ (UNY159) cells expressing Atg9-3xGFP and RFP-Ape1 were grown in rich medium to mid-log phase and shifted to nitrogen-starvation medium for 0.5 h after which cells were examined by fluorescence microscopy. Arrowheads indicate the position of RFP-Ape1 at the PAS, and double arrowheads show overlaps between Atg9-3xGFP and RFP-Ape1. (C) Quantification of colocalization. The error bars represent SEM from three independent experiments; n = 102 for the wild type, and 116 for the mutant. (D) Representative images showing the transport of Atg9 after knocking out ATG1 in wild-type (UNY171) or tlg2Δ (UNY170) cells. (E) The average percentage of cells showing more than one Atg9-3xGFP punctum. The error bars are SEM from three independent experiments; n = 114 for the wild type, and 121 for the mutant. Scale bar, 5 μm. (F) Cells expressing GST-Tlg2 or GST-Ufe1 under the control of the GAL1 promoter (UNY168 and UNY180, respectively) were transformed with the indicated plasmids; CUP1 promoter-driven PA-Sso1 and GFP-Sec9 in the case of the experimental strain, or CUP1 promoter-driven PA alone and empty vector for the control strain. Cells were grown in SMG medium to OD600 = 1.0 and shifted to SG-N for 1 h. Spheroplasts prepared from these cells were subjected to DSP cross linking as described in the Supplement. Cell lysates were prepared and subjected to affinity isolation with IgG sepharose. See also Figure S6.
Fig. 7
Fig. 7. Autophagy Is Impaired in the Absence of Sec1, Sec18, and the R/v-SNAREs Sec22 and Ykt6
(A, C and F) The indicated mutant and the corresponding wild-type (BY4742) strains were analyzed with the GFP-Atg8 processing assay in rich or nitrogen starvation conditions. R, recovery. (B, D and G) vam3ts (UNY162), atg1Δ (TYY164), sec18-1, sec22-1 and ykt6ts cells expressing GFP-Atg8 were pre-incubated at 34°C for 30 min, shifted to starvation conditions for 1 h at the NPT and examined for sensitivity to proteinase K (PK) with or without 0.2% Triton X-100 (TX) as described in the Supplement. (E and H) Cells expressing GST-Sec9 under the control of the GAL1 promoter (UNY172) were transformed with the indicated plasmids; CUP1 promoter-driven PA-Sec22, PA-Ykt6 or PA-Gos1, and GFP-Sso1, or CUP1 promoter-driven PA alone and GFP-Sso1. Cells were grown in SMG medium medium to OD600 = 1.0 and shifted to SG-N for 1 h. Spheroplasts prepared from these cells were subjected to DSP cross linking as described in the Supplement. Cell lysates were prepared and subjected to affinity isolation with IgG sepharose. The asterisk indicates a non-specific band. See also Figure S7.
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References

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