SNARE Proteins Are Required for Macroautophagy Usha Nair, 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  Cell  Volume 146, Issue 2, Pages 290-302 (July 2011) DOI: 10.1016/j.cell.2011.06.022 Copyright © 2011 Elsevier Inc. Terms and Conditions

Cell 2011 146, 290-302DOI: (10.1016/j.cell.2011.06.022) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 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 Extended Experimental Procedures. (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. So that fusion could be followed, 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 Extended Experimental Procedures, 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. Cell 2011 146, 290-302DOI: (10.1016/j.cell.2011.06.022) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 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 hr. R, recovery; cells were returned to 24°C after the 34°C incubation for an additional 2 hr. (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 hr. The atg1Δ (TYY127) strain was used as a negative control. Error bars represent the standard deviation (SD) from three independent experiments. (C) The strains from (A) were examined by pulse-chase at the 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 hr starvation. Scale bars represent 500 nm. (E) Cultures of vam3ts (UNY162), atg1Δ (TYY164), sec9-4 and sso1Δ/2ts (H603) cells were preincubated at 34°C for 30 min, shifted to starvation conditions for 1 hr at the NPT, and analyzed for sensitivity to proteinase K (PK) with or without 0.2% Triton X-100 (TX) as described in the Extended Experimental Procedures. See also Figure S2. Cell 2011 146, 290-302DOI: (10.1016/j.cell.2011.06.022) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 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 the NPT for 0.5 hr. Incubation was continued for 0.5 hr 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 represent the 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 represent the SEM from three independent experiments; n = 150 for the wild-type and the mutant. (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 the NPT for 30 min, and incubated in nitrogen-starvation medium at the NPT for 1 hr. Cells were fixed and fluorescence microscopy was performed. (F) Quantification of colocalization. The error bars indicate the SEM; n = 256 for the wild-type and 310 for the mutant. Scale bars represent 2.5 μm. DIC, differential interference contrast. See also Figure S3. Cell 2011 146, 290-302DOI: (10.1016/j.cell.2011.06.022) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 4 Ultrastructural Analysis of the Atg9-Containing Membranes and of the prApe1 Oligomer Wild-type (WT, UNY151) (E) and sso1Δ/2ts (UNY141) (A–D and F–H) cells were grown to exponential phase at the PT (A and B) before being shifted to the NPT (C–H) in either rich (YPD) (A–C, F, and G) or nitrogen-starvation (SD-N) (D, E, and H) medium for 1.5 hr. Cells were processed for immuno-EM as described in the Extended Experimental Procedures before and after the temperature and the medium changes. Cryosections shown in (A)–(D) were immunolabeled for GFP to detect Atg9-GFP, while those presented in (E)–(H) were immunolabeled for prApe1. (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 and D) The Atg9-containing vesicles are more dispersed throughout the cytoplasm in the sso1Δ/2ts mutant shifted to the 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 the NPT in rich medium. (H) In the sso1Δ/2ts cells nitrogen starved at the 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 bars represent 200 nm. AB, autophagic body; CW, cell wall; ER, endoplasmic reticulum; PM, plasma membrane; V, vacuole. See also Figure S4. Cell 2011 146, 290-302DOI: (10.1016/j.cell.2011.06.022) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 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 hr. (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 hr 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. The scale bar represents 5 μm. See also Figure S5. Cell 2011 146, 290-302DOI: (10.1016/j.cell.2011.06.022) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 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 represent the 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 hr 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 the 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 represent the SEM from three independent experiments; n = 114 for the wild-type, and 121 for the mutant. (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 hr. Spheroplasts prepared from these cells were subjected to DSP crosslinking as described in the Extended Experimental Procedures. Cell lysates were prepared and subjected to affinity isolation with IgG sepharose. Scale bars represent 5 μm. See also Figure S6. Cell 2011 146, 290-302DOI: (10.1016/j.cell.2011.06.022) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 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 preincubated at 34°C for 30 min, shifted to starvation conditions for 1 hr at the NPT and examined for sensitivity to proteinase K (PK) with or without 0.2% Triton X-100 (TX) as described in the Extended Experimental Procedures. (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 to OD600 = 1.0 and shifted to SG-N for 1 hr. Spheroplasts prepared from these cells were subjected to DSP crosslinking as described in the Extended Experimental Procedures. Cell lysates were prepared and subjected to affinity isolation with IgG sepharose. The asterisk indicates a nonspecific band. See also Figure S7. Cell 2011 146, 290-302DOI: (10.1016/j.cell.2011.06.022) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure S1 Atg8/LC3–PE Is Not Sufficient to Drive Membrane Hemifusion under Physiological Conditions, Related to Figure 1 (A) Tethering measured by turbidity assay. Full spectrum analysis of reactions as in Figure 1A, run with or without all reactants reveals a coupling-dependent turbidity signal. The spectrophotometer was first blanked against a no Atg3 reaction sample. (B) 30% PE liposomes do not support Atg8-mediated fusion. At least five independent experiments are shown for each protein concentration and at least two independent preparations of liposomes and expression/purifications of protein were tested. Even the highest concentrations of Atg8 were insufficient to drive fusion of 30% liposomes. The inherent fusogenicity of unreacted negative controls in the presence of 55% PE was at least three-fold higher than the highest rates observed in any experiment with lower PE, regardless of Atg8 concentration. The highest rate of fusion observed is plotted for each experiment. Blue dots are full reaction conditions, and red dots are negative controls lacking Atg7. The highest rate rather than initial rate is plotted because each experiment typically displayed a 5 min delay in the onset of fusion. Rate is more meaningful than endpoint because the most robust experimental conditions reach a plateau in as little as 20 min. (C) To mimic the enzyme-dependent ubiquitin-like coupling of a PE to the C terminus of Atg8, we introduced a C-terminal cysteine residue in place of the normally lipid-associated C-terminal glycine to generate Atg8G116C. Liposomes were prepared with 5 mole % of a maleimide-associated lipid, which spontaneously reacts with the cysteine when Atg8G116C is introduced. In this way, coupling of Atg8 to the liposomes is essentially independent of molar PE concentration. Quenching of the reactive group with β-mercaptoethanol (β-Me) serves as a negative control. (D) Purified recombinant LC3 with a C-terminal cysteine (LC3-C) or glycine (LC3-WT) were conjugated in vitro in the presence of the indicated concentrations of PE and maleimide PE (malPE). (E) A dequenching fusion reaction was carried out with the indicated version of LC3 conjugated to liposomes containing various concentrations of PE. A sample with only labeled liposomes (labeled alone), which is not able to undergo dequenching, is a negative control. Error bars correspond to standard deviation (SD) from 3 independent assays. Cell 2011 146, 290-302DOI: (10.1016/j.cell.2011.06.022) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure S2 The Exocytic Q/t-SNAREs but Not v-SNARES Are Required for Autophagy, Related to Figure 2 (A) Complementation of autophagy activity at the NPT in the sso1Δ/2ts and sec9-4 strains. The empty vector with the CUP1 promoter (pCu416), or the plasmid pCuHASec9(416) or pCuSso1(416) was transformed together with pGFP-Atg8(414) into sso1Δ/2ts (H603) or sec9-4 (JGY243) cells. Transformants were cultured and examined by immunoblotting as described in Figure 2A. (B) Autophagic activity in the snc1Δ snc2Δ strain. Cell lysates from wild-type (WT; TN124), atg1Δ (UNY5) and snc1Δ snc2Δ (UNY130) strains were used to measure Pho8Δ60 activity. Cells were cultured in SMD medium until OD600 = 0.8 and then incubated in SD-N for 2 hr. Error bar, SD from three independent experiments. (C) Mutations in Sso1/2 affect the vesicle formation step. Cultures of pep4Δ (JGY248), atg1Δ (TYY164), and sso1Δ/2ts (H603) cells were pre-incubated at 34°C for 30 min and then subjected to a protease protection assay as described in the Supplemental Experimental Procedures. In the wild-type (pep4Δ) cells, prApe1 was protected from cleavage by proteinase K alone and was only digested in the presence of detergent. The protease protection pattern in the sso1Δ/2ts strain was similar to that seen in atg1Δ cells. T, total; S5, 5,000 x g supernatant fraction; P5, 5,000 x g pellet fraction; TX, Triton X-100; PK, proteinase K. The cytosolic marker protein Pgk1 was only detected in the total and supernatant fractions, indicating efficient spheroplast lysis. Cell 2011 146, 290-302DOI: (10.1016/j.cell.2011.06.022) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure S3 The Recruitment of Atg9-3xGFP to the PAS Is Defective in the sec9-4 Mutant, Related to Figure 3 (A) Wild-type (Atg9-3xGFP RFP-Ape1; UNY145) or sec9-4 (Atg9-3xGFP RFP-Ape1 sec9-4; UNY132) cells were grown in rich medium to mid-log phase, shifted to NPT for 0.5 hr to inactivate Sec9, and incubated in nitrogen-starvation medium for another 0.5 hr. The cells were fixed and subjected to fluorescence microscopy. Sixteen Z-section images were captured and projected to visualize all the puncta present throughout the cells. (A) Representative projected images. Arrowheads denote the position of RFP-Ape1 at the PAS, and double arrowheads indicate overlaps of Atg9-3xGFP and RFP-Ape1. (B) The quantification of the percentage of cells showing colocalization between Atg9-3xGFP and RFP-Ape1. Error bars, SEM from three independent experiments; n = 218 for the wild-type, and n = 156 for the mutant. DIC, differential interference contrast. Scale bar, 2.5 μm. (C–F) The colocalization between RFP-Ape1 and GFP-Atg11 (C and D) or GFP-Atg1 (E and F) is not affected in the sso1Δ/2ts mutant. Wild-type (UNY146) or sso1Δ/2ts (UNY147) cells were transformed with plasmids bearing GFP-Atg11 or GFP-Atg1. The cells were grown in rich medium to mid-log phase and shifted to NPT for 0.5 hr to inactivate Sso2, after which RFP-Ape1 and GFP-Atg11 localization was examined by fluorescence microscopy. For examining the localization of RFP-Ape1 and GFP-Atg1, cells were cultured as mentioned above; however, prior to visualization by fluorescence microscopy, incubation was continued for another 0.5 hr in nitrogen-starvation medium. (C and E) Representative images showing the colocalization between RFP-Ape1 and GFP-Atg11 or GFP-Atg1, respectively; and (D and F) the corresponding quantification of colocalization. The error bars represent standard error of the mean (SEM) from three independent experiments. For the graph shown in D, n = 146 for the wild-type, and n = 139 for the mutant; and for the graph shown in F, n = 124 for the wild-type and n = 158 for the mutant. For images shown in C and E, scale bar, 5 μm. Cell 2011 146, 290-302DOI: (10.1016/j.cell.2011.06.022) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure S4 The sso1Δ/2ts Mutant Is Defective in Atg8 Localization, Related to Figure 4 Wild-type (pep4Δ; JGY248) or sso1Δ/2ts pep4Δ (UNY142) cells were transformed with a plasmid expressing GFP-Atg8 (pCUP1-GFP-ATG8(416)). Cells were grown in SMD-Ura until mid-log phase at PT and shifted for 30 min to NPT in the same medium to inactivate sso2. In order to facilitate the formation of autophagosomes, the cells were washed in SD-N and incubation was continued for 1.5 hr at NPT. Cells were processed for immuno-electron microscopy, and labeled with anti-YFP followed by immunogold. (A and B) In agreement with previously published data (Kirisako et al., 1999), in wild-type cells we detected GFP-Atg8 in autophagic bodies (black arrow), and on phagophores (white arrow). (C and D) In sso1Δ/2ts pep4Δ vps4Δ cells autophagic bodies were never observed in the vacuole; white arrowheads mark dispersed GFP-Atg8, and the white arrow represents a phagophore membrane decorated with GFP-Atg8. Scale bar, 250 nm. Cell 2011 146, 290-302DOI: (10.1016/j.cell.2011.06.022) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure S5 Components of the Exocyst Complex, but Not Genes Necessary for Endosomal Function, Are Required for Autophagy, Related to Figure 5 (A) Wild-type (WT), sec3-2, sec8-9, sec15-1 or atg1Δ cells transformed with a plasmid expressing the endogenous ATG8 promoter-driven GFP-Atg8, were cultured in rich medium to mid-log phase at 24°C. The cultures were shifted to 37°C for 30 min to inactivate the sec mutations, and then starved at the same temperature for 2 hr. For recovery (R), the cells were shifted back to 24°C, and incubation was continued for another 2 hr. Samples were collected before (+) and after (−) starvation, or after recovery. TCA precipitated cell lysates were resolved by SDS-PAGE, and GFP-Atg8 processing was analyzed. Pgk1 was used as a loading control. (B) Wild-type (WT), rvs167Δ, end3Δ or atg1Δ cells in the BY4742 strain background were transformed with a plasmid expressing the endogenous ATG8 promoter-driven GFP-Atg8. The cells were grown in rich medium to mid-log phase, and then shifted to nitrogen-starvation medium for the indicated time points. Approximately 1 OD600 unit of cells was collected by centrifugation, TCA precipitated, washed with acetone and resolved by SDS-PAGE. GFP-Atg8 processing was analyzed by immunoblotting with anti-YFP antibody and anti-Pgk1 (as a loading control) antiserum. The positions of full-length GFP-Atg8 and free GFP are indicated. Cell 2011 146, 290-302DOI: (10.1016/j.cell.2011.06.022) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure S6 Tlg2 Cannot Drive In Vitro Proteoliposome Fusion with Sso1 and Sec9, Related to Figure 6 (A) A Tlg2-Sso1-Sec9 complex does not promote liposome fusion in vitro. A kinetic fusion graph representing the percent of maximum fluorescence during a two-hour time course. An in vitro fusion assay was carried out as described in Supplemental Experimental Procedures. (B) The same reaction was carried out as in (A) with the addition of 3.5 nmol of a peptide corresponding to the C terminus of Snc2 (Paumet et al., 2001). Similar results were obtained with Sso1-His6. Cell 2011 146, 290-302DOI: (10.1016/j.cell.2011.06.022) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure S7 Analysis of the N-Ethylmaleimide-Sensitive Fusion Protein (NSF) Sec18, and the R/v-SNAREs Sec22 and Ykt6 in the Cvt and Autophagy Pathways, Related to Figure 7 (A and B) The Cvt pathway is defective in sec18-1 and sec22-1 mutants. The sec18-1, sec22-1 and corresponding wild-type strains were examined by pulse-chase at the NPT (32°C and 37°C for sec18-1 and sec22-1, respectively) and immunoprecipitated with anti-Ape1 antiserum. (C) The anterograde movement of Atg9 is affected in the sec22-1 mutant. Representative projected images showing the transport of Atg9 after knocking out ATG1 in the wild-type (UNY171) or sec22-1 (UNY165) cells expressing Atg9-3xGFP and RFP-Ape1. DIC, differential interference contrast. Scale bar, 2.5 μm. (D and E) Representative images showing the colocalization between Atg9-3xGFP and RFP-Sec22 or RFP-YKT6, respectively, in strain W303-1B. Cell 2011 146, 290-302DOI: (10.1016/j.cell.2011.06.022) Copyright © 2011 Elsevier Inc. Terms and Conditions