1. Volume 64, Issue 2, Pages 221-235 (October 2016)
Two Independent Pathways within Selective Autophagy Converge to Activate Atg1 Kinase at the Vacuole 
Raffaela Torggler, Daniel Papinski, Thorsten Brach, Levent Bas, Martina Schuschnig, Thaddäus Pfaffenwimmer, Sabrina Rohringer, Tamara Matzhold, David Schweida, Andrea Brezovich, Claudine Kraft 
Molecular Cell 
Volume 64, Issue 2, Pages (October 2016)
DOI: /j.molcel
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2. Molecular Cell 2016 64, 221-235DOI: (10.1016/j.molcel.2016.09.008)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1 Bypass of Atg19 Cargo Receptor Function in the Cvt Pathway
(A) iPass. Wild-type cells assemble a trimeric complex, A-B-C, where B bridges the binding of A and C. If B is deleted, then A is unable to interact with C, and the function of the complex is lost. Induced tethering of A to C can restore function when the only non-redundant role of B is to link A to C.
(B) Schematic of the experimental iPass setup. Atg11 is inducibly tethered to cargo (Ape1) in cells where the Cvt cargo receptor Atg19 is deleted.
(C) The indicated strains containing FKBP-Ape1 were grown to mid-log phase and treated with rapamycin (rapa) to induce tethering where indicated. Trichloroacetic acid (TCA) extracts were prepared, and Ape1 processing was monitored by anti-Ape1 western blotting. See also Figure S7A.
(D) Mid-log phase cells containing BFP-Ape1 or FKBP-BFP-Ape1 were treated with FM4-64 and analyzed for Ape1 localization with or without tethering by rapamycin (rapa) treatment for 15 min. The mean fraction of perivacuolar BFP-Ape1 puncta from three independent experiments is shown. At least 25 Ape1 puncta for each strain were analyzed per experiment. Error bars, SD. Asterisks, statistically significant difference between samples is indicated by vertical or horizontal bars (∗p < 0.05, ∗∗p < 0.01). For exact p values, see Statistical Analysis. See also Figures S1B and S6A.
(E) Mid-log phase cells containing FKBP-BFP-Ape1 were imaged after treatment with rapamycin (rapa). Scale bar, 5 μm. See also Figures S1C and S1D.
(F) Mid-log phase cells containing FKBP-Ape1 were treated with FK506 (FK) where indicated. TCA extracts were analyzed as in (C). See also Figure S7B.
(G) TCA samples of mid-log phase cells were analyzed as in (C). See also Figure S7C.
(H) Cells were analyzed as in (G). See also Figure S7D.
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4. Figure 2 Bypass of Atg36 Cargo Receptor Function in Pexophagy
(A) Schematic of the experimental setup. Atg11 is inducibly tethered to Pex3 on peroxisomes (perox) in cells lacking the pexophagy cargo receptor Atg36.
(B) Strains were grown to mid-log phase followed by 4- to 6-hr starvation and FK506 treatment. TCA samples were analyzed for Pex11-GFP processing by anti-GFP western blotting.
(C) Strains were treated and analyzed as in (B).
(D) Pex3-mCherry-FKBP localization was analyzed during log phase growth or after 2.5 hr of nitrogen starvation with and without FK506 treatment. Arrows, vacuoles positive for Pex3-mCherry-FKBP. Scale bar, 5 μm.
(E) Cells as indicated were grown to mid-log phase, overexpression of pCUP1-Ape1 was induced by addition of 250 μM CuSO4 for 3 hr, and isolation membrane expansion was enhanced by treatment with 300 nM rapamycin for 1 hr. Scale bar, 2 μm. See also Figure S3D.
(F) Representative electron micrographs of the indicated cells labeled with anti-Ape1. Gold particles, 6 nm. Scale bar, 200 nm. See also Figure S2E.
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5. Figure 3 Atg1 Is Activated by Simple Clustering
(A) Atg1 was immunoprecipitated and analyzed by silver staining. The starvation response was triggered by rapamycin treatment for 1 hr (‘starv’). The indicated co-purifying bands were excised and analyzed by mass spectrometry. HC, IgG heavy chain; LC, IgG light chain.
(B) Atg1 was immunoprecipitated from mid-log phase cells, and kinase activity was measured by detecting Atg1 autophosphorylation (auto-p) and substrate phosphorylation (substrate-p, Atg19cterm; Pfaffenwimmer et al., 2014) in the presence of radioactive ATP. The amount of immunoprecipitated Atg1 was analyzed by anti-protein A (PAP) western blotting. See also Figure S7E.
(C) Atg1 kinase activity was assessed as in (B). Ape1 maturation was assessed in cell extracts by anti-Ape1 western blotting. See also Figure S7F.
(D) Tethering in log-phase cells was induced by rapamycin (rapa) addition. In wild-type cells, the starvation response (+‘starv’) was triggered by rapamycin. A kinase assay was performed as in (B). Atg13 and Ape1 were analyzed in cell extracts, and phosphorylated (pT737) and total Sch9 was analyzed in TCA samples by western blotting. See also Figure S7G.
(E) Cells were treated and analyzed as in (D). See also Figure S7H.
(F) Cells were treated and analyzed as in (D). See also Figure S7I.
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6. Figure 4
Tethering of Atg1 to the Vacuole Bypasses the Need for Atg13 in the Cvt Pathway
(A) Mid-log phase cells were stained with FM4-64. Formation and localization of Atg1-mNeonGreen and Atg11-GFP-FRB puncta were quantified over time after inducing tethering by rapamycin addition. Top: total and perivacuolar Atg1-mNeonGreen puncta per cell. Bottom: total and perivacuolar Atg11-GFP-FRB puncta per cell. Data were normalized to the 30-min time point. Lines represent fits with a logistic function. Half-maximum times of vacuolar and total puncta: Atg1, 14.2 and 13.3 min, respectively; Atg11, 12.7 and 8.6 min, respectively. A minimum of 35 cells (35–208) from at least three independent experiments was analyzed at each time-point. See also Figure S6B.
(B) Cells were grown and treated as in Figure 3D. Atg1 was isolated using GFP-Trap MA beads. Atg1 and co-precipitating Atg11 was assessed by anti-GFP and anti-Atg11 western blotting. See also Figure S7J.
(C) Atg1 was isolated and analyzed as in (B). See also Figure S7J.
(D) Strains were analyzed as in Figure 1F. See also Figures S6C and S7K.
(E) Strains were analyzed as in Figure 1C. See also Figures S6C and S7L.
(F) Illustration of the M-track assay.
(G) Protein A-H3-tagged Atg1 was co-expressed with Tco89-HKMT in wild-type and atg13Δ cells or as a control with myc-HKMT or an empty plasmid in wild-type cells. Extracts were prepared from mid-log phase cells, and protein A-H3 tagged proteins were isolated on IgG magnetic beads, followed by tobacco etch virus (TEV) protease cleavage as described in Brezovich et al., Methylation was assessed by western blotting with an anti-trimethylation-specific antibody, and the amount of cleaved protein A-H3 on beads with anti-protein A (PAP). See also Figure S7M.
(H) An M-track assay was performed with the indicated strains as described in (G). See also Figure S7M.
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7. Figure 5 Atg1 Is Activated at the Vacuole
(A) Domain structure of Atg13 and known interacting proteins. Asterisks, interactions enhanced upon (∗) or limited to (∗∗) starvation.
(B) Co-localization of the indicated GFP fusion proteins with BFP-Ape1 was analyzed in mid-log phase cells. The mean fraction of BFP-Ape1 puncta positive for GFP from three independent experiments is shown. At least 18 Ape1 puncta for each strain were analyzed per experiment. Error bars, SD. Asterisks, statistically significant difference between Atg1 and Atg1-13-C (∗∗∗p < 0.001). For exact p values, see Statistical Analysis. See also Figure S6D.
(C) Cells were analyzed as in (B). At least 41 Ape1 puncta for each strain were analyzed per experiment. Error bars, SD. Asterisks, statistically significant difference between Atg1 and Atg1-13FV-M-GFP in each strain (∗∗∗p < 0.001). For exact p values, see Statistical Analysis. See also Figure S6D.
(D) Mid-log phase cells were analyzed for Ape1 maturation. See also Figure S7N.
(E–G) Atg1 kinase activity was assessed as in Figure 3B. See also Figures S7O–S7Q.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
The Presence of Cargo Allows Atg1 Activation at the Vacuole but Is Not Required for Atg1 Localization to the Vacuole.
(A) M-track was performed as in Figure 4G. See also Figures S5C and S5D.
(B and C) Atg1 kinase activity was analyzed as in Figure 3B. See also Figures S7R (B) and S7S (C).
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7
Atg13 and Cargo Complexes Cooperate in the Spatial and Temporal Control of Atg1 Activation
In selective autophagy, Atg1 kinase is activated by the convergence of two independent pathways. Atg1 is recruited to the vacuole by Atg13, and cargo complexes are recruited independently to the vacuole by Atg11. Only at the vacuole can Atg1 bind to and cluster on the cargo to become active, resulting in tight control of kinase activation in both space and time.
Molecular Cell  , DOI: ( /j.molcel )
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