P52A/R67A. The plasmids expressing AD-fused Atg1 variants were constructed as follows: DNA

Transcription

1 SUPPLEMENTAL DATA The Autophagy-Related Protein Kinase Atg1 Interacts with the Ubiquitin-Like Protein Atg8 via the Atg8 Family Interacting Motif to Facilitate Autophagosome Formation Hitoshi Nakatogawa, Shiran Ohbayashi, Machiko Sakoh-Nakatogawa, Soichiro Kakuta, Sho W. Suzuki, Hiromi Kirisako, Chika Kondo-Kakuta, Nobuo N. Noda, Hayashi Yamamoto, and Yoshinori Ohsumi SUPPLEMENTAL METHODS Yeast Strains The yeast strains used in this study are listed in Table S1. Gene disruption and tagging were carried out by a PCR-based method using pfa6a and pym plasmids (1) as templates and appropriate pairs of oligonucleotides as primers (Table S2) to construct most of the strains. To construct YNH261, 273, and 741, the parental strains were transformed with PCR products prepared using pbs-atg8-kan, pbs-atg8 P52A/R67A -kan, and pbs-3 FLAG ATG8-kan as templates, respectively, and Atg8-integ-Fw/Rv as primers. Atg1 was C-terminally-tagged with GFP in these strains using pym25 and ATG1-GFP-Fw/Rv for the preparation of PCR products, resulting in YNH695 and 696. To construct the strains chromosomally expressing the AIM mutant of Atg1 and the corresponding wild-type strain (YNH736 and 737), BY4741 (2) was transformed with PCR products prepared using pbs-atg1-kan and pbs-atg1 AIM mut -kan and ATG1-integ-Fw/Rv as primers. These strains were transformed with PCR products obtained using pym25 and ATG1-GFP-Fw/Rv to construct the strains expressing Atg1 GFP and Atg1 AIM mut GFP (YNH742 and 743). To construct the strains expressing Pho8Δ60 (YNH621, 738, and 739), DNA fragments amplified by PCR using pym-n15 and GPDpro-pho8Δ60-Fw/Rv were introduced into the parental strains. The GFP tagging of the chromosomal ATG8 gene was performed in YNH736 and YNH737 to construct YNH744 and 745 as described previously (3). Plasmids The single-copy plasmids expressing Atg1 WT GFP, Atg1 AIM mut GFP, and Atg1 Y878A/R885A GFP were constructed as follows: DNA fragments containing the promoter and open reading frame of ATG1 were amplified by PCR using oligo DNA XhoI-ATG1pro-Fw/ATG1-BamHI-Rv (Table S2) as primers and yeast genomic DNA as a template, digested with XhoI and BamHI, and inserted into the same sites in the centromeric vector prs316 (4). This plasmid was sequentially ligated with DNA fragments encoding GFP and the ATG9 terminator, which were prepared by PCR using pegfp(gfpplus) ATG8-hphNT1 (3) and yeast genomic DNA as templates and BamHI-GFP-Fw/GFP-HindIII-Rv and 1

2 HindIII-ATG9ter-Fw/ATG9ter-SacI-Rv as primers, respectively, resulting in prs316-atg1 GFP. prs316-atg1 AIM mut GFP and prs316-atg1 Y878A/R885A GFP were constructed using the QuickChange site-directed mutagenesis kit (Stratagene) with prs316-atg1 GFP and ATG1-AIM mut-fw/rv and ATG1-Y878A/R885A-Fw/Rv, respectively. To construct pbs-atg1-kan, DNA fragments containing the TEF terminator and the TEF promoter followed by the open reading frame of the kan gene, which were prepared using pfa6a-kanmx4 as a common template and BamHI-TEFter-Fw/TEFter-NdeI-Rv and NdeI-TEFpro-Fw/kan-XbaI-Rv (Table S2) as primers, respectively, were sequentially ligated with the pbluescript II SK+ vector (Stratagene), resulting in pbs-t-p-kan. DNA fragments amplified by PCR using yeast genomic DNA and XhoI-ATG1-Fw/ATG1-BamHI-Rv were inserted into the XhoI and BamHI sites in pbs-t-p-kan, resulting in pbs-atg1-kan. This plasmid was subjected to the site-directed mutagenesis using ATG1-AIM mut-fw/rv to construct pbs-atg1 AIM mut -kan. pgbd-c1-atg8 was constructed by inserting DNA fragments encoding wild-type Atg8 (amino acid residues 2-117) amplified by PCR using yeast genomic DNA as a template and BamHI-ATG8-Fw/ATG8-PstI-Rv (Table S2) as primers into the BamHI and PstI sites in the pgbd-c1 vector (5). The P52A and R67A mutations were sequentially introduced into this plasmid using ATG8-P52A-Fw/Rv and ATG8-R67A-Fw/Rv, resulting in pgbd-c1-atg8 P52A/R67A. The plasmids expressing AD-fused Atg1 variants were constructed as follows: DNA fragments encoding the linker sequence (Gly-Ser) 5 followed by the sequences encoding full-length Atg1 (amino acid residues 2-897) containing the kinase-dead mutation D211A, the N-terminal (2-332), middle ( ), and C-terminal ( ) regions, and Atg1 D211A lacking these regions were prepared by PCR using appropriate DNA primers, digested with NdeI and BamHI, and inserted into the same sites into the pgadt7 vector (Clontech). pbs-atg8-kan and pbs-atg8 P52A/R67A -kan were constructed by inserting DNA fragments amplified by PCR using pgbd-c1-atg8 and pgbd-c1-atg8 P52A/R67A as templates, respectively, and EcoRI-ATG8-Fw/ATG8-BamHI-Rv (Table S2) as primers into the EcoRI and BamHI sites in pbs-t-p-kan. To construct pbs-3 FLAG ATG8-kan, oligo DNA SalI-3 FLAG-EcoRI-Fw and -Rv were annealed, treated with T4 polynucleotide kinase, and ligated with pbs-atg8-kan digested with SalI and EcoRI. Media Yeast cells without plasmids were cultured in SD+CA+ATU media (0.17% yeast nitrogen base without amino acids and ammonium sulfate, 0.5% ammonium sulfate, 0.5% casamino acid, 0.002% adenine sulfate, 0.002% tryptophan, 0.002% uracil, and 2% glucose), and cells harboring prs316-atg1 GFP plasmids were cultured in the same media without uracil. AH109 cells carrying both pgbd and pgad plasmids were grown in SC-LT media (0.17% yeast nitrogen base without amino acids and ammonium sulfate, 0.5% ammonium 2

3 sulfate, and 2% glucose, supplemented with 0.002% adenine sulfate, 0.002% uracil, and appropriate amino acids without leucine and tryptophan). To induce autophagy, yeast cells were treated with 0.2 μg/ml rapamycin in nutrient rich media or shifted to nitrogen-deprived media SD-N (0.17% yeast nitrogen base without amino acids and ammonium sulfate and 2% glucose). SUPPLEMENTAL REFERENCES 1. Janke, C., Magiera, M. M., Rathfelder, N., Taxis, C., Reber, S., Maekawa, H., Moreno-Borchart, A., Doenges, G., Schwob, E., Schiebel, E., and Knop, M. (2004) Yeast 21, Brachmann, C. B., Davies, A., Cost, G. J., Caputo, E., Li, J., Hieter, P., and Boeke, J. D. (1998) Yeast 14, Nakatogawa, H., Ishii, J., Asai, E., and Ohsumi, Y. (2012) Autophagy 8, Sikorski, R. S., and Hieter, P. (1989) Genetics 122, James, P., Halladay, J., and Craig, E. A. (1996) Genetics 144, Shintani, T., and Klionsky, D. J. (2004) J. Biol. Chem. 279,

4 TABLE S1. Yeast strains used in this study Name Genotype Reference or source BY4741 MATa his3-δ1 leu2-δ0 met15-δ0 ura3-δ0 (2) BJ3505 MATa pep4::his3 prb1-δ1.6r his3-δ200 ura3-52 GAL can1 Yeast Genetic Stock Center YNH512 BY4741, ATG1-EGFP-kanMX6 (3) YNH731 YNH512, atg14δ::natnt2 This study YNH732 YNH512, atg11δ::natnt2 This study YNH733 YNH512, atg19δ::natnt2 This study YNH734 YNH512, atg34δ-atg19δ::natnt2 This study YNH735 YNH512, atg32δ::natnt2 This study YNH261 BY4741, ATG8-kan This study YNH273 BY4741, ATG8 P52A/R67A -kan This study YNH695 YNH261, ATG1-yeGFP-hphNT1 This study YNH696 YNH273, ATG1-yeGFP-hphNT1 This study YNH736 BY4741, ATG1-kan This study YNH737 BY4741, ATG1 AIM mut -kan This study YNH738 YNH736, natnt2-gpdpro-pho8δ60 This study YNH739 YNH737, natnt2-gpdpro-pho8δ60 This study YNH621 BY4741, natnt2-gpdpro-pho8δ60 atg1δ::natnt2 This study YNH204 BY4741, atg1δ::natnt2 (3) YNH740 BJ3505, atg2δ::natnt2 atg1δ::hphnt1 This study YNH741 BJ3505, atg2δ::natnt2 atg1δ::hphnt1 3 FLAG-ATG8-kanMX4 This study YNH742 BY4741, ATG1-yeGFP-hphNT1 This study YNH743 BY4741, ATG1 AIM mut -yegfp-hphnt1 This study YNH744 YNH736, EGFP-ATG8-hphNT1 This study YNH745 YNH737, EGFP-ATG8-hphNT1 This study YNH746 YNH736, atg13δ::natnt2 This study YNH747 YNH737, atg13δ::natnt2 This study 4

5 TABLE S2. Oligo DNA used in this study Name Sequence atg14δ-fw 5'-tagaaggataacgagtagagaaaaagggaagtaaaagttaaaaactagaatcctagtatgaccgtacgctgcaggtcgac-3' atg14δ-rv 5'-ctgactacatgcaactttatacacacggcaggaaaaaaagtgcgcactctagcctaccacgatcgatgaattcgagctcg-3' atg11δ-fw 5'-attattttagtgtactgttgttgttcggaaagtacttcttttattttcttttatacatcatgcgtacgctgcaggtcgac-3' atg11δ-rv 5'-gttaaatagatacataattaaaatcttgtcatttgtgacaaacgtttagcactgttcaaacatcgatgaattcgagctcg-3' atg19δ-fw 5'-gaatcgaggtaattgcggcggcacttgcttcagtaacgcccaaaggagagttctggtaaatgcgtacgctgcaggtcgac-3' atg19δ-rv 5'-gtatgtgaaaaggtactcattgctgtataaaaatagagtttgacctagagttcttcccaagatcgatgaattcgagctcg-3' atg34δ-fw 5 -agttaaataagtactatagccaaagaaactggaagaatataaaaaagcatatcgatgaattcgagctc-3 atg32δ-fw 5'-gataagcaatattgaagtcctaatcacaaaagcaaaaaaaatctgccaggaacagtaaacatcgtacgctgcaggtcgac-3' atg32δ-rv 5'-aacagaagtgatagtaaaaaagtgagtaggaacgtgtatgtttgtgtatattggaaaaaggatcgatgaattcgagctcg-3' atg1δ-fw 5'-tttgaagctaccccatattttcaaatctcttttacaacaccagacgagaaattaagaaaatgcgtacgctgcaggtcgac-3' atg1δ-rv 5'-aagatacttgaaaatatagcaggtcatttgtacttaataagaaaaccatattatgcatcacatcgatgaattcgagctcg-3' atg2δ-fw 5'-gaaagcataaagattaaagcaaattaagaggaaccctttttttttttgatttcgatacaatgcgtacgctgcaggtcgac-3' atg2δ-rv 5'-aaaaaaaaaaaaagagataaaatatgaattgaatatatatcaaaaatgtctgcaaaaatttatcgatgaattcgagctcg-3' atg13δ-fw 5'-ccttccaggctcaagtcttgaaaagaaagcagaacatacagcccggttgaatagcatgagtccgtacgctgcaggtcgac-3' atg13δ-rv 5'-gattatttttctttagttgtgccctttaaaataaaactttaccatttttaaccttctttagatcgatgaattcgagctcg-3' ATG8-integ-Fw 5 -ataattgtaaagttgagaaaatcataataaaaataattactagagacatgaagtctacatttaagtctga-3 ATG8-integ-Rv 5 -atactggaacaatagatggctaatgagtccctataatttcgattttagatttagaaaaactcatcgagca-3 GPDpro-pho8Δ60-Fw 5 -ggctacataaacatttacatatcagcatacgggacattatttgaacgcgcattagcagccgtacgctgcaggtcgac-3 GPDpro-pho8Δ60-Rv 5 -ggtcccatcccatccgtcacgaagaatatgacattcttcttcttgtgtgatgcagacatcgatgaattctctgtcgtcc-3 ATG1-integ-Fw 5 -gaagctaccccatattttcaaatctcttttacaacaccagacgagaaattaagaaaatgggagacattaaaaataaagat-3 ATG1-integ-Rv 5 -cttgaaaatatagcaggtcatttgtacttaataagaaaaccatattatgcatcacttagaaaaactcatcgagcatcaaa-3 ATG1-GFP-Fw 5 -gatagtattgcaaacaggttgaaaatattgaggcagaagatgaaccaccaaaatggtggtgcagcaggaggatcg-3 ATG1-GFP-Rv 5 -cttgaaaatatagcaggtcatttgtacttaataagaaaaccatattatgcatcacttaatcgatgaattcgagctcg-3 BamHI-TEFter-Fw 5 -ggccggatcctcagtactgacaataaaaagattct-3 TEFter-NdeI-Rv 5 ccggcatatggagctcgttttcgacactggatggc-3 NdeI-TEFpro-Fw 5 -ccggcatatgccgggttaattaaggcgcgccagat-3 kan-xbai-rv 5 -ccggtctagattagaaaaactcatcgagcatcaaa-3 XhoI-ATG1-Fw 5 -ccggctcgagatgggagacattaaaaataaagatc-3 ATG1-BamHI-Rv 5 -ggccggatccttaattttggtggttcatcttctgc-3 XhoI-ATG1pro-Fw 5 -gggaaccctcgagaaataagatacattgccgctgc-3 BamHI-GFP-Fw 5 -ggccggatccggttctggttctggttctggttctggttctggttctggttctggttctggtgtgagcaaaggagaagaac-3 GFP-HindIII-Rv 5 -ccccaagcttttattaccccccgggagaacctttgtagagttcatccatgccgagtgtaatcccagcagc-3 HindIII-ATG9ter-Fw 5 -caagaagtctgacgtcggaagaggatcctaatgatagcccaagcttggcagactgtgtcgtgtacatc-3 ATG9ter-SacI-Rv 5 -ggccgagctcaaagagaaagaaaactacgtggcgg-3 ATG1-AIM mut-fw 5 -atagaagctttgaaagagaa-gctgtggtggct-gagaagaaatcggttgaagt-3 ATG1-AIM mut-rv 5 -acttcaaccgatttcttctc-agccaccacagc-ttctctttcaaagcttctat-3 ATG1-Y878A/R885A-Fw 5 -ggtggttcatcttctgcctcaatattttcaaggcgtttgcaatactatcaatggcc-3 ATG1-Y878A/R885A-Rv 5 -gacagaataatcataagaaaggccattgatagtattgcaaacgccttgaa-3 BamHI-ATG8-Fw 5'-aaaggatccaagtctacatttaagtctg-3 ATG8-PstI-Rv 5'-aaactgcagctacctgccaaatgtattttc-3 SalI-3 FLAG-EcoRI-Fw 5 -tcgacgactacaaagaccatgacggtgattataaagatcatgacatcgattacaaggatgacgatgacaagg-3 SalI-3 FLAG-EcoRI-Rv 5 -aattccttgtcatcgtcatccttgtaatcgatgtcatgatctttataatcaccgtcatggtctttgtagtcg-3 5

6 SUPPLEMENTAL FIGURES FIGURE S1. Atg1 is degraded in the vacuole. A, Wild-type cells expressing Atg1 GFP (YNH512) treated with rapamycin in the presence or absence of 1 mm PMSF for the indicated time periods were subjected to immunoblotting analysis using anti-gfp antibodies. B, Wild-type (YNH512) and atg14δ (YNH731) cells expressing Atg1 GFP were analyzed as described in Fig. 1B, except for the use of anti-atg1 antibodies instead of anti-gfp antibodies. FIGURE S2. Known autophagic adaptor and receptors are not important for the vacuolar transport of Atg1. The vacuolar transport of Atg1 GFP in wild-type (YNH512), atg11δ (YNH732), atg19δ (YNH733), atg34δ atg19δ (YNH734), and atg32δ (YNH735) cells was examined as described in Fig. 1B. 6

7 FIGURE S3. Analysis of interaction between Atg1 and Atg8 by the yeast two-hybrid assay. AH109 transformed with plasmids expressing the full-length (F), N, M, or C region of Atg1 fused with the AD; AD Atg1 lacking either the N, M, or C region (ΔN, ΔM, or ΔC, respectively); or an empty vector (vec) in combination with either a plasmid expressing BD-fused Atg8 or an empty vector were grown on SC-LTH+3AT (-His), SC-LTA (-Ade), and SC-LT (control) agar plates. FIGURE S4. Autophagic activity of ATG1 AIM mut cells. A, ATG1 WT AIM mut (YNH738), ATG1 (YNH739), and atg1δ (YNH621) cells were grown in SD+CA+ATU media, treated with rapamycin for the indicated time periods, and subjected to the ALP assay. The same experiments were repeated three times; data are represented as means with standard deviations. B and C, AIM mut The vacuolar transport of GFP Atg8 was examined to assess autophagic activity of ATG1 cells. ATG1 WT (YNH744) and ATG1 AIM mut (YNH745) cells were treated with rapamycin for the indicated time periods and analyzed by immunoblotting using anti-gfp antibodies (B). GFP denote the GFP fragments generated following the vacuolar transport and degradation of GFP Atg8 via autophagy (6). The same experiments were repeated three times, and the intensities of the bands of GFP Atg8 and GFP at the time point of 4 h were measured to estimate the efficiency of the production of GFP fragments (the intensities of GFP were divided by the sum of those of GFP Atg8 and GFP ); results are represented as means with standard deviations (C). 7

8 FIGURE S5. PAS localization of the Atg1 AIM mutant and that of Atg8 in the Atg1 mutant cells. Representative images obtained by fluorescence microscopy of ATG1 WT GFP (YNH742), ATG1 AIM mut GFP (YNH743), ATG1 WT GFP ATG8 (YNH744), and ATG1 AIM mut GFP ATG8 (YNH745) cells treated with rapamycin for 1 h are shown. Arrowheads indicate dots representing the PAS. 8

9 FIGURE S6. PAS localization of the Atg1 Y878A/R885A mutant coexpressed with wild-type Atg1. A, Wild-type cells (BY4741) expressing Atg1 WT GFP or Atg1 Y878A/R885A GFP from single-copy plasmids were treated with rapamycin for 1 h and observed under a fluorescence microscope. Arrowheads indicate dots representing the PAS. B, Using the images obtained in A, cells with GFP dots were counted; the percentages of total cell numbers are shown (the numbers of cells examined were 262 and 218, respectively). FIGURE S7. Putative AIMs in Arabidopsis Atg1 Homologs. The sequence alignment of Atg1 homologs from S. cerevisiae (ScAtg1) and Arabidopsis thaliana (AtAtg1a-c) is shown. Tyr429 and Val432 in ScAtg1, which were shown to be important for binding to Atg8, and the corresponding residues in AtAtg1s are highlighted in green. Acidic and basic residues are shown in red and blue, respectively. 9

10 SUPPLEMENTAL MOVIE LEGEND MOVIE S1. Accumulation of autophagic bodies in ATG1 AIM mut cells. The vacuolar protease-deficient strain BJ3505 lacking chromosomal ATG1 and expressing Atg1 WT GFP or Atg1 AIM mut GFP from a single-copy plasmid was incubated in SD-N media for 2 h to induce autophagy and observed under a light microscope. Arrowheads indicate visible vacuoles. In these strains, the autophagic body, an inner-membrane vesicle released into the vacuole upon fusion between the autophagosomal outer membrane and the vacuolar membrane, accumulates in the vacuole. The results show that ATG1 WT cells accumulate autophagic bodies more abundantly than ATG1 AIM mut cells; most vacuoles are filled with autophagic bodies, which are aggregated and do not move vigorously, in ATG1 WT cells. 10