Table 1.(A-E) Yeast cells transfected with HA-Bcl2-L-13 or empty vector were collected 72 h after induction of mitophagy in SD-G medium and subjected to western blotting for mCherry. Generation of free mCherry is indicated by an arrow-head. Yeast strains are atg32∆, atg3∆/atg32∆, atg4∆/atg32∆, atg5∆/atg32∆, atg6∆/atg32∆ or atg8∆/atg32∆ in (A), atg32∆, atg10∆/atg32∆, atg12∆/atg32∆, atg16∆/atg32∆, atg18∆/atg32∆ or atg19∆/atg32∆ in (B), atg32∆, atg20∆/atg32∆, atg21∆/atg32∆, atg23∆/atg32∆ or atg24∆/atg32∆ in (C), atg32∆, atg27∆/atg32∆, atg29∆/atg32∆, atg31∆/atg32∆ or atg33∆/atg32∆ in (D). Quantitative analysis for free mCherry in (A-D) is shown in (E). The value for atg32∆ cells transfected with HA-Bcl2-L-13 in each experiment was set equal to 1 (n = 3). *P < 0.05 versus all other groups. Bars in graphs indicate P < 0.05. Results are shown as mean ± SEM. (A-G) Yeast cells transfected with HA-Bcl2-L-13 or empty vector were collected 48 h after induction of mitophagy in SD-N medium and subjected to western blotting for mCherry. Generation of free mCherry is indicated by an arrow-head. Yeast strains are atg32∆, atg1∆, atg2∆, atg9∆ or atg14∆ in (A), atg32∆, atg3∆/atg32∆, atg4∆/atg32∆, atg5∆/atg32∆ or atg6∆/atg32∆ in (B), atg32∆, atg7∆ or atg13∆/atg32∆ in (C), atg32∆, atg8∆/atg32∆, atg10∆/atg32∆, atg12∆/atg32∆ or atg16∆/atg32∆ in (D), atg32∆, atg18∆/atg32∆, atg19∆/atg32∆, atg20∆/atg32∆, atg21∆/atg32∆ or atg23∆/atg32∆ in (E), atg32∆, atg24∆/atg32∆, atg27∆/atg32∆, atg29∆/atg32∆, atg31∆/atg32∆ or atg33∆/atg32∆ in (F). Quantitative analysis for free mCherry in (A-F) is shown in (G). The value from atg32∆ cells transfected with HA-Bcl2-L-13 in each experiment was set equal to 1 (n = 3). *P < 0.05 versus all other groups. Bars in graphs indicate P < 0.05. Results are shown as mean ± SEM. 

(E) Conceivable

Models of Atg32-mediated and Bcl2-L-13-mediated Mitophagy. Atg32-mediated mitophagy: When mitophagy is induced, Atg11 interacts with Atg32 in yeast. The Atg11-Atg32 complex recruits mitochondria to the PAS. Mitochondria are surrounded by the phagophore through an interaction between Atg32 and Atg8 via AIM. Bcl2-L-13-mediated mitophagy: Upon mitophagy induction, LC3B is recruited to Bcl2-L-13, while the assembly of the ULK1 complex and the interaction between ULK1 and LC3B through the LIR motif take place. How Bcl2-L-13 interacts with the ULK1 complex is unclear, but it is possible that they interact though LC3B multimerization. 
Table S2. 



  Figure S1. Results of Mitophagy Assay in Glycerol Medium, Related to Figure 1 and Table 1.(A-E) Yeast cells transfected with HA-Bcl2-L-13 or empty vector were collected 72 h after induction of mitophagy in SD-G medium and subjected to western blotting for mCherry. Generation of free mCherry is indicated by an arrow-head. Yeast strains are atg32∆, atg3∆/atg32∆, atg4∆/atg32∆, atg5∆/atg32∆, atg6∆/atg32∆ or atg8∆/atg32∆ in (A), atg32∆, atg10∆/atg32∆, atg12∆/atg32∆, atg16∆/atg32∆, atg18∆/atg32∆ or atg19∆/atg32∆ in (B), atg32∆, atg20∆/atg32∆, atg21∆/atg32∆, atg23∆/atg32∆ or atg24∆/atg32∆ in (C), atg32∆, atg27∆/atg32∆, atg29∆/atg32∆, atg31∆/atg32∆ or atg33∆/atg32∆ in (D). Quantitative analysis for free mCherry in (A-D) is shown in (E). The value for atg32∆ cells transfected with HA-Bcl2-L-13 in each experiment was set equal to 1 (n = 3). *P < 0.05 versus all other groups. Bars in graphs indicate P < 0.05. Results are shown as mean ± SEM. 




  Figure S2. Results of Mitophagy Assay in Nitrogen-Starvation Medium, Related to Figure 1.(A-G) Yeast cells transfected with HA-Bcl2-L-13 or empty vector were collected 48 h after induction of mitophagy in SD-N medium and subjected to western blotting for mCherry. Generation of free mCherry is indicated by an arrow-head. Yeast strains are atg32∆, atg1∆, atg2∆, atg9∆ or atg14∆ in (A), atg32∆, atg3∆/atg32∆, atg4∆/atg32∆, atg5∆/atg32∆ or atg6∆/atg32∆ in (B), atg32∆, atg7∆ or atg13∆/atg32∆ in (C), atg32∆, atg8∆/atg32∆, atg10∆/atg32∆, atg12∆/atg32∆ or atg16∆/atg32∆ in (D), atg32∆, atg18∆/atg32∆, atg19∆/atg32∆, atg20∆/atg32∆, atg21∆/atg32∆ or atg23∆/atg32∆ in (E), atg32∆, atg24∆/atg32∆, atg27∆/atg32∆, atg29∆/atg32∆, atg31∆/atg32∆ or atg33∆/atg32∆ in (F). Quantitative analysis for free mCherry in (A-F) is shown in (G). The value from atg32∆ cells transfected with HA-Bcl2-L-13 in each experiment was set equal to 1 (n = 3). *P < 0.05 versus all other groups. Bars in graphs indicate P < 0.05. Results are shown as mean ± SEM. 




  Figure S3. ULK1-LC3B (Atg1-Atg8) Interaction through the LIR (AIM) Motif Is Important for Bcl2-L-13-mediated Mitophagy, Related to Figure 2. (A) ATG1 WT/atg32∆ and atg1 AIM mutant/atg32∆ cells expressing GFP-Atg8 were grown to mid-log phase in rich dextrose medium, incubated for the indicated time points in starvation medium (SD-N), and subjected to western blotting. Generation of free GFP (indicated by an arrow-head) indicates transport of autophagosomes to the vacuole. (B) ATG1 WT/atg32∆ and atg1 AIM mutant KI/atg32∆ cells transfected with HA-Bcl2-L-13 or empty vector were collected 8 h after induction of mitophagy in SD-N medium and subjected to western blotting for mCherry. (C) Endogenous interaction between Bcl2-L-13 and ULK1. HEK292A cells were treated with 15 µM CCCP for 6 h. One hundred nM bafilomycin A1 was added 3 h before sampling. Then, cells were lysed and immunoprecipitated with anti-Bcl2-L-13 antibody. Co-precipitated ULK1 was detected by immunoblotting. ULK1 is indicated by an arrow-head. (D and E) Colocalization of autophagosomes and mitochondria in HEK293A cells. HEK293A cells were transfected with ULK1-specific siRNA for 72 h followed by transfection of HA-Bcl2-L-13 and FLAG tagged wild-type ULK1 or ULK1 mutants. Forty-two h after transfection, cells were treated with 100 nM bafilomycin A1 for 6 h and immunostained with anti-LC3B and anti-ATP synthase antibodies. Images in the box at higher magnification are shown in the right panels. White arrows indicate colocalization of LC3B and ATP synthase double positive dots. The number of LC3B dots colocalized with ATP synthase dots per cell is shown in (E). At least 20 cells were counted for each group (n = 3). Scale bar: 10 µm. *P < 0.05. Results are shown as mean ± SEM. 




  met150 ura30 TEFp-mito-DHFR-mcherry::CgHIS3 KOY1422 BY4741 his31 leu20 met150 ura30 TEFp-mito-DHFR mcherry::CgHIS3 atg32::kanMX6 KOY1424 BY4741 his31 leu20 met150 ura30 TEFp-mito-DHFR-mcherry::CgHIS3 atg7::kanMX6 KOY1569 BY4741 his31 leu20 met150 ura30 TEFp-mito-DHFR-mcherry::CgHIS3 atg1::kanMX6 KOY1539 BY4741 his31 leu20 met150 ura30 TEFp-mito-DHFR-mcherry::CgHIS3 atg2::kanMX6 KOY1545 BY4741 his31 leu20 met150 ura30 TEFp-mito-DHFR-mcherry::CgHIS3 atg9::kanMX6 KOY1547 BY4741 his31 leu20 met150 ura30 TEFp-mito-DHFR-mcherry::CgHIS3 atg14::kanMX6 YMT7 BY4741 his31 leu20 met150 ura30 TEFp-mito-DHFR-mcherry::CgHIS3 atg11::natNT2 atg32::kanMX6 YMT9 BY4741 his31 leu20 met150 ura30 TEFp-mito-DHFR-mcherry::CgHIS3 atg13::natNT2 atg32::kanMX6 YMT11 BY4741 his31 leu20 met150 ura30 TEFp-mito-DHFR-mcherry::CgHIS3 atg17::natNT2 atg32::kanMX6 YMT13 BY4741 his31 leu20 met150 ura30 TEFp-mito-DHFR-mcherry::CgHIS3 atg24::natNT2 atg32::kanMX6 YMT55 BY4741 his31 leu20 met150 ura30 TEFp-mito-DHFR-mcherry::CgHIS3 atg3::natNT2 atg32::kanMX6 YMT57 BY4741 his31 leu20 met150 ura30 TEFp-mito-DHFR-mcherry::CgHIS3 atg4::natNT2 atg32::kanMX6 YMT59 BY4741 his31 leu20 met150 ura30 TEFp-mito-DHFR-mcherry::CgHIS3 atg5::natNT2 atg32::kanMX6 YMT39 BY4741 his31 leu20 met150 ura30 TEFp-mito-DHFR-mcherry::CgHIS3 atg6::natNT2 atg32::kanMX6 YMT61 BY4741 his31 leu20 met150 ura30 TEFp-mito-DHFR-mcherry::CgHIS3 atg8::natNT2 atg32::kanMX6 YMT69 BY4741 his31 leu20 met150 ura30 TEFp-mito-DHFR-mcherry::CgHIS3 atg10::natNT2 atg32::kanMX6 YMT25 BY4741 his31 leu20 met150 ura30 TEFp-mito-DHFR-mcherry::CgHIS3 atg12::natNT2 atg32::kanMX6 YMT27 BY4741 his31 leu20 met150 ura30 TEFp-mito-DHFR-mcherry::CgHIS3 atg16::natNT2 atg32::kanMX6 YMT29 BY4741 his31 leu20 met150 ura30 TEFp-mito-DHFR-mcherry::CgHIS3 atg18::natNT2 atg32::kanMX6 YMT67 BY4741 his31 leu20 met150 ura30 TEFp-mito-DHFR-mcherry::CgHIS3 atg19::natNT2 atg32::kanMX6 YMT47 BY4741 his31 leu20 met150 ura30 TEFp-mito-DHFR-mcherry::CgHIS3 atg20::natNT2 atg32::kanMX6 YMT51 BY4741 his31 leu20 met150 ura30 TEFp-mito-DHFR-mcherry::CgHIS3 atg23::natNT2 atg32::kanMX6 YMT65 BY4741 his31 leu20 met150 ura30 TEFp-mito-DHFR-mcherry::CgHIS3 atg27::natNT2 atg32::kanMX6 YMT35 BY4741 his31 leu20 met150 ura30 TEFp-mito-DHFR-mcherry::CgHIS3 atg29::natNT2 atg32::kanMX6 YMT37 BY4741 his31 leu20 met150 ura30 TEFp-mito-DHFR-mcherry::CgHIS3 atg31::natNT2 atg32::kanMX6 YMT77 BY4741 his31 leu20 met150 ura30 TEFp-mito-DHFR-mcherry::CgHIS3 atg33::natNT2 atg32::kanMX6 YMT71 BY4741 his31 leu20 met150 ura30 TEFp-mito-DHFR-mcherry::CgHIS3 atg1::ATG1::kanMX4 YMT73 BY4741 his31 leu20 met150 ura30 TEFp-mito-DHFR-mcherry::CgHIS3 atg1::ATG1 AIM mut::kanMX4 YMT79 BY4741 his31 leu20 met150 ura30 TEFp-mito-DHFR-mcherry::CgHIS3 atg1::ATG1::kanMX4 atg32::natNT2 YMT81 BY4741 his31 leu20 met150 ura30 TEFp-mito-DHFR-mcherry::CgHIS3 atg1::ATG1 AIM mut::kanMX4 atg32::natNT2 


Table S3 . Primers Used in This Study, Related to STAR Methods

 S3 
	Name	5´-3´ oligonucleotide
	ATG3 cassette -Fw	TATCAAGCTAGCTAGAAGTTAGGAACAAAGAAGTACAAAGGAGTAAATAC
		AATTTTATTATCCGTACGCTGCAGGTCGAC
	ATG3 cassette -Rv	TCTTGTTTATCCTGTTTTTTGACCACCTGGCTTGCAGCTAATAGTGAAAAA
		ACACAAATTTATCGATGAATTCGAGCTCG
	ATG4 cassette -Fw	CACACAATAACGTTAGTAGATGAAGAATGGACGACTTCTTATCACGTATAG
		GAGTGATATACCGTACGCTGCAGGTCGAC
	ATG4 cassette -Rv	CTCAGTTTTAGGGAATATATTAAAACAAGTATATATGCTTATGAACTAGTGA
		ATTCCTTACAATCGATGAATTCGAGCTC
	ATG5 cassette -Fw	GCGAATGAATTTTTGTTCTTTTGGTTCTAGAAGAACGGAGATAGGAAACCT
		ATGATGTAAGTCGTACGCTGCAGGTCGAC
	ATG5 cassette -Rv	AACGTCCAAGTTATTTTCTGCGATATTTGAATGACACTTTTAAATGCGTATA
		TAACAGCTCATCGATGAATTCGAGCTCG
	ATG6 cassette -Fw	AGTTCAGTTACGTTTTATGGCAGTCACTGTTTTCGCAAAGACTCCCAGACA
		CGGGCATTAAACGTACGCTGCAGGTCGAC
	ATG6 cassette -Rv	AAGTGGAAAAAGAAATTTTCCCTTTATCACATTTATGAAAAAATGCATTTAT
		ATGAACTACATCGATGAATTCGAGCTCG
	ATG8 cassette -Fw	TGATAAGAGAATCTAATAATTGTAAAGTTGAGAAAATCATAATAAAAATAAT
		TACTAGAGACCGTACGCTGCAGGTCGAC
	ATG8 cassette -Rv	CCTATAATTTCGATTTTAGATGTTAACGCTTCATTTCTTTTCATATAAAAGA
		CTACCTGCCATCGATGAATTCGAGCTCG
	ATG10 cassette -Fw	ATGAGAAGAGAACACCATGAAAAAAAAAAAAAAAGGGCTAAAAAACAGAA
		TTATCAGACTTGCGTACGCTGCAGGTCGAC
	ATG10 cassette -Rv	ATATATATATATATTTACATAGATGATTGCATAGTGTTTTAAAAAGCTTTCCT
		AGGTTAAGATCGATGAATTCGAGCTCG
	ATG11-flk[942-1003]-S1FW	ATTATTTTAGTGTACTGTTGTTGTTCGGAAAGTACTTCTTTTATTTTCTTTTA
		TACATCATGCGTACGCTGCAGGTCGAC
	ATG11-flk[4592-4532]-S2RV GTTAAATAGATACATAATTAAAATCTTGTCATTTGTGACAAACGTTTAGCAC
		TGTTCAAACATCGATGAATTCGAGCTCG
	ATG12 cassette -Fw	TGACGAAAGCTCCATCATATCCACAAATAATGGAACGGCAATGGAAAGAT
		CCAGAAATAATCCGTACGCTGCAGGTCGAC
	ATG12 cassette -Rv	TCGTACGGGATTTTTGATCGACTGTAGGTTTTCTTCTTAGACCATTCCAGC
		GCCCGGGTATATCGATGAATTCGAGCTC
	ATG13-flk[939-1000]-S1FW	CCTTCCAGGCTCAAGTCTTGAAAAGAAAGCAGAACATACAGCCCGGTTGA
		ATAGCATGAGTCCGTACGCTGCAGGTCGAC
	ATG13-flk[3264-3204]-S2RV GATTATTTTTCTTTAGTTGTGCCCTTTAAAATAAAACTTTACCATTTTTAACC
		TTCTTTAGATCGATGAATTCGAGCTCG
	ATG16 cassette -Fw	CAAGGAGGCCCATTTAAATGAGTTGTTTCAAGATAATAGTGGCGCCATTG
		GTGGCAACATTGCGTACGCTGCAGGTCGAC
	ATG16 cassette -Rv	TGCACCACAATGATTTTATTTTCTTTTGTATGCATTTTGTGACGATTTGACA
		ACTGATGCAATCGATGAATTCGAGCTCG
	ATG17-flk[942-1003]-S1FW	GTCGCATAACGTACGATAAATTCGATACTGCGAGGATATTATCAACGTATT
		TAACACCTATGCGTACGCTGCAGGTCGAC
	ATG17-flk[2296-2236]-S2RV CAATTATTGAATCTTTGTACCGTATCCTTTTTTTCCTTTTTTCTAAGGATTCT
		TCACGTTGATCGATGAATTCGAGCTCG
	ATG18 cassette -Fw	TTAATCAGTTAGTAATAGTGTTCCAGTTAACTCTGTATCCTTTTCTTCTTCG
		GCCTGACACGTACGCTGCAGGTCGAC
	ATG18 cassette -Rv	TATAAACTATATTGGTATGCGTTGTGACGTACGGAAGGCAGCGCGAGACA
		CTTCCGTGAATCGATGAATTCGAGCTCG
	ATG19 cassette -Fw	GAATCGAGGTAATTGCGGCGGCACTTGCTTCAGTAACGCCCAAAGGAGA
		GTTCTGGTAAATGCGTACGCTGCAGGTCGAC
	ATG19 cassette -Rv	GTATGTGAAAAGGTACTCATTGCTGTATAAAAATAGAGTTTGACCTAGAGT
		TCTTCCCAAGATCGATGAATTCGAGCTCG

Table S3 . Primers Used in This Study (continued)

 S3 
	Name	5´-3´ oligonucleotide
	ATG20 cassette -Fw	CGAATACTGGTGGTGCAATTGAACATACCATCTAGAAAAGTTGTCTTCTAC
		AGCACACAATGCGTACGCTGCAGGTCGAC
	ATG20 cassette -Rv	ACGTTGTTTATATATATATATATCAAGTATGCTATAACGCTAAAAAAAAATG
		CTCATAATAATCGATGAATTCGAGCTCG
	ATG21 cassette -Fw	AATTTGACCAACAAAAGACAATTCCACTCCTTTGGATTTGAAATAGACAGA
		TAGAAAAGGATCGTACGCTGCAGGTCGAC
	ATG21 cassette -Rv	TAGCAATTCACACGTGAATACGTACAATATCTATTAAGATTATGAAAACTG
		CACATATGCAATCGATGAATTCGAGCTCG
	ATG23 cassette -Fw	TCTTGTTCCACATTTTAACCTCGTTGTTCTATAAGGTAACAAAATAAAGTGA
		AGAAGTAAATCGTACGCTGCAGGTCGAC
	ATG23 cassette -Rv	GTTTACTACACTTTCAGAAATGCCAATCTTTGGTGGCGATTTGCTAGCAAC
		CAAGAATGAGATCGATGAATTCGAGCTCG
	ATG24-flk[942-1003]-S1FW	TAAGCCGTTATTAAACTTTATTTACGGTATACCACAATACTGCTCTTTTTGT
		TGAGGATATGCGTACGCTGCAGGTCGAC
	ATG24-flk[2333-2273]-S2RV ATCAGATAATCGTAGTGCCCAAGGTATTATCAGTAGTAATGGGAAAACATT
		AAGAGCACCAATCGATGAATTCGAGCTCG
	ATG27 cassette -Fw	TGTAGATTTATATCTGAGATCTTCAATCGATGCGATAGATAAAGGTAAGGA
		AAGCTTTCACGCGTACGCTGCAGGTCGAC
	ATG27 cassette -Rv	GCCTAAGATTCTCGAAACCACTTCCTTGCAGAATTCAGGTAAGGAAGTGA
		GAAATTGAGTAATCGATGAATTCGAGCTCG
	ATG29 cassette -Fw	ATACTTATCAAGAAGAGACATACATAATTGTACTGCTGTGCATTTTCCTACT
		TGACTTTCCGTACGCTGCAGGTCGAC
	ATG29 cassette -Rv	AAACGCCGGGCCTTTTTGGCCGACAGTTGGTTTTTTGATTGTGCTTGTGA
		AAGATGTAAAATCGATGAATTCGAGCTCG
	ATG31 cassette -Fw	TAAACGTCGCATTCCCATTTTCTTATTAAGCCGGTAAACATTGCTGAAATC
		TGCGAACAGGACGTACGCTGCAGGTCGAC
	ATG31 cassette -Rv	AGAACATATACCTACATAAACAAGTTAAGAGAGTCTCATCCATGCGGCTTC
		ATTTTTGCTTATCGATGAATTCGAGCTCG
	ATG32 cassette -Fw	TAAGCAATATTGAAGTCCTAATCACAAAAGCAAAAAAAATCTGCCAGGAAC
		AGTAAACATATGCGTACGCTGCAGGTCGAC
	ATG32 cassette -Rv	ACAGAAGTGATAGTAAAAAAGTGAGTAGGAACGTGTATGTTTGTGTATATT
		GGAAAAAGGATCGATGAATTCGAGCTC
	ATG1-integ-Fw	GAAGCTACCCCATATTTTCAAATCTCTTTTACAACACCAGACGAGAAATTA
		AGAAAATGGGAGACATTAAAAATAAAGAT
	ATG1-integ-Rv	CTTGAAAATATAGCAGGTCATTTGTACTTAATAAGAAAACCATATTATGCAT
		CACTTAGAAAAACTCATCGAGCATCAAA
	ULK1 K46N-Fw	GACCTGGAGGTGGCCGTCAACTGCATTAACAAGAAGAAC
	ULK1 K46N-Rv	GTTCTTCTTGTTAATGCAGTTGACGGCCACCTCCAGGTC
	ULK1F357A V360A-Fw	TGTGACACAGATGACGCTGTCATGGCCCCAGCCCAGTTTCCA
	ULK1 F357A V360A-Rv	TGGAAACTGGGCTGGGGCCATGACAGCGTCATCTGTGTCACA

Table S1. Comparison between ATG Gene Requirements for Bcl2-L-13-mediated Mitophagy and for Canonical Yeast Mitophagy, Related to Figure 1 and Table 1. Bcl2-L-13-mediated mitophagy was assayed by protein degradation assay using mitochondria targeted mCherry-DHFR expressing cells grown in glycerol medium for 72 h. The requirement of certain ATG genes for the canonical yeast mitophagy has previously been described (Okamoto et al., 2009). Phenotypes in Bcl2-L-12-mediated mitophagy are indicated with plus and minus signs as follows: ++, a similar to or higher level than that in atg32; +, a significantly higher level than the corresponding empty vector-transfected strain and lower level than atg32; -, a similar level to the corresponding vector-transfected strain. Phenotypes in canonical yeast mitophagy are indicated with plus and minus signs as follows: +++, wild-type-like; ++, slightly defective; +, partially defective; -, completely defective.