Phospholipid methylation controls Atg32 ‐ mediated mitophagy and Atg8 recycling by Kaori Sakakibara, Akinori Eiyama, Sho W Suzuki, Machiko Sakoh ‐ Nakatogawa, Nobuaki Okumura, Motohiro Tani, Ayako Hashimoto, Sachiyo Nagumo, Noriko Kondo ‐ Okamoto, Chika Kondo ‐ Kakuta, Eri Asai, Hiromi Kirisako, Hitoshi Nakatogawa, Osamu Kuge, Toshifumi Takao, Yoshinori Ohsumi, and Koji Okamoto EMBO J. Volume 34(21): November 3, 2015 ©2015 by European Molecular Biology Organization

Cells lacking Opi3 exhibit severe mitophagy defects Representative images of mitochondria ‐ targeted GFP (mito ‐ GFP) and vacuole ‐ targeted mCherry (Vph1–mCherry) patterns. Kaori Sakakibara et al. EMBO J. 2015;34: ©2015 by European Molecular Biology Organization

Loss of Opi3 leads to minor defects in bulk autophagy, pexophagy, and the Cvt pathway Reintroduction of an OPI3 gene into the opi3 ‐ null mutant rescues mitophagy. Kaori Sakakibara et al. EMBO J. 2015;34: ©2015 by European Molecular Biology Organization

PMME and GSH increase in opi3 ‐ null cells under mitophagy ‐ inducing conditions Wild ‐ type, opi3Δ, cho2Δ, and opi3Δ cho2Δ cells expressing mito ‐ DHFR–mCherry were cultured and analyzed as in Fig 1B. Kaori Sakakibara et al. EMBO J. 2015;34: ©2015 by European Molecular Biology Organization

Atg32 is not induced in cells lacking Opi3 Wild ‐ type, opi3Δ, cho2Δ, and opi3Δ cho2Δ cells expressing a chromosomally integrated Atg32–HA were generated from a vacuolar protease ‐ deficient strain (pep4Δ prb1Δ), cultured, and analyzed as in Fig 1B. Kaori Sakakibara et al. EMBO J. 2015;34: ©2015 by European Molecular Biology Organization

Overexpression of Atg32 only slightly restores mitophagy in opi3 ‐ null cells Atg32 properly targets to mitochondria in the absence of Opi3. atg32Δ and atg32Δ opi3Δ cells expressing mito ‐ DHFR–mCherry and a plasmid ‐ encoded GFP–Atg32 were grown in glycerol med... Kaori Sakakibara et al. EMBO J. 2015;34: ©2015 by European Molecular Biology Organization

Loss of Opi3 causes drastic lipidation of Atg8 AA schematic diagram of post ‐ translational modification for Atg8. Kaori Sakakibara et al. EMBO J. 2015;34: ©2015 by European Molecular Biology Organization

Deletion of the OPI3 gene alters autophagy flux, Cho2 repression, and Atg32 induction Atg4 expression is not altered in the absence of Opi3. Kaori Sakakibara et al. EMBO J. 2015;34: ©2015 by European Molecular Biology Organization

Atg8 is conjugated to PMME but cannot be efficiently delipidated A schematic diagram of Atg8 conjugation and deconjugation reactions in vitro. Kaori Sakakibara et al. EMBO J. 2015;34: ©2015 by European Molecular Biology Organization

Yeast and human Atg8 family proteins are conjugated to PMME in vitro Atg8 is conjugated to PMME in cells lacking Opi3. Kaori Sakakibara et al. EMBO J. 2015;34: ©2015 by European Molecular Biology Organization

Mitophagy is strongly impaired in Atg8 deconjugation ‐ deficient cells Wild ‐ type or opi3Δ cells expressing full ‐ length Atg8, and atg4Δ or atg4Δ opi3Δ cells expressing Atg8G116 were generated from a strain for mitophagy assays, cultured, and analyzed as in Fi... Kaori Sakakibara et al. EMBO J. 2015;34: ©2015 by European Molecular Biology Organization

Atg32 overexpression and choline treatment partially rescue mitophagy in opi3 ‐ null cells Wild ‐ type and opi3Δ cells expressing mito ‐ DHFR–mCherry and a chromosomally encoded Atg32–HA were transformed with two Atg32–HA ‐ expressing plasmids or empty vectors, cu... Kaori Sakakibara et al. EMBO J. 2015;34: ©2015 by European Molecular Biology Organization

Cho2 expression affects Atg8–PMME formation and mitophagy Choline supplementation partially rescues mitophagy in cells lacking Opi3. Kaori Sakakibara et al. EMBO J. 2015;34: ©2015 by European Molecular Biology Organization