Omics analysis of degradation targets by mitochondrial autophagy
Kentaro FURUKAWA
Niigata University Graduate School of Medical and Dental Sciences
Report:Genetic screen using a yeast knockout library identified a novel protein Atg44 essentialfor mitophagy. Detailed analyses of Atg44 revealed that Atg44 is dispensable forbulk/selective autophagy other than mitophagy and that Atg44 contributes to mitophagy byfunctioning as a mitochondrial fission factor.
Research aims
Accumulation of dysfunctional mitochondria during energy production causes harmful
effects such as impaired fermentation productivity/stress resistance in yeast andneurodegenerative disease/aging in human. Mitochondrial degradation by autophagy(mitophagy) is a widely conserved quality control system of mitochondria. When mitophagy
is induced upon starvation or under stationary phase of respiratory growth in yeast, themitochondrial outer membrane protein Atg32 is phosphorylated by casein kinase 2 and thenphosphorylated Atg32 accumulates on mitochondria dependently of the adaptor protein
Atg11 1),2). This Atg32-accumulated site is recognized as a mitochondrial degradation target(Fig. 1). However, the fission factor required for mitophagy and the contents ofmitochondrial degradation target (presence/absence of mutated mitochondrial DNA anddisordered proteins, etc) remain unknown 3). The aim of this study is to reveal the molecularmechanism of mitophagy and authentic mitochondrial degradation targets by genetic,biochemical, and omics approaches.
Methods
When yeast cells expressing Idh1(Isocitrate DeHydrogenase 1)-GFP are cultured in
respiratory growth medium until stationary phase, mitochondria including Idh1-GFP aredegraded in the vacuole following mitophagy induction. However, GFP itself, which isstable even in the vacuole, can be detected as a mitophagy signal by western blot. Employing
this system in the yeast knockout library, mitophagy-defective mutants were screened. The
following analyses regarding the identified mitophagy-defective mutant (atg44∆) wereperformed; ① Whether atg44∆ affects bulk/selective autophagy other than mitophagy. ②
Localization analysis of Atg44 using anti-Atg44 antibody. ③ Morphological analysis ofmitochondria in atg44∆. ④ Conservation of Atg44 in different organisms.

Results
Screen of mitophagy-defective mutants identified an unknown factor, and it was named
Atg44 (AuTophaGy related 44). ① atg44∆ showed a strong defect in mitophagy, but notthat in other autophagic pathways including bulk autophagy, pexophagy, and the Cvt
pathway (Fig. 2). ② Western blot analysis using anti-Atg44 antibody detected Atg44 in a
mitochondrial fraction purified from yeast cell lysates, confirming that Atg44 is a
mitochondrial protein. ③ Analysis using transmission electron microscope revealed thatatg44∆ has enlarged mitochondria, which are often observed in mitochondrial
fission-deficient mutants (Fig. 3). ④ Other yeast species including the fission yeast (S.
pombe) and methylotrophic yeast (P. pastoris) also possess the Atg44 orthologues, which
suppress the mitophagy defect of S. cerevisiae atg44∆, suggesting that Atg44 is widelyconserved.
Conclusion
This study suggests that Atg44 functions as a mitochondrial fission factor essential formitophagy. Further study using Atg44-GFP is needed to demonstrate that Atg44 indeedaccumulates on the mitochondrial fission site. In addition, the proteomics analysis of thecontents of mitochondrial degradation targets will be performed.
References
1) Kanki, T., Furukawa, K., and Yamashita, SI. (2015) Mitophagy in yeast: Molecularmechanisms and physiological role. Biochim. Biophys. Acta. 1853: 2756-2765.
2) Furukawa, K. et al. (2018) The PP2A-like protein phosphatase Ppg1 and the Far
complex cooperatively counteract CK2-mediated phosphorylation of Atg32 to inhibit
mitophagy. Cell Rep. 23: 3579-3590.
3) Yamashita, SI. et al. (2016) Mitochondrial division occurs concurrently withautophagosome formation but independently of Drp1 during mitophagy. J. Cell Biol.
215: 649-665.

Figure 1: Model for the regulatory mechanism of mitophagy in yeast.
(Furukawa et al., Cell Rep, 2018)
Figure 2: Atg44 is required
specifically for mitophagy.
(A)
Monitoring
mitophagy(Idh1-GFP processing assay). (B)Monitoring
bulkautophagy(alkaline phosphatase Pho80
assay). (C) Monitoring pexophagy(Pex14-GFP processing assay).
(D) Monitoring the Cvt pathway(Ape1 maturation assay). atg1∆
was used as a negative control in
all assays.
Figure 3: atg44∆ has enlarged
mitochondria.
Mitochondria in wild type andatg44∆ were observed usingtransmission electron microscope(TEM). atg44∆ has enlarged
mitochondria, suggesting that
Atg44 plays a critical role inmitochondria fission. The whitearrows indicate mitochondria.