The yeast mitophagy receptor Atg32 is ubiquitinated and degraded by the proteasome

Abstract

Mitophagy, the process that degrades mitochondria selectively through autophagy, is involved in the quality control of mitochondria in cells grown under respiratory conditions. In yeast, the presence of the Atg32 protein on the outer mitochondrial membrane allows for the recognition and targeting of superfluous or damaged mitochondria for degradation. Post-translational modifications such as phosphorylation are crucial for the execution of mitophagy. In our study we monitor the stability of Atg32 protein in the yeast S. cerevisiae and show that Atg32 is degraded under normal growth conditions, upon starvation or rapamycin treatment. The Atg32 turnover can be prevented by inhibition of the proteasome activity, suggesting that Atg32 is also ubiquitinated. Mass spectrometry analysis of purified Atg32 protein revealed that at least lysine residue in position 282 is ubiquitinated. Interestingly, the replacement of lysine 282 with alanine impaired Atg32 degradation only partially in the course of cell growth, suggesting that additional lysine residues on Atg32 might also be ubiquitinated. Our results provide the foundation to further elucidate the physiological significance of Atg32 turnover and the interplay between mitophagy and the proteasome.

Degradation Mechanism of Autophagy-Related Proteins and Research Progress

“…In contrast, the turnover of unmodified Atg32 is mediated by an unknown protease independent of proteasome and vacuolar proteases [54]. However, studies by Camougrand et al [55] demonstrated that the degradation of Atg32 is associated with the UPS. Ubiquitination of Atg32 occurs at least on Lys282 residues and is mediated by several E3 ligases, including Rsp5, which is a relatively complicated process [55].…”

“…However, studies by Camougrand et al [55] demonstrated that the degradation of Atg32 is associated with the UPS. Ubiquitination of Atg32 occurs at least on Lys282 residues and is mediated by several E3 ligases, including Rsp5, which is a relatively complicated process [55]. To sum up, apart from the different strains used in the experimental materials, these two contradictory observations may be attributed to the differences in growth conditions or mitophagy induction, leading to the coordinated regulation of Atg32 levels by multiple pathways.…”

Mitophagy in Yeast: Molecular Mechanism and Regulation

“…This suggests that Yme1 is important for the recruitment of the mitochondria to the PAS, but the factors that trigger this proteolytic modification remain unclear [ 24 , 59 ]. It was also reported that ubiquitination of at least Lys282 of Atg32 is necessary for Atg32 degradation by proteasomes [ 60 ].…”

Mitophagy in Yeast: Decades of Research

“…An additional mechanism for conducting mitophagy induction is Atg32 phosphorylation [ 54 , 55 , 56 ]. In addition to phosphorylation, Atg32p endures other forms of post-translational modifications, such as proteolytic processing [ 57 , 58 ], ubiquitination [ 59 ] and other post-translational modifications not identified yet [ 60 ]. Molecular mechanisms of mitophagy in yeast are discussed in the review proposed by Innokentev and Kanki (2021), which is a part of this Special Issue, entitled: “Mitophagy in yeast: molecular mechanism and regulation”.…”

“…Moreover, it was demonstrated that Atg32 phosphorylation, which is required for mitophagy facilitation, is altered in the breathing natA Δ mutant [ 53 ]. It has also been shown that the Atg32 protein was ubiquitously degraded by the proteasome in the stationary growth phase, which controls the mitophagy level [ 59 ]. This regulation by phosphorylation and ubiquitination is also reported for the mitophagy receptor FUNDC1 [ 114 , 115 , 116 , 117 ].…”