Studies on the molecular mechanisms regulating initiation of selective mitochondrial degradation in budding yeast

概要

Mitophagy is an evolutionarily conserved process that selectively degrades mitochondria via autophagy, contributing to the mitochondrial quality and quantity control critical for cell homeostasis. In the budding yeast Saccharomyces cerevisiae, Atg32, a mitochondrial surface-anchored protein, is induced and phosphorylated to promote mitophagy under respiratory conditions. Atg32 forms the mitophagy initiation complex with Atg8, a ubiquitin-like modifier localized to the autophagosome, and Atg11, a selective autophagy-specific scaffold protein acting in the assembly of core Atg proteins required for autophagosome formation. Currently, molecular mechanisms underlying mitophagy initiation remain obscure.

In this study, I obtained the data suggesting that the TORC1 (target of rapamycin complex 1) signaling pathway regulates mitophagy in budding yeast through SEACIT (Seh1-associated complex inhibiting TORC1). During prolonged respiratory growth, cells lacking SEACIT exhibited strong defects in mitophagy. Under the same conditions, other autophagy-related events were only slightly affected. I also found that the inactivation of TORC1 by pharmacological and genetic approaches almost completely restored respiration-induced mitophagy in cells lacking SEACIT. Importantly, the interaction between Atg32 and Atg11 was strongly impaired in the absence of SEACIT, while Atg32 phosphorylation was hardly affected. These results support the idea that SEACIT promotes Atg32-Atg11 interactions and mitophagy through inactivating TORC1 during prolonged respiration.

In addition, using NanoLuc Binary Technology (NanoBiT), I developed a novel assay to quantitatively analyze Atg32-Atg11 interactions in live cells. A systematic approach for Atg32-Atg11 interactions in 18 core Atg mutants revealed that Atg1, a protein kinase interacting with Atg8 and Atg11, is the sole core Atg protein critical for efficient mitophagy initiation. I also found that Atg1 acts in Atg32 phosphorylation. Expression of an Atg1 variant defective in Atg1-Atg8 and Atg1-Atg11 interactions reduced Atg32 phosphorylation and mitophagy, suggesting that Atg8 and Atg11 serve to recruit Atg1 to the mitophagy initiation complex. Notably, these defects were restored by deletion of Ppg1, a protein phosphatase that negatively regulates Atg32-mediated mitophagy.

Furthermore, based on AlphaFold2 protein structural predictions and mutational analyses, I identified a conserved motif of Atg32(202-210) essential for Atg32-Atg11 interactions and efficient mitophagy. Collectively, these findings imply that mitophagy initiation in budding yeast is regulated through kinases acting upstream and downstream of Atg32-Atg11 interactions.