Atg43, a novel autophagy-related protein, serves as a mitophagy receptor to bridge mitochondria with phagophores in fission yeast

ABSTRACT

Mitophagy is a selective type of autophagy in which damaged or unnecessary mitochondria are sequestered by double-membranous structures called phagophores and delivered to vacuoles/lysosomes for degradation. The molecular mechanisms underlying mitophagy have been studied extensively in budding yeast and mammalian cells. To gain more diverse insights, our recent study identified Atg43 as a mitophagy receptor in the fission yeast Schizosaccharomyces pombe. Atg43 is localized on the mitochondrial outer membrane through the Mim1–Mim2 complex and binds to Atg8, a ubiquitin-like protein conjugated to phagophore membranes. Artificial tethering of Atg8 to mitochondria can bypass the requirement of Atg43 for mitophagy, suggesting that the main role of Atg43 in mitophagy is to stabilize phagophore expansion on mitochondria by interacting with Atg8. Atg43 shares no sequence similarity with mitophagy receptors in other organisms and has a mitophagy-independent function, raising the possibility that Atg43 has acquired the mitophagic function by convergent evolution.

Mitochondria generate energy by oxidative phosphorylation, with reactive oxygen species (ROS) as inevitable byproducts. ROS cause mitochondrial damage followed by further ROS production, potentially leading to cell dysfunction and death. To avoid this situation, the quantity and quality of mitochondria are thought to be controlled mostly by mitochondrial biogenesis, fission/fusion, and autophagy. Mitochondrial autophagy (mitophagy) eliminates dysfunctional or superfluous mitochondria via selective autophagy. Selective autophagy depends on the receptor proteins that reside on the surface of the cargo. The receptors interact with the ubiquitin-like core autophagy protein Atg8 or its mammalian counterparts (LC3/GABARAP proteins) conjugated onto phagophore membranes. The interaction is mediated by a motif (W/F/Y-x-x-L/I/V) called the Atg8-family-interacting motif (AIM) or LC3-interacting region (LIR) contained in the receptors. Apart from the Atg8-family proteins, some receptors interact with other core autophagy proteins, linking cargo to the autophagy machinery.

The fission yeast Schizosaccharomyces pombe has recently been used as a model to study autophagy, in addition to budding yeast and mammals. To identify the factors involved in mitophagy in S. pombe, we recently screened a genome-wide knockout library for mutants defective in mitophagy induced by nitrogen starvation [1]. We found that mitophagy was completely blocked in a mutant with a deletion in SPAC14C4.01c, the product of which was named Atg43. Atg43 is a mitophagy-specific factor; bulk and endoplasmic reticulum (ER) autophagy occur normally in cells lacking Atg43. Consistent with its role in starvation-induced mitophagy, Atg43 localizes to the mitochondrial outer membrane (MOM) as a transmembrane protein with its N terminus facing the cytosol, and its expression is upregulated upon nitrogen starvation under the control of target of rapamycin complex 1 (TORC1).

Atg43 consists of 244 amino acids (aa), among which only a 40 aa region (aa 185–224) near the C terminus containing a putative transmembrane domain (aa 187–211) and a 20 aa region near the N terminus (aa 21–40) are essential for mitophagy (Figure 1A). The C-terminal region is necessary and sufficient for mitochondrial localization of Atg43, indicating that a primary role for the region is to anchor Atg43 to the MOM. The N-terminal region contains an AIM (Y₂₈ELI₃₁) that is necessary for mitophagy. Indeed, Atg43 interacts with Atg8 in a manner dependent on the AIM, as in the case of other selective autophagy receptors. Remarkably, the function of Atg43 in mitophagy can be replaced by the artificial tethering of the AIM-containing 20 aa region to the MOM, but also by that of Atg8. We thus propose that Atg43 acts as a selective autophagy receptor to promote mitophagy by stabilizing phagophore expansion on mitochondria through direct interactions with Atg8 (Figure 1B).

Figure 1.

Atg43 tethers phagophores to the MOM for the efficient engulfment of mitochondria. (A) Schematic representation of the primary structure of Atg43. The C-terminal region that is conserved among fungi is marked. Regions involved in the individual processes are indicated. TM, transmembrane domain. (B) Schematic model for mitophagy promoted by Atg43. The extending phagophore is stabilized on the MOM by the interaction between Atg8 on the phagophore membrane and the N-terminal AIM of Atg43. Atg43 is also involved in an unknown cellular process that facilitates cell growth, possibly with the MIM complex

Apart from mitophagy during starvation, Atg43 facilitates vegetative cell growth in nutrient-rich conditions. In S. pombe, neither mitophagy nor autophagy defects are associated with growth phenotypes. Moreover, AIM-mutated or N-terminal truncated forms of Atg43 are mitophagy-defective but proficient in proliferation. Therefore, Atg43 plays a mitophagy-independent cellular function that facilitates normal cell growth. This mitophagy-independent role is likely to be exerted through mitochondria because the C-terminal truncated form of Atg43, which is defective in mitochondrial localization, causes a growth defect.

As Atg43-binding proteins, we identified Mim1 and Mim2, which comprise the mitochondrial import (MIM) complex, mediating the mitochondrial insertion of MOM proteins. Indeed, mitochondrial localization of Atg43 requires the MIM complex, and its absence causes a mitophagy defect that can be suppressed by the forced loading of Atg43 onto the MOM. Thus, the MIM complex contributes to mitophagy by facilitating the mitochondrial localization of Atg43. Unexpectedly, the aa region of Atg43 that is required for the interaction with the MIM complex (aa 165–184) is dispensable for mitochondrial localization of Atg43 and mitophagy, but necessary for normal cell growth (Figure 1A). Hence, the observed interaction between Atg43 and the MIM complex may reflect not merely a transient interaction during the MIM complex-mediated mitochondrial loading of Atg43 but also the formation of a stable protein complex. Possibly, the MIM complex acts together with Atg43 to promote the mitophagy-independent role of Atg43 on the MOM.

Atg43 does not share sequence homology with mitophagy receptors of budding yeast or mammals. Conversely, Atg43-like sequences can be found among fungi, but the homology is limited to the C-terminal region that contains the transmembrane domain and the sequence required for binding with the MIM complex. Thus, the mitophagy-independent function, but not the mitophagic function, appears to be conserved. We speculate that Atg43 on the MOM has acquired the mitophagic function at a later stage of evolution, and that organisms might have selected various MOM proteins for use as mitophagy receptors according to their needs.

Mitophagy is thought to play a key role in controlling the quality and quantity of mitochondria, but the underlying mechanisms remain to be elucidated. In the case of S. pombe, mitophagy-defective atg43 mutants lose the ability to resume proliferation after long-term starvation. Further phenotypic analyses of the atg43 mutants will shed light on how mitophagy contributes to mitochondrial functions. Collectively, our findings on Atg43 provide a new model system to study the molecular mechanisms and physiological roles of mitophagy in eukaryotic cells.

Funding Statement

This work was partially supported by grants from the Japan Society for the Promotion of Science [20K06552 to TF] and the Takeda Science Foundation [to TF].

Disclosure statement

The authors declare no conflict of interests.