Mitochondrial fission facilitates mitophagy in Saccharomyces cerevisiae

Abstract

As a highly dynamic organelle, mitochondria undergo constitutive fusion and fission as well as biogenesis and degradation. Mitophagy, selective mitochondrial degradation through autophagy, is a conserved cellular process used for the elimination of excessive and damaged mitochondria in eukaryotes. Despite the significance of mitophagy in cellular physiology and pathophysiologies, the underlying mechanism of this process is far from clear. In this report, we studied the role of mitochondrial fission during mitophagy, and uncover a direct link between the fission complex and mitophagy machinery in Saccharomyces cerevisiae.

In eukaryotic cells, macroautophagy (hereafter autophagy) is a catabolic pathway degrading cytosol, protein aggregates, organelles and even invading pathogens. Two major types of autophagy have been characterized: nonselective bulk autophagy and selective autophagy. Nonselective autophagy mediates bulk degradation and recycling of cytoplasm to support cell survival upon nutrient deprivation. In contrast, selective autophagy participates in a wider array of processes, recognizing and targeting specific cargos or organelles for degradation, allowing cellular adaption to a variety of extracellular environments, and eliminating deleterious factors, including damaged organelles and pathogens. Mitophagy is one of the selective types of autophagy, with mitochondria being the particular cargos targeted for degradation.

Mitochondria are double-membrane organelles that participate in various aspects of cell metabolism, including energetics, amino acid biogenesis and programmed cell death. In recent years, mitophagy has attracted increasing attention due to its protective role in preventing mitochondria-dependent cell death that may contribute, for example, to certain types of neurodegeneration such as Parkinson disease; mitophagy is generally thought to promote cell survival. In addition, mitophagy is used to remove mitochondria during erythrocyte development, and sperm-derived mitochondria after fertilization, thus playing a role in normal developmental processes.

Due to its genetic tractability, the budding yeast Saccharomyces cerevisiae is an excellent system for studying autophagy as well as mitophagy. In this organism, mitophagy is dramatically induced upon changing the culture conditions from nutrient-rich medium with lactic acid to nitrogen starvation medium with glucose. Genomic screening enabled us to identify a group of proteins involved in mitophagy, including the mitophagy receptor Atg32 and the upstream mitogen-activated protein kinases Slt2 and Hog1.

Atg32 is a mitochondrial outer membrane protein, and it recruits the scaffold protein Atg11 to mitochondria when mitophagy is induced. Atg11 links the cargo (degrading mitochondria) to other Atg proteins and promotes the formation of mitochondria-specific autophagosomes that ultimately leads to degradation of the organelle in the vacuole. To specifically indicate the degrading mitochondria on the mitochondrial reticulum, we took advantage of the bimolecular fluorescence complementation (BiFC) assay, and showed that the fluorescent YFP puncta resulting from the interaction between Atg32 and Atg11 represent degrading mitochondria through the entire process of mitophagy.

Previous work indicated that during selective types of autophagy cargos with a large size cannot be efficiently engulfed by phagophores. The majority of yeast mitochondria exist as an extended and reticular structure; therefore, it is conceivable that these large organelles have to be divided into smaller pieces first, and then sequestered by phagophores. Along these lines, the DNM1 gene, which encodes a dynamin-related GTPase controlling mitochondrial fission, was identified in our genomic screen for mitophagy-defective mutants.

To maintain cellular homeostasis, appropriate mitochondrial morphology must be achieved through mitochondrial fusion and fission. In budding yeast, four subunits of the fission complex have been identified—Dnm1, Fis1, Mdv1, and Caf4. Of the four proteins, Dnm1 and Fis1 are highly conserved from yeast to human. Fis1 is an integral membrane protein and is required for the proper localization of Dnm1 and Mdv1 on mitochondria. Dnm1 is a dynamin-related GTPase that assembles specifically at the sites where mitochondrial fission occurs. Mdv1 and Caf4 redundantly bridge the interaction between Fis1 and Dnm1.

Deletion of DNM1 or FIS1 results in a significant decrease of mitophagy activity. Due to the functional redundancy of MDV1 and CAF4, single deletion of MDV1 or CAF4 has limited effects on mitophagy; however, double deletion of both genes causes a strong defect in mitophagy. Therefore, the intact fission complex is required for efficient mitophagy. Fluorescence microscopy shows that Dnm1, Mdv1, and Caf4 are recruited to the degrading mitochondria when mitophagy is induced, suggesing that the fission complex might directly participate in mitophagy.

To uncover the molecular mechanism behind this phenomenon, we used co-immunoprecipitation and showed that a mitophagy-specific fission complex is formed, which includes Atg32, Atg11, Dnm1, Fis1, Mdv1 and Caf4. Our BiFC analysis suggests that a direct interaction occurs between Atg11 and Dnm1. Both Atg11 and Dnm1 are cytosolic proteins, and their recruitment to the mitochondria is dependent on Atg32 and Fis1, respectively. The BiFC interaction dots formed by Atg11 and Dnm1 only constitute a small population of the overall Dnm1 puncta. This finding suggests that only a portion of these proteins will interact with Atg11 and Atg32 to form the mitophagy-specific fission complex, whereas the majority of Dnm1 and the other components of the fission complex still drive constitutive (homeostatic) mitochondrial fission.

Further truncation and mutagenesis analyses enabled us to identify two Dnm1 mutants, Dnm1^E728R and Dnm1^D729R. These two mutants retain normal Dnm1 function including the ability to interact with other members of the fission complex, but lose interaction with Atg11 or Atg32. As expected, these mutants show mitophagy defects, even though they allow cells to carry out homeostatic mitochondrial fission, in agreement with the suggestion that there are at least two separate populations of the fission complex.

In this study, we answer several questions in the mitophagy field. First, we generated a marker that specifically indicates degrading mitochondria during mitophagy. Second, we revealed part of the molecular mechanism through which mitochondrial fission participates in mitophagy. Third, we unveiled another role of Atg11 as a scaffold protein during selective autophagy: recruiting the mitochondrial fission machinery.

Mao K, Wang K, Liu X, Klionsky DJ. The scaffold protein atg11 recruits fission machinery to drive selective mitochondria degradation by autophagy. Dev Cell. 2013;26:9–18. doi: 10.1016/j.devcel.2013.05.024.

10.4161/auto.25804

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.