Mitophagy In Yeast Research Articles

The TORC1 signaling pathway regulates respiration-induced mitophagy in yeast

Mitophagy is an evolutionarily conserved autophagy process that selectively degrades mitochondria. This catabolic event is considered to be a mitochondrial quality control system crucial for cell homeostasis, however, mechanisms regulating mitophagy remain largely unknown. Here we show that the TORC1 (target of rapamycin complex 1) signaling pathway regulates mitophagy in budding yeast via SEACIT (Seh1-associated complex inhibiting TORC1) consisting of Iml1, Npr2, and Npr3. Cells lacking SEACIT displayed significant reductions in mitophagy during prolonged respiratory growth, while the other selective autophagy processes are less affected. Under the same conditions, mitophagy defects were strikingly rescued in the SEACIT mutants (1) treated with rapamycin, a specific TOR kinase inhibitor, (2) lacking Gtr1, a TORC1-stimulating Rag family GTPase downstream of SEACIT, and (3) devoid of Pib2, a phosphatidylinositol 3-phosphate-binding TORC1 activator. Notably, loss of Npr2 exacerbated mitophagy defects in cells lacking Atg13, a TORC1 effector crucial for activation of autophagy-related processes, suggesting additional mitophagy-specific regulator(s) downstream of TORC1. Finally, we found that npr2-null cells failed to stabilize the interaction of Atg32 with Atg11, a scaffold protein essential for mitophagy. Collectively, our data implicate SEACIT-mediated inactivation of TORC1 signaling as a critical step to promote respiration-induced mitophagy.

The ER membrane insertase Get1/2 is required for efficient mitophagy in yeast

Mitophagy is an evolutionarily conserved autophagy pathway that selectively eliminates mitochondria to control mitochondrial quality and quantity. Although mitophagy is thought to be crucial for cellular homeostasis, how this catabolic process is regulated remains largely unknown. Here we demonstrate that mitophagy during prolonged respiratory growth is strongly impaired in yeast cells lacking Get1/2, a transmembrane complex mediating insertion of tail-anchored (TA) proteins into the endoplasmic reticulum (ER) membrane. Under the same conditions, loss of Get1/2 caused only slight defects in other types of selective and bulk autophagy. In addition, mitophagy and other autophagy-related processes are mostly normal in cells lacking Get3, a cytosolic ATP-driven chaperone that promotes delivery of TA proteins to the Get1/2 complex. We also found that Get1/2-deficient cells exhibited wildtype-like induction and mitochondrial localization of Atg32, a protein essential for mitophagy. Notably, Get1/2 is important for Atg32-independent, ectopically promoted mitophagy. Together, we propose that Get1/2-dependent TA protein(s) and/or the Get1/2 complex itself may act specifically in mitophagy.

Mitophagy: A Mechanism for Plant Growth and Survival.

Mitophagy is a conserved cellular process that is important for autophagic removal of damaged mitochondria to maintain a healthy mitochondrial population. Mitophagy also appears to occur in plants and has roles in development, stress response, senescence, and programmed cell death. However, many of the genes that control mitophagy in yeast and animal cells are absent from plants, and no plant proteins marking defunct mitochondria for autophagic degradation are yet known. New insights implicate general autophagy-related proteins in mitophagy, affecting the senescence of plant tissues. Mitophagy control and its importance for energy metabolism, survival, signaling, and cell death in plants are discussed. Furthermore, we suggest mitochondrial membrane proteins containing ATG8-interacting motifs, which might serve as mitophagy receptor proteins in plant mitochondria.

Mechanisms and Physiological Roles of Mitophagy in Yeast.

Mitochondria are responsible for supplying of most of the cell’s energy via oxidative phosphorylation. However, mitochondria also can be deleterious for a cell because they are the primary source of reactive oxygen species, which are generated as a byproduct of respiration. Accumulation of mitochondrial and cellular oxidative damage leads to diverse pathologies. Thus, it is important to maintain a population of healthy and functional mitochondria for normal cellular metabolism. Eukaryotes have developed defense mechanisms to cope with aberrant mitochondria. Mitochondria autophagy (known as mitophagy) is thought to be one such process that selectively sequesters dysfunctional or excess mitochondria within double-membrane autophagosomes and carries them into lysosomes/vacuoles for degradation. The power of genetics and conservation of fundamental cellular processes among eukaryotes make yeast an excellent model for understanding the general mechanisms, regulation, and function of mitophagy. In budding yeast, a mitochondrial surface protein, Atg32, serves as a mitochondrial receptor for selective autophagy that interacts with Atg11, an adaptor protein for selective types of autophagy, and Atg8, a ubiquitin-like protein localized to the isolation membrane. Atg32 is regulated transcriptionally and post-translationally to control mitophagy. Moreover, because Atg32 is a mitophagy-specific protein, analysis of its deficient mutant enables investigation of the physiological roles of mitophagy. Here, we review recent progress in the understanding of the molecular mechanisms and functional importance of mitophagy in yeast at multiple levels.

The Nem1-Spo7 protein phosphatase complex is required for efficient mitophagy in yeast

Mitochondria-targeted selective autophagy, termed mitophagy, is an evolutionarily conserved process that contributes to mitochondrial quantity and quality control. Mitophagy requires elaborate membrane biogenesis of autophagosomes surrounding mitochondria, although how this process is regulated remains obscure. We show here that mitophagy is strongly suppressed in yeast cells lacking Nem1 or Spo7, two proteins forming a heterodimeric protein phosphatase complex known to be important for proper shaping of the nucleus and endoplasmic reticulum (ER). Under the same conditions, selective degradation of the ER and peroxisomes was also suppressed strongly and to a lesser extent, respectively, whereas autophagy and the cytoplasm to vacuole targeting (Cvt) pathway were only slightly affected in those mutants. We also found that mitochondrial sequestration in the cytoplasm and their degradation in the vacuole, a lytic compartment in yeast, occurred poorly but did not completely arrest. Notably, deletion of the INO2 gene in the nem1-or spo7-null mutant partially rescued nuclear/ER membrane shaping and mitophagy. Together, our data suggest that Nem1-Sop7-mediated regulation of membrane biogenesis is needed to promote mitophagy in yeast.

The mitochondrial phosphatidylserine decarboxylase Psd1 is involved in nitrogen starvation-induced mitophagy in yeast.

Mitophagy, the selective degradation of mitochondria by autophagy, is a central process that is essential for the maintenance of cell homeostasis. It is implicated in the clearance of superfluous or damaged mitochondria and requires specific proteins and regulators to perform. In yeast, Atg32, an outer mitochondrial membrane protein, interacts with the ubiquitin-like Atg8 protein, promoting the recruitment of mitochondria to the phagophore and their sequestration within autophagosomes. Atg8 is anchored to the phagophore and autophagosome membranes thanks to a phosphatidylethanolamine tail. In Saccharomyces cerevisiae, several phosphatidylethanolamine synthesis pathways have been characterized, but their contribution to autophagy and mitophagy are unknown. Through different approaches, we show that Psd1, the mitochondrial phosphatidylserine decarboxylase, is involved in mitophagy induction only after nitrogen starvation, whereas Psd2, which is located in vacuole, Golgi and endosome membranes, is required preferentially for mitophagy induction in the stationary phase of growth but also to a lesser extent for nitrogen starvation-induced mitophagy. Our results suggest that the mitophagy defect observed in Δpsd1 yeast cells after nitrogen starvation may be due to a failure of Atg8 recruitment to mitochondria.This article has an associated First Person interview with the first author of the paper.

Molecular mechanism and physiological functions of mitophagy in yeast

Molecular mechanism and physiological functions of mitophagy in yeast

FKBP8 protects the heart from hemodynamic stress by preventing the accumulation of misfolded proteins and endoplasmic reticulum-associated apoptosis in mice

Protein quality control in cardiomyocytes is crucial to maintain cellular homeostasis. The accumulation of damaged organelles, such as mitochondria and misfolded proteins in the heart is associated with heart failure. During the process to identify novel mitochondria-specific autophagy (mitophagy) receptors, we found FK506-binding protein 8 (FKBP8), also known as FKBP38, shares similar structural characteristics with a yeast mitophagy receptor, autophagy-related 32 protein. However, knockdown of FKBP8 had no effect on mitophagy in HEK293 cells or H9c2 myocytes. Since the role of FKBP8 in the heart has not been fully elucidated, the aim of this study is to determine the functional role of FKBP8 in the heart. Cardiac-specific FKBP8-deficient (Fkbp8−/−) mice were generated. Fkbp8−/− mice showed no cardiac phenotypes under baseline conditions. The Fkbp8−/− and control wild type littermates (Fkbp8+/+) mice were subjected to pressure overload by means of transverse aortic constriction (TAC). Fkbp8−/− mice showed left ventricular dysfunction and chamber dilatation with lung congestion 1week after TAC. The number of apoptotic cardiomyocytes was dramatically elevated in TAC-operated Fkbp8−/− hearts, accompanied with an increase in protein levels of cleaved caspase-12 and endoplasmic reticulum (ER) stress markers. Caspase-12 inhibition resulted in the attenuation of hydrogen peroxide-induced apoptotic cell death in FKBP8 knockdown H9c2 myocytes. Immunocytological and immunoprecipitation analyses indicate that FKBP8 is localized to the ER and mitochondria in the isolated cardiomyocytes, interacting with heat shock protein 90. Furthermore, there was accumulation of misfolded protein aggregates in FKBP8 knockdown H9c2 myocytes and electron dense deposits in perinuclear region in TAC-operated Fkbp8−/− hearts. The data suggest that FKBP8 plays a protective role against hemodynamic stress in the heart mediated via inhibition of the accumulation of misfolded proteins and ER-associated apoptosis.

Balanced CoQ6 biosynthesis is required for lifespan and mitophagy in yeast.

Coenzyme Q is an essential lipid with redox capacity that is present in all organisms. In yeast its biosynthesis depends on a multiprotein complex in which Coq7 protein has both catalytic and regulatory functions. Coq7 modulates CoQ6 levels through a phosphorylation cycle, where dephosphorylation of three amino acids (Ser/Thr) by the mitochondrial phosphatase Ptc7 increases the levels of CoQ6. Here we analyzed the role of Ptc7 and the phosphorylation state of Coq7 in yeast mitochondrial function. The conversion of the three Ser/Thr to alanine led to a permanently active form of Coq7 that caused a 2.5-fold increase of CoQ6 levels, albeit decreased mitochondrial respiratory chain activity and oxidative stress resistance capacity. This resulted in an increase in endogenous ROS production and shortened the chronological life span (CLS) compared to wild type. The null PTC7 mutant (ptc7∆) strain showed a lower biosynthesis rate of CoQ6 and a significant shortening of the CLS. The reduced CLS observed in ptc7Δ was restored by the overexpression of PTC7 but not by the addition of exogenous CoQ6. Overexpression of PTC7 increased mitophagy in a wild type strain. This finding suggests an additional Ptc7 function beyond the regulation of CoQ biosynthesis. Genetic disruption of PTC7 prevented mitophagy activation in conditions of nitrogen deprivation. In brief, we show that, in yeast, Ptc7 modulates the adaptation to respiratory metabolism by dephosphorylating Coq7 to supply newly synthesized CoQ6, and by activating mitophagy to remove defective mitochondria at stationary phase, guaranteeing a proper CLS in yeast.

Ubiquitination of ERMES components by the E3 ligase Rsp5 is involved in mitophagy

ABSTRACTMitochondria are dynamic organelles that undergo permanent fission and fusion events. These processes play an essential role in maintaining normal cellular function. In the yeast Saccharomyces cerevisiae, the endoplasmic reticulum-mitochondrial encounter structure (ERMES) is a marker of sites of mitochondrial division, but it is also involved in a plethora of other mitochondrial functions. However, it remains unclear how these different functions are regulated. We show here that Mdm34 and Mdm12, 2 components of ERMES, are ubiquitinated by the E3 ligase Rsp5. This ubiquitination is not involved in mitochondrial dynamics or in the distribution and turnover of ERMES. Nevertheless, the ubiquitination of Mdm34 and Mdm12 was required for efficient mitophagy. We thus report here the first identification of ubiquitinated substrates participating in yeast mitophagy.

Mitophagy in Yeast: A Screen of Mitophagy-Deficient Mutants.

Mitochondrial autophagy (mitophagy) is a process that selectively degrades mitochondria via autophagy. Recent studies have shown that mitophagy plays an important role in mitochondrial homeostasis by degrading damaged or excess mitochondria. The budding yeast Saccharomyces cerevisiae is a powerful model organism that has been employed to study several biological phenomena. Recently, there has been significant progress in the understanding of mitophagy in yeast following the identification of Atg32, a mitochondrial outer membrane receptor protein for mitophagy. In this chapter, we describe protocols to study mitophagy in yeast via a genome-wide screen for mitophagy-deficient mutants using fluorescence microscopy and immunoblotting.

Assays for Mitophagy in Yeast.

Elimination of damaged or surplus mitochondria is crucial to maintain cellular integrity and an energy supply-demand balance. Mitophagy serves to selectively catabolize mitochondria in a manner dependent on autophagy, and contributes to mitochondrial quality and quantity control. This degradation system is highly conserved among eukaryotes including the budding yeast, Saccharomyces cerevisiae. Therefore, analyses of mitophagy using yeast have the potential to provide insights into the common mechanisms of mitophagy. Here, we introduce experimental approaches with fluorescence microscopy and western blotting to validate mitophagy in yeast. The former is useful to visualize transport of mitochondria to the vacuole, a lytic compartment, and formation of mitochondria-containing autophagosomes. The latter allows us to quantify mitochondria degradation.

Investigation of Yeast Mitophagy with Fluorescence Microscopy and Western Blotting.

Selective clearance of superfluous or dysfunctional mitochondria is a fundamental process that depends on the autophagic membrane trafficking pathways found in many cell types. This catabolic event, called mitophagy, is conserved from yeast to humans and serves to control mitochondrial quality and quantity. In budding yeast, degradation of mitochondria occurs under various physiological conditions, such as respiration at stationary phase, or starvation in a prolonged period. During these events, the transmembrane protein Atg32 localizes to the mitochondrial surface and plays a specific and essential role in yeast mitophagy. In this chapter, we describe methods to observe transport of mitochondria to the vacuole, a lytic compartment in yeast, using fluorescence microscopy, and semi-quantify the progression of Atg32-mediated mitophagy by Western blotting.

酵母におけるマイトファジーの分子機構と生理的役割

酵母におけるマイトファジーの分子機構と生理的役割

Phospholipid methylation controls Atg32-mediated mitophagy and Atg8 recycling.

Degradation of mitochondria via selective autophagy, termed mitophagy, contributes to mitochondrial quality and quantity control whose defects have been implicated in oxidative phosphorylation deficiency, aberrant cell differentiation, and neurodegeneration. How mitophagy is regulated in response to cellular physiology remains obscure. Here, we show that mitophagy in yeast is linked to the phospholipid biosynthesis pathway for conversion of phosphatidylethanolamine to phosphatidylcholine by the two methyltransferases Cho2 and Opi3. Under mitophagy-inducing conditions, cells lacking Opi3 exhibit retardation of Cho2 repression that causes an anomalous increase in glutathione levels, leading to suppression of Atg32, a mitochondria-anchored protein essential for mitophagy. In addition, loss of Opi3 results in accumulation of phosphatidylmonomethylethanolamine (PMME) and, surprisingly, generation of Atg8-PMME, a mitophagy-incompetent lipid conjugate of the autophagy-related ubiquitin-like modifier. Amelioration of Atg32 expression and attenuation of Atg8-PMME conjugation markedly rescue mitophagy in opi3-null cells. We propose that proper regulation of phospholipid methylation is crucial for Atg32-mediated mitophagy.

BCL2L13 is a mammalian homolog of the yeast mitophagy receptor Atg32

Although Atg32 is essential for mitophagy in yeast, no mammalian homolog has been identified. Here, we demonstrate that BCL2L13 (BCL2-like 13 [apoptosis facilitator]) is a functional mammalian homolog of Atg32. First, we hypothesized that a mammalian mitophagy receptor will share certain molecular features with Atg32. Using the molecular profile of Atg32 as a search tool, we screened public databases for novel Atg32 functional homologs and identified BCL2L13. BCL2L13 induces mitochondrial fragmentation and mitophagy in HEK293 cells. In BCL2L13, the BH domains are important for fragmentation, whereas the WXXI motif, an LC3 interacting region, is needed for mitophagy. BCL2L13 induces mitochondrial fragmentation and mitophagy even in the absence of DNM1L/Drp1 and PARK2/Parkin, respectively. BCL2L13 is indispensable for mitochondrial damage-induced fragmentation and mitophagy. Furthermore, BCL2L13 induces mitophagy in Atg32-deficient yeast. Induction and/or phosphorylation of BCL2L13 may regulate its activity. Our findings thus open a new chapter in mitophagy research.

Protein N-terminal Acetylation by the NatA Complex Is Critical for Selective Mitochondrial Degradation

Mitophagy is an evolutionarily conserved autophagy pathway that selectively degrades mitochondria. Although it is well established that this degradation system contributes to mitochondrial quality and quantity control, mechanisms underlying mitophagy remain largely unknown. Here, we report that protein N-terminal acetyltransferase A (NatA), an enzymatic complex composed of the catalytic subunit Ard1 and the adaptor subunit Nat1, is crucial for mitophagy in yeast. NatA is associated with the ribosome via Nat1 and acetylates the second amino acid residues of nascent polypeptides. Mitophagy, but not bulk autophagy, is strongly suppressed in cells lacking Ard1, Nat1, or both proteins. In addition, loss of NatA enzymatic activity causes impairment of mitochondrial degradation, suggesting that protein N-terminal acetylation by NatA is important for mitophagy. Ard1 and Nat1 mutants exhibited defects in induction of Atg32, a protein essential for mitophagy, and formation of mitochondria-specific autophagosomes. Notably, overexpression of Atg32 partially recovered mitophagy in NatA-null cells, implying that this acetyltransferase participates in mitophagy at least in part via Atg32 induction. Together, our data implicate NatA-mediated protein modification as an early regulatory step crucial for efficient mitophagy.

Bcl-2-like protein 13 is a mammalian Atg32 homologue that mediates mitophagy and mitochondrial fragmentation

Damaged mitochondria are removed by mitophagy. Although Atg32 is essential for mitophagy in yeast, no Atg32 homologue has been identified in mammalian cells. Here, we show that Bcl-2-like protein 13 (Bcl2-L-13) induces mitochondrial fragmentation and mitophagy in mammalian cells. First, we hypothesized that unidentified mammalian mitophagy receptors would share molecular features of Atg32. By screening the public protein database for Atg32 homologues, we identify Bcl2-L-13. Bcl2-L-13 binds to LC3 through the WXXI motif and induces mitochondrial fragmentation and mitophagy in HEK293 cells. In Bcl2-L-13, the BH domains are important for the fragmentation, while the WXXI motif facilitates mitophagy. Bcl2-L-13 induces mitochondrial fragmentation in the absence of Drp1, while it induces mitophagy in Parkin-deficient cells. Knockdown of Bcl2-L-13 attenuates mitochondrial damage-induced fragmentation and mitophagy. Bcl2-L-13 induces mitophagy in Atg32-deficient yeast cells. Induction and/or phosphorylation of Bcl2-L-13 may regulate its activity. Our findings offer insights into mitochondrial quality control in mammalian cells.

Synthetic quantitative array technology identifies the Ubp3-Bre5 deubiquitinase complex as a negative regulator of mitophagy.

Mitophagy is crucial to ensuring mitochondrial quality control. However, the molecular mechanism and regulation of mitophagy are still not fully understood. Here, we developed a quantitative methodology termed synthetic quantitative array (SQA) technology, which allowed us to perform a genome-wide screen for modulators of rapamycin-induced mitophagy in S.cerevisiae. SQA technology can be easily employed for other enzyme-based reporter systems and widely applied in yeast research. We identified 86 positive and 10 negative regulators of mitophagy. Moreover, SQA-based analysis of non-selective autophagy revealed that 63 of these regulators are specific for mitophagy and 33 regulate autophagy in general. The Ubp3-Bre5 deubiquitination complex was found to inhibit mitophagy but, conversely, topromote other types of autophagy, including ribophagy. This complex translocates dynamically to mitochondria upon induction of mitophagy. These findings point to a role of ubiquitination in mitophagy in yeast and suggest a reciprocal regulation of distinct autophagy pathways.

Receptor-mediated mitophagy in yeast and mammalian systems.

Mitophagy, or mitochondria autophagy, plays a critical role in selective removal of damaged or unwanted mitochondria. Several protein receptors, including Atg32 in yeast, NIX/BNIP3L, BNIP3 and FUNDC1 in mammalian systems, directly act in mitophagy. Atg32 interacts with Atg8 and Atg11 on the surface of mitochondria, promoting core Atg protein assembly for mitophagy. NIX/BNIP3L, BNIP3 and FUNDC1 also have a classic motif to directly bind LC3 (Atg8 homolog in mammals) for activation of mitophagy. Recent studies have shown that receptor-mediated mitophagy is regulated by reversible protein phosphorylation. Casein kinase 2 (CK2) phosphorylates Atg32 and activates mitophagy in yeast. In contrast, in mammalian cells Src kinase and CK2 phosphorylate FUNDC1 to prevent mitophagy. Notably, in response to hypoxia and FCCP treatment, the mitochondrial phosphatase PGAM5 dephosphorylates FUNDC1 to activate mitophagy. Here, we mainly focus on recent advances in our understanding of the molecular mechanisms underlying the activation of receptor-mediated mitophagy and the implications of this catabolic process in health and disease.