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.

Mitochondrial degradation during starvation is selective and temporally distinct from bulk autophagy in yeast.

Selective degradation of mitochondria is a fundamental process that depends on formation of autophagy-related double-membrane vesicles exclusive to mitochondria, and is thus termed mitophagy. In yeast, mitophagy is induced by a shift from respiration to starvation, or prolonged respiratory growth. Here we show that mitochondrial degradation in yeast also occurs selectively under starvation conditions even without respiration. Induction of mitophagy takes place much later than that of bulk autophagy, requiring Atg11 and Atg32 essential for mitophagy as well as Atg17, Atg29, and Atg31 specific for bulk autophagy. We propose that these two discrete protein complexes cooperatively activate starvation-induced mitophagy.

Autophagy-related protein 32 acts as autophagic degron and directly initiates mitophagy.

Autophagy-related degradation selective for mitochondria (mitophagy) is an evolutionarily conserved process that is thought to be critical for mitochondrial quality and quantity control. In budding yeast, autophagy-related protein 32 (Atg32) is inserted into the outer membrane of mitochondria with its N- and C-terminal domains exposed to the cytosol and mitochondrial intermembrane space, respectively, and plays an essential role in mitophagy. Atg32 interacts with Atg8, a ubiquitin-like protein localized to the autophagosome, and Atg11, a scaffold protein required for selective autophagy-related pathways, although the significance of these interactions remains elusive. In addition, whether Atg32 is the sole protein necessary and sufficient for initiation of autophagosome formation has not been addressed. Here we show that the Atg32 IMS domain is dispensable for mitophagy. Notably, when anchored to peroxisomes, the Atg32 cytosol domain promoted autophagy-dependent peroxisome degradation, suggesting that Atg32 contains a module compatible for other organelle autophagy. X-ray crystallography reveals that the Atg32 Atg8 family-interacting motif peptide binds Atg8 in a conserved manner. Mutations in this binding interface impair association of Atg32 with the free form of Atg8 and mitophagy. Moreover, Atg32 variants, which do not stably interact with Atg11, are strongly defective in mitochondrial degradation. Finally, we demonstrate that Atg32 forms a complex with Atg8 and Atg11 prior to and independent of isolation membrane generation and subsequent autophagosome formation. Taken together, our data implicate Atg32 as a bipartite platform recruiting Atg8 and Atg11 to the mitochondrial surface and forming an initiator complex crucial for mitophagy.

Mitochondria and autophagy: critical interplay between the two homeostats.

BACKGROUND: Mitochondria are dynamic organelles that frequently change their number, size, shape, and distribution in response to intra- and extracellular cues. After proliferated from pre-existing ones, fresh mitochondria enter constant cycles of fission and fusion that organize them into two distinct states - "individual state" and "network state". When compromised with various injuries, solitary mitochondria are subjected to organelle degradation. This clearance pathway relies on autophagy, a self-eating process that plays key roles in manifold cell activities. Recent studies reveal that defects in autophagic degradation selective for mitochondria (mitophagy) are associated with neurodegenerative diseases, highlighting the physiological relevance to cellular functions. SCOPE OF REVIEW: Here we review recent progress regarding a link between mitochondria and autophagy in yeast and multicellular eukaryotes. In particular, fundamental principles underlying mitophagy, and mitochondrial quality control are emphasized. Accumulating evidence also implicates nonselective autophagy in the management of mitochondrial fitness. Conversely, mitochondria are suggested to serve as signaling platforms vital for regulating autophagy. These interdependent relationships are likely to coordinate metabolic plasticity in the cell. MAJOR CONCLUSIONS: Mitochondria and autophagy are elaborately linked homeostatic elements that act in response to changes in cellular environment such as energy, nutrient, and stress. How cells integrate these double membrane-bound systems still remains elusive. GENERAL SIGNIFICANCE: Interplay between mitochondria and autophagy seems to be evolutionarily conserved. Defects in one of these elements could simultaneously impair the other, resulting in risk increments for various human diseases. This article is part of a Special Issue entitled Biochemistry of Mitochondria.

A landmark protein essential for mitophagy: Atg32 recruits the autophagic machinery to mitochondria.

Degradation of mitochondria is a fundamental process conserved from yeast to humans that utilizes the machinery of autophagy. In contrast to starvation-induced, nonselective autophagy responsible for nutrient recycling, selective autophagy, which involves particular cues and receptors required for induction and cargo recognition, respectively, mediates mitochondria-specific breakdown. Although numerous studies highlight that mitochondria autophagy (mitophagy) contributes to homeostatic control of mitochondria, the molecular mechanisms underlying this selective clearance process are poorly understood. Using a genome-wide visual screen, we identified Atg32, a protein essential for mitophagy in budding yeast. During respiratory growth, Atg32 is highly expressed, likely in response to oxidative stress, and anchored on the surface of mitochondria. We also demonstrate that Atg32 interacts with Atg8 and Atg11, proteins critical for recognition of cargo receptors. Notably, Atg32 contains WXXI/L/V, a conserved motif that serves as a binding site for the Atg8 family members. Our recent findings suggest that Atg32 is a transmembrane receptor that directs autophagosome formation to mitochondria.

Mitochondria-anchored receptor Atg32 mediates degradation of mitochondria via selective autophagy.

Mitochondria are essential organelles that produce most of the energy for a cell, but concomitantly accumulate oxidative damage. Degradation of damaged mitochondria is critical for cell homeostasis, and this process is thought to be mediated by mitophagy, an autophagy-related pathway specific for mitochondria. However, whether mitochondria are selectively degraded, and how the autophagic machinery is targeted to mitochondria, remain largely unknown. Here we demonstrate that, in post-log phase cells under respiratory conditions, a substantial fraction of mitochondria are exclusively sequestered as cargoes and transported to the vacuole, a lytic compartment in yeast, in an autophagy-dependent manner. Interestingly, we found Atg32, a mitochondria-anchored protein essential for mitophagy that is induced during respiratory growth. In addition, our data suggest that Atg32 interacts with Atg8 and Atg11, autophagy-related proteins critical for recognition of cargo receptors. We propose that Atg32 acts as a mitophagy-specific receptor and regulates selective degradation of mitochondria.

Tetratricopeptide repeat proteins Tom70 and Tom71 mediate yeast mitochondrial morphogenesis.

The maintenance of correct mitochondrial shape requires numerous proteins that act on the surface or inside of the organelle. Although the soluble F-box protein Mfb1 was recently found to associate peripherally with mitochondria and to regulate organelle connectivity in budding yeast, how it localizes to mitochondria is unknown. Here, we show that two tetratricopeptide repeat proteins-the general preprotein import receptor Tom70 (a component of translocase of the outer membrane) and its paralogue Tom71-are required for Mfb1 mitochondrial localization. Mitochondria in cells lacking Tom70 and Tom71 form short tubules and aggregates, aberrant morphologies similar to those observed in the mfb1-null mutant. In addition, Mfb1 interacts with Tom71 in vivo, and binds to mitochondria through Tom70 in vitro. Our data indicate an unexpected role for Tom70 in recruitment of soluble proteins to the mitochondrial surface, and indicate that Tom71 has a specialized role in Mfb1-mediated mitochondrial morphogenesis.

The novel F-box protein Mfb1p regulates mitochondrial connectivity and exhibits asymmetric localization in yeast.

Although it is clear that mitochondrial morphogenesis is a complex process involving multiple proteins in eukaryotic cells, little is known about regulatory molecules that modulate mitochondrial network formation. Here, we report the identification of a new yeast mitochondrial morphology gene called MFB1 (YDR219C). MFB1 encodes an F-box protein family member, many of which function in Skp1-Cdc53/Cullin-F-box protein (SCF) ubiquitin ligase complexes. F-box proteins also act in non-SCF complexes whose functions are not well understood. Although cells lacking Mfb1p contain abnormally short mitochondrial tubules, Mfb1p is not essential for known pathways that determine mitochondrial morphology and dynamics. Mfb1p is peripherally associated with the mitochondrial surface. Coimmunoprecipitation assays reveal that Mfb1p interacts with Skp1p in an F-box-dependent manner. However, Mfb1p does not coimmunoprecipitate with Cdc53p. The F-box motif is not essential for Mfb1p-mediated mitochondrial network formation. These observations suggest that Mfb1p acts in a complex lacking Cdc53p required for mitochondrial morphogenesis. During budding, Mfb1p asymmetrically localizes to mother cell mitochondria. By contrast, Skp1p accumulates in the daughter cell cytoplasm. Mfb1p mother cell-specific asymmetry depends on the F-box motif, suggesting that Skp1p down-regulates Mfb1p mitochondrial association in buds. We propose that Mfb1p operates in a novel pathway regulating mitochondrial tubular connectivity.

Mmm1p spans both the outer and inner mitochondrial membranes and contains distinct domains for targeting and foci formation.

In the yeast Saccharomyces cerevisiae, the integral membrane protein Mmm1p is required for maintenance of mitochondrial morphology and retention of mitochondrial DNA (mtDNA). Mmm1p localizes to discrete foci on mitochondria that are adjacent to mtDNA nucleoids in the matrix, raising the possibility that this protein plays a direct role in organizing, replicating, or segregating mtDNA. Although Mmm1p has been shown to cross the outer membrane with its C terminus facing the cytoplasm, the location of the N terminus has not been resolved. Here we show that Mmm1p spans both the outer and inner mitochondrial membranes, exposing its N terminus to the matrix. Surprisingly, deletion of the N-terminal extension decreased steady-state levels of the Mmm1 protein but did not affect mitochondrial morphology or mtDNA maintenance. Moreover, expression of Neurospora crassa MMM1, which naturally lacks a long N-terminal extension, substituted for loss of Mmm1p in budding yeast. These results indicate that the matrix-exposed portion of Mmm1p is not essential for mtDNA nucleoid maintenance. Additional studies revealed that the transmembrane segment and C-terminal domain of Mmm1p are required for foci formation and mitochondrial targeting, respectively. Our data suggest that the double membrane-spanning topology of Mmm1p at the membrane contact site is critical for formation of tubular mitochondria.