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.

The autophagy-related protein kinase Atg1 interacts with the ubiquitin-like protein Atg8 via the Atg8 family interacting motif to facilitate autophagosome formation.

In autophagy, a cup-shaped membrane called the isolation membrane is formed, expanded, and sealed to complete a double membrane-bound vesicle called the autophagosome that encapsulates cellular constituents to be transported to and degraded in the lysosome/vacuole. The formation of the autophagosome requires autophagy-related (Atg) proteins. Atg8 is a ubiquitin-like protein that localizes to the isolation membrane; a subpopulation of this protein remains inside the autophagosome and is transported to the lysosome/vacuole. In the budding yeast Saccharomyces cerevisiae, Atg1 is a serine/threonine kinase that functions in the initial step of autophagosome formation and is also efficiently transported to the vacuole via autophagy. Here, we explore the mechanism and significance of this autophagic transport of Atg1. In selective types of autophagy, receptor proteins recognize degradation targets and also interact with Atg8, via the Atg8 family interacting motif (AIM), to link the targets to the isolation membrane. We find that Atg1 contains an AIM and directly interacts with Atg8. Mutations in the AIM disrupt this interaction and abolish vacuolar transport of Atg1. These results suggest that Atg1 associates with the isolation membrane by binding to Atg8, resulting in its incorporation into the autophagosome. We also show that mutations in the Atg1 AIM cause a significant defect in autophagy, without affecting the functions of Atg1 implicated in triggering autophagosome formation. We propose that in addition to its essential function in the initial stage, Atg1 also associates with the isolation membrane to promote its maturation into the autophagosome.

Two distinct mechanisms target the autophagy-related E3 complex to the pre-autophagosomal structure.

In autophagy, Atg proteins organize the pre-autophagosomal structure (PAS) to initiate autophagosome formation. Previous studies in yeast revealed that the autophagy-related E3 complex Atg12-Atg5-Atg16 is recruited to the PAS via Atg16 interaction with Atg21, which binds phosphatidylinositol 3-phosphate (PI3P) produced at the PAS, to stimulate conjugation of the ubiquitin-like protein Atg8 to phosphatidylethanolamine. Here, we discover a novel mechanism for the PAS targeting of Atg12-Atg5-Atg16, which is mediated by the interaction of Atg12 with the Atg1 kinase complex that serves as a scaffold for PAS organization. While autophagy is partially defective without one of these mechanisms, cells lacking both completely lose the PAS localization of Atg12-Atg5-Atg16 and show no autophagic activity. As with the PI3P-dependent mechanism, Atg12-Atg5-Atg16 recruited via the Atg12-dependent mechanism stimulates Atg8 lipidation, but also has the specific function of facilitating PAS scaffold assembly. Thus, this study significantly advances our understanding of the nucleation step in autophagosome formation.

Localization of Atg3 to autophagy-related membranes and its enhancement by the Atg8-family interacting motif to promote expansion of the membranes.

The E2 enzyme Atg3 conjugates the ubiquitin-like protein Atg8 to phosphatidylethanolamine (PE) to drive autophagosome formation in Saccharomyces cerevisiae. In this study, we show that Atg3 localizes to the pre-autophagosomal structure (PAS) and the isolation membrane (IM), providing crucial evidence that Atg8-PE conjugates are produced on these structures. We also find that mutations in the Atg8-family interacting motif (AIM) of Atg3 significantly impairs the PAS/IM localization of Atg3, resulting in inefficient IM expansion. It is suggested that the AIM-mediated PAS/IM localization of Atg3 facilitates membrane expansion in these structures probably by ensuring active production of Atg8-PE on the membranes.

A novel role for 12/15-lipoxygenase in regulating autophagy.

12/15-Lipoxygenase (LOX) enzymatically generates oxidized phospholipids in monocytes and macrophages. Herein, we show that cells deficient in 12/15-LOX contain defective mitochondria and numerous cytoplasmic vacuoles containing electron dense material, indicating defects in autophagy or membrane processing, However, both LC3 expression and lipidation were normal both basally and on chloroquine treatment. A LOX-derived oxidized phospholipid, 12-hydroxyeicosatetraenoic acid-phosphatidylethanolamine (12-HETE-PE) was found to be a preferred substrate for yeast Atg8 lipidation, versus native PE, while both native and oxidized PE were effective substrates for LC3 lipidation. Last, phospholipidomics demonstrated altered levels of several phospholipid classes. Thus, we show that oxidized phospholipids generated by 12/15-LOX can act as substrates for key proteins required for effective autophagy and that cells deficient in this enzyme show evidence of autophagic dysfunction. The data functionally link phospholipid oxidation with autophagy for the first time.

Hrr25 triggers selective autophagy-related pathways by phosphorylating receptor proteins.

In selective autophagy, degradation targets are specifically recognized, sequestered by the autophagosome, and transported into the lysosome or vacuole. Previous studies delineated the molecular basis by which the autophagy machinery recognizes those targets, but the regulation of this process is still poorly understood. In this paper, we find that the highly conserved multifunctional kinase Hrr25 regulates two distinct selective autophagy-related pathways in Saccharomyces cerevisiae. Hrr25 is responsible for the phosphorylation of two receptor proteins: Atg19, which recognizes the assembly of vacuolar enzymes in the cytoplasm-to-vacuole targeting pathway, and Atg36, which recognizes superfluous peroxisomes in pexophagy. Hrr25-mediated phosphorylation enhances the interactions of these receptors with the common adaptor Atg11, which recruits the core autophagy-related proteins that mediate the formation of the autophagosomal membrane. Thus, this study introduces regulation of selective autophagy as a new role of Hrr25 and, together with other recent studies, reveals that different selective autophagy-related pathways are regulated by a uniform mechanism: phosphoregulation of the receptor-adaptor interaction.

Atg12-Atg5 conjugate enhances E2 activity of Atg3 by rearranging its catalytic site.

Two autophagy-related ubiquitin-like systems have unique features: the E2 enzyme Atg3 conjugates the ubiquitin-like protein Atg8 to the lipid phosphatidylethanolamine, and the other ubiquitin-like protein conjugate Atg12-Atg5 promotes that conjugase activity of Atg3. Here, we elucidate the mode of this action of Atg12-Atg5 as a new E3 enzyme by using Saccharomyces cerevisiae proteins. Biochemical analyses based on structural information suggest that Atg3 requires a threonine residue to catalyze the conjugation reaction instead of the typical asparagine residue used by other E2 enzymes. Moreover, the catalytic cysteine residue of Atg3 is arranged in the catalytic center such that the conjugase activity is suppressed; Atg12-Atg5 induces a reorientation of the cysteine residue toward the threonine residue, which enhances the conjugase activity of Atg3. Thus, this study reveals the mechanism of the key reaction that drives membrane biogenesis during autophagy.

Roles of protein-disulfide isomerase-mediated disulfide bond formation of yeast Mnl1p in endoplasmic reticulum-associated degradation.

The endoplasmic reticulum (ER) has a strict protein quality control system. Misfolded proteins generated in the ER are degraded by the ER-associated degradation (ERAD). Yeast Mnl1p consists of an N-terminal mannosidase homology domain and a less conserved C-terminal domain and facilitates the ERAD of glycoproteins. We found that Mnl1p is an ER luminal protein with a cleavable signal sequence and stably interacts with a protein-disulfide isomerase (PDI). Analyses of a series of Mnl1p mutants revealed that interactions between the C-terminal domain of Mnl1p and PDI, which include an intermolecular disulfide bond, are essential for subsequent introduction of a disulfide bond into the mannosidase homology domain of Mnl1p by PDI. This disulfide bond is essential for the ERAD activity of Mnl1p and in turn stabilizes the prolonged association of PDI with Mnl1p. Close interdependence between Mnl1p and PDI suggests that these two proteins form a functional unit in the ERAD pathway.