Takao Hanada

The Atg12-Atg5 Conjugate Has a Novel E3-like Activity for Protein Lipidation in Autophagy

Autophagy is a bulk degradation process in eukaryotic cells; autophagosomes enclose cytoplasmic components for degradation in the lysosome/vacuole. Autophagosome formation requires two ubiquitin-like conjugation systems, the Atg12 and Atg8 systems, which are tightly associated with expansion of autophagosomal membrane. Previous studies have suggested that there is a hierarchy between these systems; the Atg12 system is located upstream of the Atg8 system in the context of Atg protein organization. However, the concrete molecular relationship is unclear. Here, we show using an in vitro Atg8 conjugation system that the Atg12-Atg5 conjugate, but not unconjugated Atg12 or Atg5, strongly enhances the formation of the other conjugate, Atg8-PE. The Atg12-Atg5 conjugate promotes the transfer of Atg8 from Atg3 to the substrate, phosphatidylethanolamine (PE), by stimulating the activity of Atg3. We also show that the Atg12-Atg5 conjugate interacts with both Atg3 and PE-containing liposomes. These results indicate that the Atg12-Atg5 conjugate is a ubiquitin-protein ligase (E3)-like enzyme for Atg8-PE conjugation reaction, distinctively promoting protein-lipid conjugation.

The Crystal Structure of Atg3, an Autophagy-related Ubiquitin Carrier Protein (E2) Enzyme that Mediates Atg8 Lipidation

Atg3 is an E2-like enzyme that catalyzes the conjugation of Atg8 and phosphatidylethanolamine (PE). The Atg8-PE conjugate is essential for autophagy, which is the bulk degradation process of cytoplasmic components by the vacuolar/lysosomal system. We report here the crystal structure of Saccharomyces cerevisiae Atg3 at 2.5-Å resolution. Atg3 has an α/β-fold, and its core region is topologically similar to canonical E2 enzymes. Atg3 has two regions inserted in the core region, one of which consists of ∼80 residues and has a random coil structure in solution and another with a long α-helical structure that protrudes from the core region as far as 30Å. In vivo and in vitro analyses suggested that the former region is responsible for binding Atg7, an E1-like enzyme, and that the latter is responsible for binding Atg8. A sulfate ion was bound near the catalytic cysteine of Atg3, suggesting a possible binding site for the phosphate moiety of PE. The structure of Atg3 provides a molecular basis for understanding the unique lipidation reaction that Atg3 carries out.

Structure of the Atg12–Atg5 conjugate reveals a platform for stimulating Atg8–PE conjugation

Atg12 is conjugated to Atg5 through enzymatic reactions similar to ubiquitination. The Atg12–Atg5 conjugate functions as an E3‐like enzyme to promote lipidation of Atg8, whereas lipidated Atg8 has essential roles in both autophagosome formation and selective cargo recognition during autophagy. However, the molecular role of Atg12 modification in these processes has remained elusive. Here, we report the crystal structure of the Atg12–Atg5 conjugate. In addition to the isopeptide linkage, Atg12 forms hydrophobic and hydrophilic interactions with Atg5, thereby fixing its position on Atg5. Structural comparison with unmodified Atg5 and mutational analyses showed that Atg12 modification neither induces a conformational change in Atg5 nor creates a functionally important architecture. Rather, Atg12 functions as a binding module for Atg3, the E2 enzyme for Atg8, thus endowing Atg5 with the ability to interact with Atg3 to facilitate Atg8 lipidation.

Antibiotic susceptibility of mammalian mitochondrial translation

All medically useful antibiotics should have the potential to distinguish between target microbes (bacteria) and host cells. Although many antibiotics that target bacterial protein synthesis show little effect on the translation machinery of the eukaryotic cytoplasm, it is unclear whether these antibiotics target or not the mitochondrial translation machinery. We employed an in vitro translation system from bovine mitochondria, which consists of mitochondrial ribosomes and mitochondrial elongation factors, to estimate the effect of antibiotics on mitichondrial protein synthesis. Tetracycline and thiostrepton showed similar inhibitory effects on both Escherichia coli and mitochondrial protein synthesis. The mitochondrial system was more resistant to tiamulin, macrolides, virginiamycin, fusidic acid and kirromycin than the E. coli system. The present results, taken together with atomic structure of the ribosome, may provide useful information for the rational design of new antibiotics having less adverse effects in humans and animals.

Structure-function relationship of Atg12, a ubiquitin-like modifier essential for autophagy.

Atg12, a post-translational modifier, is activated and conjugated to Atg5 by a ubiquitin-like conjugation system, though it has no obvious sequence homology to ubiquitin. The Atg12-Atg5 conjugate is essential for autophagy, an intracellular bulk degradation process. Here, we show that the carboxyl-terminal region of Atg12 that is predicted to fold into a ubiquitin-like structure is necessary and sufficient for both conjugation and autophagy, which indicates that the domain essential for autophagy resides in the ubiquitin-fold region. We further show that two hydrophobic residues within the ubiquitin-fold region are important for autophagy: mutation at Y149 affects conjugate formation catalyzed by Atg10, an E2-like enzyme, while mutation at F154 has no effect on Atg12-Atg5 conjugate formation but its hydrophobic nature is essential for autophagy. In response to the F154 mutation, Atg8-PE conjugation, the other ubiquitin-like conjugation in autophagy, is severely reduced and autophagosome formation fails. Gel filtration analysis suggests that F154 plays a critical role in the assembly of a functional Atg12-Atg5?Atg16 complex that is requisite for autophagosome formation.

Translation ability of mitochondrial tRNAsSer with unusual secondary structures in an in vitro translation system of bovine mitochondria

Background Metazoan mitochondrial (mt) tRNAs are structurally quite different from the canonical cloverleaf secondary structure. The mammalian mt tRNASerGCU for AGY codons (Y = C or U) lacks the entire D arm, whereas tRNASerUGA for UCN codons (N = A, G, C or U) has an extended anti‐codon stem. It has been a long‐standing problem to prove experimentally how these tRNAsSer work in the mt translation system.

The amino-terminal region of Atg3 is essential for association with phosphatidylethanolamine in Atg8 lipidation

MINT‐7010457: ATG8 (uniprotkb:P38182) and ATG3 (uniprotkb:P40344) bind (MI:0407) by biochemical (MI:0401)

Structural and Functional Compensation by Proteins for the RNA Deficit of Animal Mitochondrial Translation Systems

Compared to the more usual bacterial translation systems, animal mitochondrial translation systems are unique in their use of extremely simplified RNAs. It seems that the RNA deficit of these mitochondrial systems is compensated by the presence of novel proteins and/or bacterial homologues that have longer N- or C-terminal regions, certain of which may compensate for missing regions in animal mitochondrial RNA. On the basis of two distinct cases we discuss the possibility that certain structures and functions of bacterial RNAs were transferred to proteins during the evolutionary process from ancestral to extant organisms. Our analyses may provide appropriate models for an experimental verification of the concept of transition from the “RNA world” to the “RNP world” in the early process of the evolution of life.