Showing papers by "Hitoshi Nakatogawa published in 2013"

Recruitment of the autophagic machinery to endosomes during infection is mediated by ubiquitin

Abstract: Although ubiquitin is thought to be important for the autophagic sequestration of invading bacteria (also called xenophagy), its precise role remains largely enigmatic. Here we determined how ubiquitin is involved in this process. After invasion, ubiquitin is conjugated to host cellular proteins in endosomes that contain Salmonella or transfection reagent–coated latex (polystyrene) beads, which mimic invading bacteria. Ubiquitin is recognized by the autophagic machinery independently of the LC3–ubiquitin interaction through adaptor proteins, including a direct interaction between ubiquitin and Atg16L1. To ensure that invading pathogens are captured and degraded, Atg16L1 targeting is secured by two backup systems that anchor Atg16L1 to ubiquitin-decorated endosomes. Thus, we reveal that ubiquitin is a pivotal molecule that connects bacteria-containing endosomes with the autophagic machinery upstream of LC3.

Two ubiquitin-like conjugation systems that mediate membrane formation during autophagy

Abstract: In autophagy, the autophagosome, a transient organelle specialized for the sequestration and lysosomal or vacuolar transport of cellular constituents, is formed via unique membrane dynamics. This process requires concerted actions of a distinctive set of proteins named Atg (autophagy-related). Atg proteins include two ubiquitin-like proteins, Atg12 and Atg8 [LC3 (light-chain 3) and GABARAP (γ-aminobutyric acid receptor-associated protein) in mammals]. Sequential reactions by the E1 enzyme Atg7 and the E2 enzyme Atg10 conjugate Atg12 to the lysine residue in Atg5, and the resulting Atg12-Atg5 conjugate forms a complex with Atg16. On the other hand, Atg8 is first processed at the C-terminus by Atg4, which is related to ubiquitin-processing/deconjugating enzymes. Atg8 is then activated by Atg7 (shared with Atg12) and, via the E2 enzyme Atg3, finally conjugated to the amino group of the lipid PE (phosphatidylethanolamine). The Atg12-Atg5-Atg16 complex acts as an E3 enzyme for the conjugation reaction of Atg8; it enhances the E2 activity of Atg3 and specifies the site of Atg8-PE production to be autophagy-related membranes. Atg8-PE is suggested to be involved in autophagosome formation at multiple steps, including membrane expansion and closure. Moreover, Atg4 cleaves Atg8-PE to liberate Atg8 from membranes for reuse, and this reaction can also regulate autophagosome formation. Thus these two ubiquitin-like systems are intimately involved in driving the biogenesis of the autophagosomal membrane.

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

Abstract: In the yeast autophagy system, the Atg12–Atg5 conjugate acts as an E3 to promote the E2 activity of Atg3, which conjugates Atg8 to phosphatidylethanolamine. Now structural and biochemical analyses reveal that Atg12–Atg5 induces a rearrangement in the catalytic center of Atg3, which employs a threonine residue in addition to the active cysteine to catalyze the conjugation reaction.