Atg5 is covalently modified with a ubiquitin-like modifier, Atg12, and the Atg12-Atg5 conjugate further forms a complex with the multimeric protein Atg16. The Atg12-Atg5⅐Atg16 multimeric complex plays an essential role in autophagy, the bulk degradation system conserved in all eukaryotes. We have reported here the crystal structure of Atg5 complexed with the N-terminal region of Atg16 at 1.97 Å resolution. Atg5 comprises two ubiquitin-like domains that flank a helix-rich domain. The N-terminal region of Atg16 has a helical structure and is bound to the groove formed by these three domains. In vitro analysis showed that Arg-35 and Phe-46 of Atg16 are crucial for the interaction. Atg16, with a mutation at these residues, failed to localize to the pre-autophagosomal structure and could not restore autophagy in Atg16-deficient yeast strains. Furthermore, these Atg16 mutants could not restore a severe reduction in the formation of the Atg8-phosphatidylethanolamine conjugate, another essential factor for autophagy, in Atg16-deficient strains under starvation conditions. These results taken together suggest that the direct interaction between Atg5 and Atg16 is crucial to the performance of their roles in autophagy.
Aniline, which causes serious environmental problems, was electrochemically treated and thereby removed from aqueous solution. This method demonstrated herein is based on the formation of polyaniline at an electrode surface by electrochemical oxidation. Initially, the electrode behavior of aniline at several conditions was investigated on a platinum, glassy carbon (GC), and carbon fiber (CF) electrode using voltammetric techniques. On the platinum electrode, aniline did not polymerize at low concentrations. However, electropolymerization of aniline was observed on both the GC and CF electrodes even at low concentrations in neutral pH solutions. The removal of aniline was carried out using a CF bundle with a large surface area. High removal efficiency for aniline was obtained by applying potentials greater than 0.8 V. Although generation of p-benzoquinone as a byproduct was observed during electrochemical treatment, its generation was suppressed by applying a potential lower than 0.9 V. The cyclic voltammograms and X-ray photoelectron spectra of the CF after the treatment for aniline solution showed that polymeric aniline existed on the CF surface. The maximum surface coverage of electropolymerized aniline was estimated to be about 1.49 x 10(-8) mol/cm2. Furthermore, the continuous treatment of aniline was demonstrated using a flow system.
Acanthamoeba isolated from environmental soil harbors the obligate intracellular symbiont Neochlamydia, which has a critical role in host amoebal defense against Legionella pneumophila infection. Here, by using morphological analysis with confocal laser scanning fluorescence microscopy and transmission electron microscopy, proteome analyses with two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and liquid chromatography-mass spectrometry (LC/MS), and transcriptome analysis with DNA microarray, we explored the mechanism by which the Neochlamydia affected this defense. We observed that when rare uptake did occur, the symbiotic amoebae allowed Legionella to grow normally. However, the symbiotic amoebae had severely reduced uptake of Legionella when compared with the aposymbiotic amoebae. Also, in contrast to amoebae carrying the endosymbiont, the actin cytoskeleton was significantly disrupted by Legionella infection in aposymbiotic amoebae. Furthermore, despite Legionella exposure, there was little change in Neochlamydia gene expression. Taken together, we concluded that the endosymbiont, Neochlamydia prevents Legionella entry to the host amoeba, resulting in the host defense against Legionella infection.
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