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内容記述 |
Macroautophagy is a bulk degradation process induced by starvation in eukaryotic cells．In yeast, Saccharomyces cerevisiae, at least 15 Apg proteins are essential for the formation of autophagosomes．Recently, several key reactions mediated by Apg proteins have been described: two ubiquitin-like conjugation systems, a phosphatidylinositol 3-kinase (PI3- kinase) system and a protein kinase system．Two ubiquitin-like systems consist of Apg12 system and Aut7/Apg8 system．Apg12p, Apg7p, Apg10p, Apg5p and Apg16p are involved in Apg12 system．This system finally produces a stable complex, namely the Apg12p-Apg5p- Apg16p complex．In Aut7 system, phosphatidylethanolamine-conjugated Aut7p (Aut7p-PE)，which is required for the progression of autophagy, is generated by the sequential reactions of Aut2p/Apg4p, Apg7p and Aut1p/Apg3p to Aut7p．Vps30p/Apg6p and Apg14p form a specific PI3-kinase complex essential for autophagy．The protein kinase complex is composed of Apg1p, Apg13p and Apg17p．The kinase activity of Apg1p is enhanced by induction of autophagy．Despite of such detailed molecular analyses, the overall network between these reactions still remains to be elucidated．<br />Electron microscopic study showed that Aut7p is 1ocalized on the membranes of autophagosomes in the course of formation, and finally in the lumen of autophagosomes．Green fluorescent protein (GFP)-fused Aut7p was constructed and its dynamics was examined by fluorescence microscopy．GFP-Aut7p labeled autophagosomes, autophagic bodies and a novel structure which I designated as the pre-autophagosomal structure．Further analyses demonstrated that this pre-autophagosomal structure, containing at least five Apg proteins, i．e．Apg1p, Apg2p, Apg5p, Aut7p and Apg16p, is localized in the vicinity of the vacuole and directly involved in autophagosome formation．Analysis of apg mutants revealed that the formation of both Aut7p-PE and the Apg12p-Apg5p conjugate is essential for the localization of Aut7p to the pre-autophagosomal structure．Vps30p and Apg14p, components of an autophagy-specific PI3-kinase complex, Apg9p and Apg16p are all required for the localization of Apg5p and Aut7p to the structure．The Apg1p protein kinase complex functions in the late stage of autophagosome formation on the pre-autophagosomal structure．Thc pre-autophagosomal structure, which is organized by concerted interactions of two conjugation systems and the PI3-kinase system, plays a pivotal role in autophagosome formation．<br />Aminopeptidase I (API) is a vacuolar hydrolase,which is selectively delivered via autophagosomes under starvation conditions and becomes mature form after processing by vacuolar proteases．Electron microscopic study demonstrated that the proform of API forms the cytosol-to-vacuole targeting (Cvt) complex, which is enwrapped by autophagosome and delivered to the vacuole．I constructed a strain expressing API-GFP and investigated the dynamics of cargo delivery by autophagosome．Fluorescence microscopy showed that API- GFP is concentrated on a dot close to the vacuole．This dot, which corresponds to the Cvt complex,mostly appears very close to the pre-autophagosomal structure．Interestingly，formation of the Cvt complex is not impaired in the absence of any Apg protein examined or Cvt19p, a receptor for the Cvt pathway．The time-lapse observation using apg1ts cells clarified that the Cvt complex stays outside the vacuole at the non-permissive temperature and is transported to the vacuole after shifting to the permissive temperature．The whole Cvt complex is delivered to the vacuole by one step,indicating the complex is engulfed into one autophagosome．The complex, which entered the vacuole, is immediately disintegrated and diffused throughout the vacuole in one minute．API-GFP enables me to analyze the localization of Cvt complexes and the dynamics of cargo delivery mediated by autophagosome．<br />In this study,I discovered and described the pre-autophagosomal structure involved in generating autophagosomes．Several Apg proteins are converged upon the structure as a consequence of interaction of Apg proteins．Moreover, I successfully visualized the process of a cargo transport to the vacuole with API-GFP．API forms a Cvt complex on the pre- autophagosomal structure．This complex is enclosed into autophagosome and finally delivered to the vacuole．Here,I described the overall image of autophagosome formation and cargo delivery during autophagy by GFP-Aut7p andAPI-GFP as markers of autophagosome and the pre-autophagosomal structure,and a cargo,respectively． |
要旨・審査要旨 / Abstract, Screening Result (256.9 kB)
