Studies on the ubiquitin-like conjugation reaction of Atg8 required for autophagosome formation
Year: 2009
Type: dissertation
Abstract: Autophagy is a major self-degradative process in eukaryotic cells that plays fundamental roles in cellular and organismal homeostasis, and is involved in many physiological and pathological situations. When autophagy is induced, cytoplasmic materials and organelles are sequestered into newly emerging double-membrane vesicles called autophagosomes, and delivered to the lysosome or the vacuole for degradation. In the past decade, many ATG (autophagy-related) genes have been identified by genetic approaches using the yeast Saccharomyces cerevisiae. Atg8, a ubiquitin-like protein (Ubl), is one of the proteins essential for autophagosome formation. The cysteine protease Atg4 first removes the C-terminal arginine of Atg8 to expose the glycine as the new terminus. This glycine forms a thioester bond with Atg7, an activating enzyme (E1), and transfers to and also forms a thioester bond with Atg3, a conjugation enzyme (E2). Atg8 is eventually conjugated to the amino group in the hydrophilic head of phosphatidylethanolamine (PE). Atg8 is anchored to isolation membrane and autophagosomal membranes probably as this lipid-modified form, and thought to directly participate in the formation of these membranes. Atg4 also catalyzes the deconjugation of Atg8-PE after it has fulfilled its role in autophagosome formation, thus Atg8 is reused. Because the details of this sequential reaction of Atg8 lipidation are unclear, I focus on the mechanism of Atg8 lipidation, in this study. The Atg8 conjugation system was reconstituted using purified proteins expressed in Escherichia coliand PE-containing liposomes in vitro. First, I successfully capture authentic thioester intermediates, Atg8-Atg7 and Atg8-Atg3, which can not have been detected because of their lability. This allows me to analyze the sequential reaction of Atg8 lipidation. It was shown that Atg8 could be conjugated with phosphatidylserine (PS) as efficiently as PE in vitro. However, PE was identified as the sole lipid conjugated to the C-terminal glycine of Atg8 in vivo. It suggests that there exists a mechanism that directs Atg8 conjugation preferentially to PE in the cell. In this study, I show that, in contrast to PE conjugation, the PS conjugation of Atg8 is markedly suppressed at physiological (neutral) pH. Then, I show that both of the Atg8-Atg7 and the Atg8-Atg3 intermediates are formed in the presence of PS liposomes as rapidly as in the presence of PE liposomes, and transfer of Atg8 from Atg3 to PS is specifically retarded at neutral pH. Furthermore, the addition of acidic phospholipids to liposomes is also suggested to result in the preferential formation of the Atg8−PE conjugate. I also show that the acidic phospholipids specifically promote the recruitment of the Atg8-Atg7 and the Atg8-Atg3 thioester intermediates to the membrane. Furthermore, .it was reported that the Atg12-Atg5 conjugate, which is formed by ubiquitin-like conjugation reaction, is indispensable for Atg8-PE production in vivo, and that recombinant Atg12-Atg5 indeed stimulates Atg8-PE and Atg8-PS production in vitro. The preferential formation of Atg8-PE can be achieved by combination of neutral pH, acidic phospholipids, and the Atg12-Atg5 conjugate. Furthermore, I show that PS is not essential for autophagosome formation even if Atg8 is conjugated to PS in vivo, because the autophagic activity of cells deficient for the PS synthesis enzyme-deficient (pss1△) cells was normal. However, in vitro, the less efficient but significant production of Atg8-PS was still observed, suggesting that the exclusive formation of Atg8-PE requires precise in vivo settings for these factors and/or other facto(s). Alternatively, this result may imply the production of Atg8-PS in vivo, although its amount should be much less than the PE oonjugate. Previously, PE was detected as the sole lipid conjugated to Atg8 and its mammalian homolog LC3 (microtubule-associated protein light chain 3) in vivo. However, lipidated Atg8 and LC3 were forced to accumulate by mutation or treatment with lysosomal inhibitors under nutrient-replete conditions. Therefore, there also remains an alternative possibility that Atg8-PS is formed in starved cells undergoing autophagy, so, I try to purify lipidated Atg8 from the cells under starvation conditions for detailed analysis of lipids conjugated to Atg8 by LC-MS/MS. Next, I perform gel filtration chromatography to know the interaction of the components in the Atg8 conjugation reaction in vitro. Atg3 interacts with Atg7 as it shown, and Atg3 also interacts with the Atg8-Atg7 thioester intermediate. Furthermore, I find the Atg8-Atg3 thioester intermediate releases from Atg7. It is reasonable because Atg7 is reused rapidly for the next cycle of the reaction. Furthermore, I show that Atg3 and Atg7 are interacted via the disulfide bond between active center cysteines in Atg3 and Atg7. I show the model of the sequential reaction of Atg8 lipidation.
Author: Kyoko Oh‐oka
Language: English
Subfield: Cell Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W753031548