Dissecting the molecular functions of the ubiquitin-like Atg8 during autophagosome biogenesis in S. cerevisiae

自噬是一种高度保守的运输途径，在营养限制的情况下，将细胞质物质甚至整个细胞器运送到液泡中进行降解。首先，双膜层囊泡，即所谓的自噬体，由自噬体前结构（PAS）形成。它们最终与液泡融合，释放出一个只有一层膜的囊泡，即自噬体，进入液泡腔，分解并再利用其成分。自噬体生物发生所需膜的来源是自噬领域的探索性问题之一。在酿酒酵母中，含有跨膜蛋白Atg9的囊泡仅将所需膜表面的约2%从非PAS池输送到PAS池。因此，其他脂质来源必须仍然未知。泛素样蛋白Atg8是自噬的关键成分之一，也是自噬体生物发生所必需的。与泛素类似，at8共价附着于磷脂酰乙醇胺上，从而附着于膜上。at8偶联的独特之处在于它催化偶联到脂质，而不是蛋白质。初步未公布的自己的数据表明，存在一个8亿美元的非考绩池。本提案的第一个目的是详细分析Atg8非PAS池及其在Atg8介导的囊泡膜转运到PAS中的潜在作用。本研究的第二个目的是关注at8在自噬体生物发生过程中所需的膜融合中的功能。at8由一个n端螺旋结构域（NHD）和一个泛素样结构域（UBL）组成。它与泛素的不同之处在于NHD对其功能至关重要。已发表的数据表明，NHD与自噬体生物发生过程中的膜融合有关。因此，特别分析了Atg8-NHD的作用。阐明其延伸和闭合机制将在理解自噬体生物发生方面取得实质性进展，这是自噬领域的另一个挑战。Atg8的数据表明，在自噬体发生的空间和时间上不同的步骤需要几个Atg8亚复合物。使用标准方法分离at8配合物不能将新鉴定的组分分配给共存的配合物之一。因此，需要通过原生电泳（蓝色原生PAGE）对不同亚配合物进行分离，进一步分析复合物组成的时空差异。这种方法应该允许建议针对时间和空间的次复杂转换的第一个模型。

Autophagy, a highly conserved transport pathway, delivers cytosolic material or even whole organelles to the vacuole for degradation upon nutrient limitation. First, double membrane-layered vesicles, the so-called autophagosomes, are formed out of the preautophagosomal structure (PAS). They finally fuse with the vacuole and release a still one membrane-layered vesicle, the autophagic body, into the vacuolar lumen for breakdown and reuse of its constituents. The source of the membrane required for autophagosome biogenesis is one of the probing questions in the autophagy field. In S. cerevisiae, vesicles containing the transmembrane protein Atg9 deliver only around 2% of the required membrane surface from the non-PAS pool to the PAS. Thus additional lipid sources must be still unknown. The ubiquitin-like protein Atg8 is one of the key components of autophagy and required for autophagosome biogenesis. Similar to ubiquitin Atg8 is covalently attached to phosphatidylethanolamine and thus to membranes. The Atg8 conjugation is unique in that it catalyzes conjugation to a lipid, not to a protein. Preliminary unpublished own data pointed to the existence of a non-PAS pool of Atg8. The first aim of this proposal is the detailed analyses of this Atg8 non-PAS pool and its potential role in Atg8-mediated vesicular membrane transport to the PAS. The second aim of this proposal focuses on the function of Atg8 in membrane fusions required during autophagosome biogenesis. Atg8 consists of a N-terminal helical domain (NHD) and a ubiquitin-like domain (UBL). It differs from ubiquitin mostly by this NHD that is crucial for its function. Own published data has brought the NHD in connection with membrane fusion during autophagosome biogenesis. Thus especially the role of the Atg8-NHD will be analyzed. Elucidating the elongation and closure mechanism would lead to substantial progress in understanding autophagosome biogenesis, another challenge for the autophagic field.The data on Atg8 indicate that several Atg8 sub-complexes are required for spatially and temporally different steps of autophagosome biogenesis. The isolation of Atg8 complexes using standard methods would not allow to assign newly identified components to one of the co-existing complexes. Therefore, the different sub-complexes should be separated by native electrophoresis (blue native PAGE) to further analyze the spatial and temporal differences in complex composition. This approach should allow the suggestion of a first model for sub-complex conversion over time and space.