Autophagic and non-autophagic functions of the S. cerevisiae PROPPINs Atg18, Atg21 and Hsv2

Atg18， Atg21和Hsv2是高度保守的PROPPINs家族的酵母成员（-结合多磷酸肌苷的螺旋桨）。它们通过周围的两个结合位点与PI3P和PI(3,5)P2结合。Atg18和Atg21最初被发现是因为它们需要巨噬（以下简称自噬）。同源Hsv2的功能尚不清楚。我们只发现了对微核吞噬的部分影响，其中液泡和核膜之间的接触部位在氮饥饿期间与细胞核的非必要部分一起被降解。PROPPINs除了在自噬中依赖pi3p的作用外，还具有其他非自噬功能。Atg18负调控液泡PI3P 5-激酶Fab1。它进一步作用于人工货物RS-ALP从液泡到高尔基体的逆行运输。Atg18和较小程度的Atg21在高渗休克时进一步参与液泡破裂。为了更好地了解PROPPINs的分子功能，我们对所有三种PROPPINs使用了接近标记BioID方法，该方法允许在活细胞中鉴定诱饵蛋白微环境中的蛋白质。在一项已经发表的研究中，我们发现Atg18是一种新型反转录复合体的一部分，该复合体在高渗胁迫下介导液泡破碎。我们进一步发现，对自噬体生物发生至关重要的整体膜蛋白Atg9的循环依赖于Atg18-retromer。利用两种类型的Atg18突变体，我们现在想要了解Atg18-retromer的精确分子功能，这两种突变体在蛋白质循环中的作用被选择性地破坏，而不是自噬。进一步的主要目的是阐明Hsv2的功能，并解剖吞噬体上atg21复合物的组织。为此，我们将对未发表的接近标记屏幕中发现的一组新颖的PROPPIN交互器进行深入分析。

Atg18, Atg21 and Hsv2 are yeast members of a highly conserved family of PROPPINs (-propeller that bind polyphosphoinositides). They bind via two binding sites at their circumference to PI3P and PI(3,5)P2. Atg18 and Atg21 were initially identified for their requirement for macroautophagy (hereafter autophagy). The function of the homologous Hsv2 remained elusive. We only found a partial effect on micronucleophagy, where the contact site between the vacuole and the nuclear envelope is degraded during nitrogen starvation together with non-essential parts of the nucleus. PROPPINs have beside their PI3P-dependent roles in autophagy additional non-autophagic functions. Atg18 negatively regulates the vacuolar PI3P 5-kinase Fab1. It further functions in retrograde transport of the artificial cargo RS-ALP from the vacuole to the Golgi. Atg18 and to a lesser extend Atg21 are further involved in vacuolar fragmentation upon hyperosmotic shock. To better understand the molecular functions of the PROPPINs, we used the proximity labeling BioID approach for all three PROPPINs, which allows in living cells the identification of proteins within the microenvironment of the bait proteins. In an already published study we found that Atg18 is part of a novel retromer complex, which mediates vacuole fragmentation upon hyperosmotic stress. We further showed that the cycling of the integral membrane protein Atg9, which is essential for autophagosome biogenesis depends on Atg18-retromer. Using two types of Atg18 mutants, which are expected to be selectively impaired in its role in protein recycling but not autophagy, we now want to understand the precise molecular function of Atg18-retromer. Further major aims are to clarify the function of Hsv2 and to dissect the organization of Atg21-complexes at the phagophore. To this end, we will carry out in depth analyses of a set of novel PROPPIN interactors found in our unpublished proximity labeling screens.