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

ATG18、ATG21和HSV2是一个高度保守的PROPPIN家族的酵母成员。PROPPIN是一种与多聚磷脂酰肌醇结合的推进器。它们通过其周长的两个结合部位与PI3P和PI(3，5)P2结合。Atg18和Atg21最初被鉴定为需要巨型自噬(以下称自噬)。同源HSV2的功能仍然难以捉摸。我们只发现了对微核吞噬的部分影响，在氮饥饿过程中，液泡和核膜之间的接触位置与核的非必要部分一起退化。PROPPIN在自噬中除了依赖于PI3P外，还有其他非自噬功能。ATG18负性调节空泡PI3P 5-激酶FAB1。它还在人工货物RS-ALP从液泡到高尔基体的逆行运输中发挥作用。Atg18和Atg21进一步参与高渗休克时的空泡碎裂。为了更好地了解PROPPIN的分子功能，我们对所有三种PROPPIN使用了邻近标记BioID方法，这允许在活细胞中识别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是一个新的逆转录复合体的一部分，该复合体在高渗透胁迫下介导液泡碎裂。我们进一步证明了自噬小体生物发生所必需的完整膜蛋白Atg9的循环依赖于Atg18-Retmer。利用两种类型的Atg18突变体，这两种突变体在蛋白质循环中的作用被选择性地削弱，但不是自噬，我们现在想要了解Atg18-Retmer的确切分子功能。进一步的主要目的是阐明HSV2的功能和剖析噬菌体中Atg21-复合体的组织结构。为此，我们将对在我们未发表的邻近标记屏幕中发现的一组新型PROPPIN交互作用进行深入分析。

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.