Protein sorting to peroxisomes via mitochondria: Characterization of the molecular mechanism and its impact on formation of organelle contact sites.

酿酒酵母的蛋白磷酸酶Ptc5含有一个针对线粒体的N端靶向信号和一个C端针对过氧化物酶体的靶向信号(PTS1)。我们最近报道了Ptc5通过一种新的靶向机制通过线粒体被分类为过氧化物体(Stehlik等人，2020)。现在我们将更详细地描述该机制(1.)。此外，我们将关注以下假设：蛋白质从线粒体移位到过氧化体中，通过分子拉锯战触发细胞器接触部位的形成，这是货物蛋白与两个隔室的输入机制同时相互作用的结果。因此，蛋白质移位本身可能是细胞器(2.)1.1.1新的易位机制所需的分子内信号将被描述。1.2在高含量筛选中发现的基因(例如Ermes组分)将被进一步研究，以确定其基因产物在Ptc5易位中的作用。此外，我们将研究线粒体输入机制的组件，这些组件通常由必需基因编码。1.3最后，我们将建立一个体外重建系统，概括我们在体内的数据。间接分选对过氧化物酶的功能影响在项目的第二部分中，我们将阐明通过竞争靶向信号进行分选如何影响线粒体和过氧酶体的物理相互作用。我们已经鉴定了几种显示这种结构域结构的酿酒酵母蛋白质(Stehlik等人，2020年)。2.1拉锯式分选对过氧化物体和线粒体关联的影响将通过研究过氧化物体导入机制成分耗尽时接触点的形成来解决。2.2我们将通过突变体分析具有竞争靶向信号的蛋白质(Cat2、Tes1和Pxp2)的生物学功能，这些突变体直接而不绕道地被转移到过氧体。它们对过氧化体和线粒体之间接触部位形成和代谢物交换的影响将被确定。2.3我们将创造一种可切换的系绳，可以用来诱导人工接触。含有这种系绳的菌株将被分析过氧化物体功能和蛋白质从线粒体到过氧化物体的移位。计划中的项目将有助于了解通过线粒体迂回输入的过氧化物体如何有助于两个细胞器的相互作用。我们相信这项研究很重要，因为过氧化物体和线粒体共享许多代谢途径，但它们如何物理上相互作用还不太清楚。由于已知哺乳动物蛋白质含有相互竞争的过氧化体和线粒体靶向信号，我们的项目可能会更好地理解过氧化物体和线粒体相互作用在人类生理中的作用。

The protein phosphatase Ptc5 from Saccharomyces cerevisiae contains an N-terminal targeting signal for mitochondria and a C-terminal targeting signal for peroxisomes (PTS1). We recently reported that Ptc5 is sorted to peroxisomes via mitochondria by a novel targeting mechanism (Stehlik et al, 2020). We now will characterize the mechanism in greater detail(1.). In addition, we will focus on the following hypothesis: translocation of proteins into peroxisomes from mitochondria triggers the formation of organelle contact sites via molecular tug-of-war, which results from simultaneous interaction of the cargo protein with the import machineries of both compartments. Thus, protein translocation per se may organelles (2.).1. Elucidation of the molecular mechanism of protein sorting to peroxisomes via mitochondria1.1 Intra-molecular signals required for the novel translocation mechanism will be characterized.1.2 Genes identified in the high-content screen (e.g. ERMES components) will be further investigated to address the roles of their gene products for Ptc5 translocation. In addition, we will examine components of the mitochondrial import machinery, which are often encoded by essential genes.1.3 Finally, we will set up an in vitro reconstitution system that recapitulates our in vivo data.2. Functional consequences of indirect sorting to peroxisomesIn the second part of the project we will elucidate how sorting via competing targeting signals influences the physical interaction of mitochondria and peroxisomes. We have identified several S. cerevisiae proteins showing this domain structure (Stehlik et al, 2020).2.1 The effect of tug-of-war like sorting on the association of peroxisomes and mitochondria will be addressed by studying contact site formation upon depletion of components of the peroxisomal import machinery.2.2 We will analyze the biological function of proteins with competing targeting signals (Cat2, Tes1 and Pxp2) via mutants that are transported to peroxisomes directly without detour. Their effect on contact site formation and metabolite exchange between peroxisomes and mitochondria will be determined.2.3 We will create a switchable tether, which can be used to induce artificial contacts. Strains containing this tether will be analyzed for peroxisome function and protein translocation from mitochondria to peroxisomes.The planned project will help to understand how peroxisomal import via a mitochondrial detour contributes to the interplay of both organelles. We are convinced that the study is important as peroxisomes and mitochondria share many metabolic pathways, but how they physically interact is less understood. Since mammalian proteins are known that contain competing peroxisomal and mitochondrial targeting signals, our project may result in a better understanding of the role of peroxisome and mitochondria interaction in human physiology.