Molecular mechanisms of the targeting of tail-anchored membrane proteins to peroxisomes and mitochondria in mammalian cells

真核细胞含有不同的膜结合细胞器，这些细胞器将特定的细胞蛋白质区隔，以实现各种基本的细胞功能。细胞器特异性蛋白的正确分类和传递对于维持细胞和生物体的细胞器功能和活力至关重要。跨膜蛋白在合成过程中通常被分类并插入到它们的靶膜中。尾部锚定（TA）蛋白是一个有趣的例外，由于它们的短膜尾，它们必须在细胞质合成后（而不是在合成过程中）进行分类和插入。出乎意料的是，我们发现越来越多的TA蛋白被过氧化物酶体（PO）和线粒体（MITO）共享（例如Fis1， Mff或GDAP1，它们有助于细胞器分裂），这表明两个细胞器之间存在更密切的功能相互关系。然而，其他TA蛋白仅在PO（如ACBD5， ALDH3A2V）， MITO或内质网（ER）上。因此，必须存在细胞器特异性靶向过程，以确保蛋白质到达正确的细胞区室以实现其适当的功能。这个重要的目标决策是如何调节的，涉及什么机制，以及PO和MITO是否共享部分机制，这些在很大程度上是未知的。目前，只有对内质网分选机制有贡献的蛋白质被更详细地表征，而关于MITO和PO TA蛋白靶向的知识仍然很少。TA蛋白完成多种需要膜锚定的基本细胞器和细胞功能（如细胞器分裂、囊泡融合、细胞凋亡、病毒防御）。PO和MITO对人类健康和发展至关重要。它们在脂质和ROS代谢中具有重要的代谢功能，从而影响神经元的发育、脂质稳态和衰老。在这个项目中，我们将研究哺乳动物细胞中TA蛋白被分类并传递到PO和MITO的分子机制。我们的初步筛选实验，导致鉴定了以前未知的PO上的TA蛋白，以及PO和MITO上的TA蛋白，已经产生了工具，现在可以首次研究细胞器特异性靶向。我们将评估PO输入受体Pex19是否对TA蛋白靶向PO至关重要。通过蛋白-蛋白相互作用研究和TA蛋白的突变分析，我们将确定Pex19与选定TA蛋白的结合，从而确定Pex19结合基序。为了确定Pex19参与TA蛋白分选的新的细胞质相互作用伙伴，我们将采用下拉策略结合定量质谱法。为了评估TA蛋白的内质网靶向机制的蛋白质（例如Sg2A、Get和热休克蛋白）是否有助于PO和MITO靶向，我们将在细胞培养中进行“敲除”研究，以沉默其功能并监测荧光TA蛋白的分选。我们将揭示Sg2A是否作为TA蛋白的一般分选站。最后，我们的目标是通过应用下拉策略，结合体外翻译，质谱分析和哺乳动物细胞培养中的表达研究，确定PO和MITO的TA蛋白进口机制的新组成部分。综上所述，我们将结合分子细胞生物学、生物化学和影像学方法来阐明TA蛋白的分选机制，并确定支持TA蛋白靶向PO和MITO的新因子。了解TA蛋白的细胞器特异性靶向是如何被控制的，将具有基本的生物学和医学意义。它具有很高的潜力，有助于发现基于TA蛋白错误靶向的新疾病和新靶点，例如抑制病毒复制。

Eukaryotic cells contain distinct membrane-bound organelles, which compartmentalize specific cellular proteins to fulfil a variety of essential cellular functions. Proper sorting and delivery of organelle-specific proteins is of fundamental importance to maintain organelle functionality and viability of the cell and the organism. Trans-membrane proteins are usually sorted to and inserted into their target membrane during their synthesis. Tail-anchored (TA) proteins represent an interesting exception, as due to their short membrane tail, they must be sorted and inserted after (and not during) their synthesis in the cytoplasm. Unexpectedly, we discovered that a growing number of TA proteins are shared by peroxisomes (PO) and mitochondria (MITO) (e.g. Fis1, Mff or GDAP1, which contribute to organelle division) suggesting a closer functional interrelationship between both organelles. However, other TA proteins are exclusively on PO (e.g. ACBD5, ALDH3A2V), on MITO or on the endoplasmic reticulum (ER). Thus, organelle-specific targeting processes must exist to ensure that the proteins reach the correct cellular compartment to fulfil their proper functions. How this important targeting decision is regulated, what machinery is involved, and if part of the machinery is shared by PO and MITO is largely unknown. At present, only proteins contributing to the ER sorting machinery have been characterized in more detail, whereas knowledge on MITO and PO TA protein targeting remains scarce. TA proteins fulfil a variety of essential organelle and cellular functions requiring membrane anchorage (e.g. organelle division, vesicle fusion, apoptosis, viral defence). PO and MITO are essential for human health and development. They have important metabolic functions in lipid and ROS metabolism, thus influencing neuronal development, lipid homeostasis and ageing.In this project we will address the molecular mechanism by which TA proteins are sorted and delivered to PO and MITO in mammalian cells. Our preliminary screening experiments, which led to the identification of previously unknown TA proteins on PO, and on both PO and MITO, have generated tools which now enable for the first time to study the organelle-specific targeting. We will assess if Pex19, a PO import receptor, is essential for TA protein targeting to PO. By protein-protein interaction studies and mutational analyses of TA proteins we will determine Pex19 binding to selected TA proteins, thereby defining the Pex19 binding motif. To identify new cytosolic interaction partners of Pex19 involved in TA protein sorting, we will apply a pull-down strategy combined with quantitative mass spectrometry. To assess if proteins of the ER targeting machinery for TA proteins (e.g. Sg2A, Get and heat shock proteins) contribute to PO and MITO targeting we will perform "knock down" studies in cell culture to silence their function and monitor sorting of fluorescent TA proteins. We will reveal if Sg2A functions as a general sorting station for TA proteins. Finally, we aim at identifying novel components of the TA protein import machinery for PO and MITO by applying pull down strategies, a combination of in vitro translation, mass spectrometry, and expression studies in mammalian cell culture.In summary, we will combine molecular cell biology, biochemical approaches and imaging to elucidate the sorting mechanism and to identify novel factors that support TA protein targeting to PO and MITO. Understanding how the organelle-specific targeting of TA proteins is controlled will be of fundamental biological and medical importance. It has high potential to contribute to the discovery of novel disorders based on mistargeting of TA proteins and new targets to e.g. inhibit virus replication.