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，其将特定的细胞蛋白质区室化以实现各种基本的细胞功能。细胞器特异性蛋白的正确分选和递送对于维持细胞器功能和细胞和生物体的活力具有根本重要性。跨膜蛋白通常在其合成过程中被分选并插入其靶膜中。尾锚定（TA）蛋白代表一个有趣的例外，因为由于它们的短膜尾，它们必须在它们在细胞质中合成之后（而不是期间）被分选和插入。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）（例如Fis 1，Mff或GDAP 1，它们有助于细胞器分裂）共享，这表明两个细胞器之间的功能相互关系更密切。然而，其他TA蛋白仅在PO（例如ACBD 5、ALDH 3A 2 V）、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。如何规范这一重要的目标选择决定，涉及哪些机制，以及PO和MITO是否共享部分机制，在很大程度上尚不清楚。目前，只有有助于ER分选机制的蛋白质已被更详细地表征，而关于MITO和PO TA蛋白质靶向的知识仍然很少。TA蛋白实现需要膜锚定的多种基本细胞器和细胞功能（例如细胞器分裂、囊泡融合、凋亡、病毒防御）。PO和MITO对人类健康和发展至关重要。TA蛋白在脂质和活性氧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。本项目将探讨TA蛋白在哺乳动物细胞中被分选并递送至PO和MITO的分子机制。我们的初步筛选实验，这导致了以前未知的TA蛋白的PO上，PO和MITO的鉴定，已经产生的工具，现在使第一次研究细胞器特异性靶向。我们将评估PO输入受体Pex 19是否是TA蛋白靶向PO所必需的。通过蛋白质-蛋白质相互作用的研究和TA蛋白的突变分析，我们将确定Pex 19与选定的TA蛋白的结合，从而确定Pex 19结合基序。为了鉴定参与TA蛋白分选的Pex 19的新的胞质相互作用伙伴，我们将应用与定量质谱相结合的下拉策略。为了评估TA蛋白的ER靶向机制的蛋白质（例如Sg 2A、Get和热休克蛋白）是否有助于PO和MITO靶向，我们将在细胞培养物中进行“敲低”研究以沉默它们的功能并监测荧光TA蛋白的分选。我们将揭示Sg 2A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，质谱和哺乳动物细胞培养中的表达研究，确定PO和MITO的TA蛋白输入机制的新组件。总之，我们将结合联合收割机分子细胞生物学，生物化学方法和成像来阐明分选机制，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控制的，将具有重要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蛋白的错误定位的新疾病和新靶点，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.