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

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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.