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

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.

In this project we will elucidate the molecular mechanism by which tail-anchored (TA) membrane proteins are sorted and delivered to peroxisomes (PO) and mitochondria (MITO) in mammalian cells. We recently showed that PO and MITO share several TA proteins, and have now identified TA proteins which are exclusively targeted to PO. How organelle-specific sorting is mediated, what machinery is involved, and if part of the sorting machinery is shared by PO and MITO is largely unknown. The molecular tools we generated now allow for the first time to address these fundamental questions in detail.
In a first part of the project, the role of Pex19, a shuttle receptor for PO trans-membrane proteins, as an essential factor in PO TA protein targeting will be analyzed. Pex19 binding to selected TA proteins will be monitored in an in vitro translation assay and by immunoprecipitation. We will use mutated versions of TA proteins and bioinformatics to define the Pex19 binding motif. Furthermore, we will use Pex19 as bait to search for additional cytosolic interaction partners in in vitro and in vivo pull down approaches. In a second part, RNAi-mediated silencing is planned to examine if Sg2A and other proteins of the ER targeting machinery (e.g. mammalian homologues of get4/5, hsp104/70/40) contribute to TA protein targeting to PO and MITO. Novel components of a potential Sg2A containing central docking complex will be identified by a combined in vitro translation/pull down approach and mass spectrometry. In a third part, we will make use of fatty aldehyde dehydrogenase ALDH3A2 (targeted to the ER) and its PO splice variant ALDH3A2V to search for common and unique binding partners involved in the targeting to PO. Furthermore, we will exploit the observation that ACBD5, an exclusively PO TA protein, is relocated to MITO in Pex19-deficient cells to identify the receptor for TA protein targeting to MITO.

This work is curiosity-driven and discovery-based, and aims to understand fundamental processes in mammalian cell biology which have great potential to be exploited in the pharmacological and public health sectors. Moreover, this work can contribute to the generation of novel tools, e.g. in the prediction of receptor binding to candidate proteins. The reason for this potential is that the mechanisms of organelle-specific targeting of proteins or organelle interplay and cross-talk are poorly understood, but are essential for cellular viability and development of the organism. In particular, mechanistic insights into the targeting of tail-anchored (TA) proteins to peroxisomes (PO) and mitochondria (MITO) have high potential to create a new field of research and are essential to understand the PO-MITO interrelationship and membrane protein import into PO but also to identify new biological functions of PO in animals and humans. Hence, novel insights generated from this work will be of interest to many aspects of cell biological research, and of particular importance for understanding protein targeting and missorting in cellular physiology and pathophysiology. TA proteins fulfil many key functions at their specific membranes, and loss of function leads to pathophysiological conditions and disease. Moreover, viruses are known to exploit TA proteins and their targeting mechanisms to ensure proper replication of their RNAs or to disturb the cellular defence. Therefore, a thorough dissection of the individual sorting steps required to target TA proteins to their correct destinations has the potential to lead to the detection of new genetic disorders caused by the mistargeting of individual TA protein families and of possible new targets e.g. to inhibit virus replication in infected individuals.
Knowledge gained from this study promises to help the identification of novel targets for drug development (of benefit to the UK and European pharmaceutical and health sectors) as well as in the diagnosis of pathophysiological conditions and disorders (public health sector). Hence, we expect opportunities for commercial exploitation. The University of Exeter has excellent links with the wider public with regular events with contributions from research staff. Researchers make regular school visits to explain their research and run events as part of National Science week. Programmes such as this and other outreach activities are critical for the long-term maintenance of the UK science base. This is also aided by the transfer of knowledge and skills between academia and industry. The PDRA and Technician will both receive full and relevant training. Several of my former PhD students are now working within the biotechnology or biomedical sector.