Peroxisomes are dynamic and multifunctional organelles, which are essential for human health and development. They are remarkably diverse, with functions that vary significantly between cells and organisms, and can dramatically change their size, shape and dynamics in response to cellular cues. In the past few years, several studies have significantly increased our understanding of the basic principles that enable peroxisome biogenesis and degradation, as well as their pivotal role in cellular signalling and homeostasis. However, several of these processes are still poorly understood. In this thesis we initially studied the peroxisome targeting mechanism of a group of C-terminally anchored membrane proteins, known as tail-anchored (TA) proteins. In order to investigate the molecular signals that enable TA protein targeting to cellular organelles, we analysed the physicochemical properties of a cohort of TA proteins both in silico and in vivo, and show that a combination of transmembrane domain (TMD) hydrophobicity and C-terminal tail charge determines organelle-specific targeting. Focusing on peroxisomes, we demonstrate that a balance between TMD hydrophobicity and high positive tail charge directs TA proteins to this organelle, and enables binding to the peroxisomal chaperone PEX19. These results allowed us to create a bioinformatical tool to predict the targeting of uncharacterised TA proteins and further develop our understanding of the molecular mechanisms involved in the targeting of this protein group. From our initial TA protein screen, we identified the TA protein MIRO1 at peroxisomes and looked at its role in the regulation of peroxisome motility. We show that endogenous MIRO1 localises to mitochondria and peroxisomes, and that dual targeting depends on the C-terminal tail. MIRO1 expression significantly increased peroxisome motility in several cell lines, and revealed a role for motility in peroxisome dynamics, by inducing organelle proliferation and elongation. These results reveal a new molecular complex at peroxisomes and provide us with a tool to further dissect the role of motility on peroxisome function.

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The biosynthesis of integral membrane proteins (IMPs) is an essential cellular process. IMPs comprise roughly 20-30% of the protein coding genes of all organisms, nearly all of which are inserted and assembled at the endoplasmic reticulum (ER). The defining structural feature of IMPs is one or more transmembrane domains (TMDs). TMDs are typically stretches of predominately hydrophobic amino acids that span the lipid bilayer of biological membranes as an alpha helix. TMDs are remarkably diverse in terms of their topological and biophysical properties. In order to accommodate this diversity, the cell has evolved different sets of machinery that cater to particular subsets of proteins. Our knowledge of how the TMDs of IMPs are selectively recognized, chaperoned into the lipid bilayer, and assembled remains incomplete. This thesis is broadly interested in investigating how TMDs are correctly inserted and assembled at the ER. To address this the biosynthesis of multi-pass IMPs was first considered. Multi-pass IMPs contain two to more than twenty TMDs, with TMDs that vary dramatically in terms of their biophysical properties such as hydrophobicity, length, and helical propensity. The beta-1 adrenergic receptor (β1-AR), a member of the G-protein-coupled receptor (GPCR) family was established as a model substrate in an in vitro system where the insertion and folding of its TMDs could be interrogated. Assembly of β1-AR is not a straightforward process, and current models of insertion fail to explain how the known translocation machinery correctly identifies, inserts, and assembles β1-AR TMDs. An in vivo screen in mammalian cells was therefore conducted to identify additional factors which may be important for multi-pass IMP assembly. The ER membrane protein complex (EMC), a well conserved ER-resident complex of unknown biochemical function, was identified as a promising hit potentially involved in this assembly process. The complexity of working with multi-pass IMPs in an in vitro system prompted the investigation of a simpler class of proteins. Tail-anchored proteins (TA) are characterized by a single C-terminal hydrophobic domain that anchors them into membranes. Though structurally simpler compared to multi-pass IMPs, the TMDs of TA proteins are similarly diverse. We found that known TA insertion pathways fail to engage low-to-moderately hydrophobic TMDs. Instead, these are chaperoned in the cytosol by calmodulin (CaM). Transient release from CaM allows substrates to sample the ER, where resident machinery mediates the insertion reaction. The EMC was shown to be necessary for the insertion of these substrates both in vivo and in vitro. Purified EMC in synthetic liposomes catalysed insertion of its TA substrates in a fully reconstituted system to near-native levels. Therefore, the EMC was rigorously established as a TMD insertase. This key functional insight may explain its critical role in the assembly of multi- pass IMPs – which is now amenable to biochemical dissection.

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Mitochondria carry out several important functions in eukaryotic cells such as energy metabolism, iron-sulfur cluster assembly, apoptosis, signaling pathways, protein quality control etc. Most mitochondrial proteins are synthesized on the cytosolic ribosomes and transported to the organelles by the cytosolic chaperones and mitochondrial protein import machinery based on specific targeting signals. Although, the basic principles of protein import have been explained, many questions remain unanswered, particularly for highly modified mitochondria such as hydrogenosomes. The aim of the study was to investigate protein translocation into hydrogenosomes of a human parasite, Trichomonas vaginalis (Tv) with a focus on the composition, function and structure of protein translocases and the role of targeting signals. The translocase of the outer membrane (TOM) is responsible for the import of most proteins into the organelle. Even though, the presence of a TOM complex in trichomonad hydrogenosomes was predicted, its components were not known. Moreover, the generic structure of the mitochondrial TOM complex was not resolved. This study showed that the TvTOM complex is highly divergent consisting of two modified core subunits - channel- forming TvTom40 isoforms and a Tom22-like protein, and two...

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