Currently the mammalian endoplasmic reticulum (ER) is known to contain at least 20 different protein disulphide isomerase (PDI) family members. The oxidoreductases in the PDI family are thought to catalyse the formation and rearrangement of disulphide bonds in newly synthesised proteins. The focus of this work was to characterise two of the PDI family members: PDI and ERp46. In vitro translation reactions of major histocompatibility complex (MHC), β1-integrin (β1-I), haemagglutinin (HA), procollagen α1(III) and preprolaction (pPL) were carried out in untreated or PDI-depleted cells. The depletion of PDI decreased the rate of folding of MHC and β1-I and also prevented the oligomerisation of HA, suggesting a role for PDI in folding these putative substrates. However, when PDI was depleted neither the folding of pPL or HA was affected, implying that they may not be substrates for PDI. To determine the role of ERp46 in the cell, a substrate-trapping approach was used. Here substrates interacting with ERp46 were trapped as mixed disulphides isolated by immunoprecipitation, separated by 2D SDS-PAGE and identified by mass spectrometry. It was demonstrated that ERp46 forms mixed disulphides with at least 23 proteins, including heavily secreted proteins such as laminins, integrins and collagens. In particular, interactions with Ero1, Prx IV, EDEM3 and ERAP2 were found and confirmed by immunoprecipitation of radiolabelled in vitro translated protein. Notably nine of these clients of ERp46 have previously been identified as substrates of ERp57 (Jessop, Watkins et al. 2009). This would support the hypothesis that several different oxidoreductases, working in concert, are required to fold certain substrate proteins. Also, it was confirmed that Prx IV and Ero1 each form a mixed disulphide with PDI. These results highlight the importance of PDI family members in recruiting co-factors to substrates. Additionally, the over-expression of ERp46 led to increased cell survival following DTT treatment, yet after depletion of ERp46, cells were less able to grow, perhaps suggesting a role for ERp46 in maintaining ER redox homeostasis and cell survival. This suggestion was supported by the finding that ERp46 is able to catalyse the reduction of Prx IV in the presence of glutathione. These results suggest that Prx IV provides a novel mechanism for the transfer of disulphide bonds to nascent proteins in the ER via PDI family members such as ERp46 and PDI.

Eukaryotic cilia are complex, highly conserved microtubule-based organelles with a broad phylogenetic distribution. Cilia were present in the last eukaryotic common ancestor and many proteins involved in cilia function have been conserved through eukaryotic diversification. The evolution of these ciliary functions may be inferred from the distribution of the molecular components from which these organelles are composed. By linking protein distribution in 45 diverse eukaryotes with organismal biology, I define an ancestral ciliary inventory. Analysis of these core proteins allows the inference that the cenancestor of the eukaryotes possessed a cilium for motility and sensory function. I show that the centriolar basal body function is ancestral, whereas the centrosome is specific to the Holozoa, and I use this information to predict a number of roles for proteins based on their phylogenetic profile. I also show that while remarkably conserved, significant divergence in ciliary protein composition has occurred in many lineages, such as the unusual centriole of Caenorhabditis elegans and the transitional changes throughout the land plants. I exemplify this divergence through ultrastructural studies of the fern Ceratopteris richardii and the liverwort Marchantia polymorpha both of which have cilia that exhibit a number of distinctive morphological features, the most conspicuous of which is a general breakdown of canonical microtubule arrangements. Cilia have also been lost multiple times in different lineages: at least twice within the land plants. During these evolutionary transitions proteins with ancestral ciliary functions may be lost or co-opted into different functions. I have interrogated genomic data to identify proteins that I predict had an ancestral ciliary role, but which have been maintained in non-ciliated land plants. I demonstrate that several of these proteins have a flagellar localisation in protozoan trypanosomes and I use expression data correlation to predict potential non-ciliary plant roles.