Every living organism possesses a genome that contains within it a unique set of genes, a substantial number of which encode proteins. Over the last 20 years, it has become apparent that organismal complexity arises not from the specific complement of genes per se, but rather from interactions between the gene products - in particular, interactions between proteins. As an inevitable consequence of the crowded cellular interior, most protein-protein interactions are fleeting. However, many are significantly more long-lived and result in stable protein complexes, in which the constituent subunits are obligately dependent on their binding partners. Despite the abundance of protein complexes and their critical importance to the cell, we currently have an incomplete understanding of the mechanisms by which the cell ensures their correct assembly. In the chapters that follow, I have attempted to improve our understanding of the regulatory systems underlying assembly of protein complexes, and the way in which assembly as a whole affects the behaviour of the cell. The thesis opens with an extended literature review covering the currently available methods for characterising protein complexes. After this introduction, chapters 2-4 are concerned with regulatory mechanisms and biological implications common to the assembly of all protein complexes. Chapter 5 diverges from this work, and describes a family of evolutionarily related proteins that regulate the behaviour of condensins and cohesins. Bacterial and archaeal genomes contain far less non-coding DNA than eukaryotes, and coding genes are often packaged into discrete units known as operons. The proteins encoded within operons are usually functionally related, either through participation in metabolic pathways or as subunits of heteromeric protein complexes. Since protein complexes assemble via ordered pathways, we reasoned that there might be a signature of assembly order present in operons, the genes of which are translated in sequential order. By comparing computationally predicted assembly pathways with gene order in operons, we demonstrated this to be the case for the large majority of operon-encoded complexes. Within operons, gene order follows assembly order, and adjacent genes are substantially more likely to share a physical interface than those further apart. This work demonstrates that efficient assembly of complexes is of sufficient importance as to have placed major constraints on the evolution of operon gene order. Following this study of bacterial operons, I present results from research investigating how patterns of protein degradation in eukaryotes are influenced by the formation of protein complexes. This showed that, whilst most proteins display exponential degradation kinetics, a sizeable minority deviate considerably from this pattern, instead being more consistent with a two-step degradation process. These proteins are predominantly members of heteromeric complexes, and their two-step decay profiles can be explained using a model under which bound and unbound subunits are degraded at different rates. Within individual complexes, we find that non-exponentially decaying proteins tend to form larger interfaces, assemble earlier, and show a higher degree of coexpression, consistent with the idea that bound subunits are degraded at a slower rate than unbound or peripheral subunits. This model also explains the behaviour of proteins in aneuploid cells where one or more chromosomes have been duplicated. In general, protein abundance scales with gene copy number, so that the immediate effect of duplicating a chromosome is to double the abundance of the proteins encoded on it. However, previous analyses of mass spectrometry data, as well as my own, have shown that the abundance of many proteins on duplicated chromosomes is significantly attenuated compared to what one would expect. These proteins, like those with non-exponential degradation patterns, are very often members of larger complexes. Since the overall concentration of a protein complex is constrained by that of its least abundant members, duplicating a single subunit will predominantly increase the unbound, unstable fraction of that subunit. The results from this work strongly suggest that the apparent attenuation of many proteins observed in aneuploid cells is indeed a consequence of the failure of these proteins to assemble into complexes. Finally, I present a study concerning an important, universally conserved family of protein complexes, namely the SMC-kleisins. Two members of this family, condensin and cohesin, are responsible for two hallmarks of eukaryotic chromatin organisation: the formation of condensed, linear chromosomes, and sister chromatid cohesion during cell division. Unlike other SMC-kleisins, condensin and cohesin possess a number of regulators containing HEAT repeats. By developing a computational pipeline for searching and clustering paralogous repeat proteins, I was able to demonstrate that these regulators form a distinct sub-family within the larger class of HEAT repeat proteins. Furthermore, these regulators arose very early in eukaryotic history, hinting at a possible role in the origin of modern condensins and cohesins.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:756971 |
| Date | January 2018 |
| Creators | Wells, Jonathan Nicholas |
| Contributors | Marsh, Joseph ; Semple, Colin |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/31552 |
Page generated in 0.0024 seconds