Understanding the role of BAR and SH3 domain-containing proteins in fungi

This thesis addresses the role of SH3 and BAR domain-containing proteins in different fungal species. SH3 domains are small modules that mediate protein-protein interactions and BAR domains are dimerization domains with membrane binding and bending properties. It is known that the ScRvs167 protein interacts via its SH3 domain with other components of the yeast endocytic machinery while its BAR domain dimerizes with the BAR domain of ScRvs161 and as a heterodimer complex is responsible for the membrane deformation that is crucial to vesicle formation as well as release of the vesicle from the plasma membrane in S. cerevisiae. In Chapter 2, we characterize a novel N-BAR heterodimer in C. albicans that consists of the Rvs162 and Rvs167-3 proteins and we show that in contrast to Rvs161/Rvs167 heterodimer it does not have an endocytic role. In Chapter 3 we demonstrate fission yeast proteins Hob1 and Hob3 (homologs of S. cerevisiae Rvs167 and Rvs161, respectively) ability to form heterodimers in vitro even though evidence suggests they have different functions. Sfp47, a novel F-BAR protein in S. pombe is the focus of Chapter 4. In the last two chapters we are focusing on SH3 domains and their interaction with peptide ligands. In Chapter 5 we investigate the evolution of SH3 domain-mediated interactions in fungi. In Chapter 6 we investigate the CaRvs167-3 SH3 domain: peptide ligand interaction. We conclude that CaRvs167-3 SH3 domain can bind to a peptide completely devoid of proline residues and we establish a novel binding sequence for the Rvs167-3 SH3

Understanding the role of BAR and SH3 domain-containing proteins in fungi

This thesis addresses the role of SH3 and BAR domain-containing proteins in different fungal species. SH3 domains are small modules that mediate protein-protein interactions and BAR domains are dimerization domains with membrane binding and bending properties. It is known that the ScRvs167 protein interacts via its SH3 domain with other components of the yeast endocytic machinery while its BAR domain dimerizes with the BAR domain of ScRvs161 and as a heterodimer complex is responsible for the membrane deformation that is crucial to vesicle formation as well as release of the vesicle from the plasma membrane in S. cerevisiae. In Chapter 2, we characterize a novel N-BAR heterodimer in C. albicans that consists of the Rvs162 and Rvs167-3 proteins and we show that in contrast to Rvs161/Rvs167 heterodimer it does not have an endocytic role. In Chapter 3 we demonstrate fission yeast proteins Hob1 and Hob3 (homologs of S. cerevisiae Rvs167 and Rvs161, respectively) ability to form heterodimers in vitro even though evidence suggests they have different functions. Sfp47, a novel F-BAR protein in S. pombe is the focus of Chapter 4. In the last two chapters we are focusing on SH3 domains and their interaction with peptide ligands. In Chapter 5 we investigate the evolution of SH3 domain-mediated interactions in fungi. In Chapter 6 we investigate the CaRvs167-3 SH3 domain: peptide ligand interaction. We conclude that CaRvs167-3 SH3 domain can bind to a peptide completely devoid of proline residues and we establish a novel binding sequence for the Rvs167-3 SH3

Evolution of the SH3 domain specificity landscape in yeasts

To explore the conservation of Src homology 3 (SH3) domain-mediated networks in evolution, we compared the specificity landscape of these domains among four yeast species, Saccharomyces cerevisiae, Ashbya gossypii, Candida albicans, and Schizosaccharomyces pombe, encompassing 400 million years of evolution. We first aligned and catalogued the families of SH3-containing proteins in these four species to determine the relationships between homologous domains. Then, we tagged and purified all soluble SH3 domains (82 in total) to perform a quantitative peptide assay (SPOT) for each SH3 domain. All SPOT readouts were hierarchically clustered and we observed that the organization of the SH3 specificity landscape in three distinct profile classes remains conserved across these four yeast species. We also produced a specificity profile for each SH3 domain from manually aligned top SPOT hits and compared the within-family binding motif consensus. This analysis revealed a striking example of binding motif divergence in a C. albicans Rvs167 paralog, which cannot be explained by overall SH3 sequence or interface residue divergence, and we validated this specificity change with a yeast two-hybrid (Y2H) assay. In addition, we show that position-weighted matrices (PWM) compiled from SPOT assays can be used for binding motif screening in potential binding partners and present cases where motifs are either conserved or lost among homologous SH3 interacting proteins. Finally, by comparing pairwise SH3 sequence identity to binding profile correlation we show that for 75% of all analyzed families the SH3 specificity profile was remarkably conserved over a large evolutionary distance. Thus, a high sequence identity within an SH3 domain family predicts conserved binding specificity, whereas divergence in sequence identity often coincided with a change in binding specificity within this family. As such, our results are important for future studies aimed at unraveling complex specificity networks of peptide recognition domains in higher eukaryotes, including mammals.LS, BW, FH and BD received funding from the European Union (Marie Curie Research Training Network Penelope; MRTN-CT-2006-036076)