Browsing by Author "Schmidt, Michael"

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Open Access
Regulation of protein turnover in fission yeast
(2008) Schmidt, Michael; Wolf, Dieter (Prof. Dr.)
Proteins are molecular machines that perform the majority of cellular functions that characterize living organisms. Protein levels within cells are the result of a delicate balance between protein synthesis and protein degradation. Eukaryotic cells are able to shift this balance to one or the other side to regulate the steady state level of each protein within a cell. If a protein or a functional group of proteins is needed to fulfill a certain duty, cells have evolved a number of pathways to increase the levels of these proteins rapidly. On the other hand, if a protein or functional group of proteins is no longer required, eukaryotes are able to rapidly decrease the concentrations of these proteins. While historically much attention and research has been devoted to how proteins are synthesized, the reverse process, i.e. how and when proteins are degraded, is not understood as well. This thesis is structured into three parts, addressing regulation of protein turnover on a molecular as well as on a system-wide level. In part one, to gain deeper insight into how eukaryotes regulate targeted protein degradation, the molecular mechanisms of regulation of Cullin-RING ubiquitin ligases (CRLs) by the COP9 signalosome (CSN), the deubiquitylating enzyme Ubp12p, and the cullin-associated protein Can1p were investigated in the fission yeast Schizosaccharomyces pombe. In a survey of eight F-box proteins, which confer target specificity to CRLs, the study uncovered the existence of variant F-box proteins lacking a critical proline residue required for efficient regulation by the CSN. The results suggest that distinctive features of the F-box motif specify the assembly of F-box proteins into CRL complexes thus destining them for regulation by the CSN through a mechanism, which can principally function independently of Can1p and Ubp12p. In part two, a large-scale biochemical approach was used to investigate whether E2 ubiquitin conjugating enzymes contribute to substrate selection. Because of the lack of a reliable method for the identification of specific ubiquitylation substrates, a new biochemical method termed SPASS allowing the study of specific E2 substrates was developed. SPASS was used to identify on a global scale ubiquitylation substrates of the S. pombe E2s Ubc7p and Ubc8p. Examination of the previously known and also the novel substrates revealed a potential new mechanism by which E2s confer substrate specificity utilizing heterodimerisation. Lastly in part three, to gain a system-wide insight on the regulation of protein expression in S. pombe, mRNA as well as protein levels of 1500 open reading frames were explored. For this purpose, a new label-free mass spectrometry based method allowing relative quantification of protein levels was developed. The fission yeast protein data showed considerable correlations with mRNA levels and with the abundance of orthologous proteins in budding yeast. Functional pathway analysis indicated that the mRNA–protein correlation is strong for proteins involved in signalling and metabolic processes, but increasingly discordant for components of protein complexes, which clustered in groups with similar mRNA-protein ratios. Self-organizing map clustering of large-scale protein and mRNA data from fission and budding yeast revealed coordinate but not always concordant expression of components of functional pathways and protein complexes.