|Ph.D Thesis||Department of Biology|
|Supervisor:||Prof. Glickman Michael|
The CSN complex is highly conserved among all multicellular eukaryotes and partakes in a number of signaling pathways. It is an essential regulator of developmental processes, cell cycle progression, and transcriptional regulation. Until recently it has been assumed that the complex is not present in S. cerevisiae. We identified a group of proteins that interact in a manner suggesting they form a CSN-like complex in S. cerevisiae. Upon disruption of the complex, Cdc53 is found only in a rubylated form. This brought up the idea that the CSN complex or its subunits act as derubylase. We found a positive interaction between Csn5 and Cdc53.
Common to the MPN domain Rpn11 in the lid and Csn5 - but not to most other MPN proteins - is the consensus sequence MPN+ motif. Single amino acid substitutions in MPN+ residues in Rpn11 all show similar phenotypes, including slow growth, sensitivity to temperature and amino acid analogs, and general proteasome-dependent proteolysis defects. We conclude that the MPN+ motif of Rpn11 is essential for proper proteolysis of ubiquitinated substrates by the proteasome. We showed that these effects are not caused by defective proteasomes, but are most likely due to a defect in proteolysis of ubiquitinated substrates. The same mutations were made in Csn5. The mutants accumulated only the rubylated form of Cdc53, suggesting that an intact MPN+ sequence in required for the de-rubylation activity of the CSN complex.
The MPN+ motif shows sequence similarity to metal-binding sites. Both the lid and the CSN each contain one MPN+ subunit, and both complexes presumably have a proteolytic function of deubiquitination or derubylation respectively. The derubylation function of the CSN may explain the complex's different functions, by substrates stabilization or targeting to degradation by SCF-dependent ubiquitination.