|Ph.D Student||Aviram Sharon|
|Subject||Characterization of the Signals and Pathways Involved in the|
Degradation of the Cyclin Pcl5 of the Yeast
|Department||Department of Medicine||Supervisor||Professor Daniel Kornitzer|
|Full Thesis text|
The ubiquitin - proteasome degradation mechanism is the major pathway by which intracellular proteins are degraded in eukaryotic cells. The canonical degradation pathway of a protein begins with its specific recognition by an ubiquitin ligase, which together with ubiquitin conjugating enzyme, and ubiquitin activating enzyme catalyzes the conjugation of a polyubiquitin chain to the protein. In its ubiquitinated form, the protein is targeted to the 26S proteasome.
Cyclin-dependent kinase (CDK) activity depends on interaction with small proteins called cyclins, where a single CDK can be activated by several cyclins. In Saccharomyces cerevisiae the CDK Pho85 is activated by ten different cyclins (called Pcls: Pho85 Cyclins), which are required for its specific functions as a regulator of the cells responses to changes in environmental conditions, and in cell cycle control.
In the cellular response to amino acids availability, Pho85 is specifically activated by the cyclin Pcl5 to regulate the stability of Gcn4, a major transcription factor involved in the expression of amino acids biosynthesis gene. Under normal growth conditions, Pho85/Pcl5 phosphorylates Gcn4, a prerequisite step for the ubiquitination of Gcn4 and its rapid proteasomal degradation. However, under amino acid starvation conditions, Gcn4 becomes stable, probably due to low activity of the Pho85/Pcl5 complex. Since Pcl5 is a highly unstable protein, it was previously suggested that by virtue of its inherent instability, Pcl5 acts as a sensor for cellular biosynthetic capacity. The aim of this study is to characterize the pathway and mechanism responsible for the high turnover rate of Pcl5.
We identified two degradation signals in Pcl5; a C-terminal degradation signal (CDS), which requires a free carboxy-terminus and is independent of Pho85, and an N-terminal degradation signal, which is dependent on Pho85 and on a specific residue, Thr32, embedded within a Pho85 consensus site. We suggest that these signals target the degradation of different Pcl5 populations; the C-terminal signal targets the degradation of free Pcl5 via a yet unknown pathway, whereas the N-terminal signal targets the degradation of Pho85-bound Pcl5 via autophosphorylation on Thr32, and subsequent ubiquitination by the SCFGrr1complex. Finally we show that under amino acid starvation conditions, two Pcl5 derivatives, Pcl5 T32A and Pcl5 T32A-13xMyc, are able to accelerate Gcn4 turnover to the rate detected under sated conditions. This result supports the suggested model where high Pcl5 turnover is necessary for the regulation of Gcn4 stability.