<p>The plant cyclin-dependent kinases (CDKs) are subjected to the regulation by the interactors/inhibitors of CDK (ICKs). Seven members of the ICK family are known in the plant <i>Arabidopsis thaliana</i>. It has been shown that the N-terminal region of ICK1 makes it unstable in plants, although the mechanism was unknown.</p>
<p>In this thesis, to determine role of the N-terminal region in other ICKs, full length ICK2 to ICK7 were compared to truncated mutants lacking the N-terminal region. Results from yeast two-hybrid studies suggest that not all the N-terminal regions in different ICKs have a role similar to the N-terminal region of ICK1. Studies of a set of HA-tagged ICK1 deletion mutants in yeast mapped the critical sequence for ICK1 degradation to the N-terminal 21<sup>st</sup> - 40<sup>th</sup> amino acid residues. Overexpression of deletion mutants in Arabidopsis also showed that deletion of the 20-amino-acid region of ICK1 lead to a high level of HA-tagged mutant protein, supporting that this region plays a major role in ICK1 degradation <i>in vivo</i>.</p>
<p>Treating yeast cells expressing HA-tagged ICK1 with the 26S proteasome inhibitor MG132 moderately increased the level of ICK1 protein, suggesting that 26S proteasome may be involved in the degradation of ICK1. To determine the possible involvement of the two E3 complexes, the Skp1-Cullin-F-box (SCF) complex and anaphase promoting complex/cyclosome (APC/C), a set of yeast mutants defective in either SCF complex or APC/C, were used to express ICK1, ICK1<sup>ÎN20</sup> (ICK1 lacking the N-terminal 20 amino acids) and ICK1<sup>ÎN40</sup>. ICK1<sup>ÎN40</sup> showed a very high level of expression in SCF defective mutants, but not in APC/C defective mutants. However, ICK1 and ICK1<sup>ÎN20</sup> did not accumulate to a high level in either of the two types of mutants. These results suggest that two pathways are involved in the degradation of ICK1.</p>
<p>Results from this study provide new understanding regarding the role of N-terminal region of ICK1 in conferring protein instability, and the differences among ICKs. They also raise new questions for future investigation on this family of plant cell cycle regulators.</p>
| Identifer | oai:union.ndltd.org:USASK/oai:usask.ca:etd-12162008-220958 |
| Date | 26 January 2009 |
| Creators | Li, Qin |
| Contributors | Wilson, Ken E., Wang, Hong, Covello, Patrick S., Warrington, Robert C., Pato, Mary D., Khandelwal, Ramji L. |
| Publisher | University of Saskatchewan |
| Source Sets | University of Saskatchewan Library |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://library.usask.ca/theses/available/etd-12162008-220958/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Saskatchewan or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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