Identification of short protein-destabilizing sequences in Arabidopsis cyclin-dependent kinase inhibitors, ICKs.

Plants have a family of cyclin-dependent kinase (CDK) inhibitors called ICKs (interactors/inhibitors of CDK) (or KRPs, Kip-related proteins). ICK proteins have important functions in cell proliferation, endoreduplication, plant growth and reproductive development and their functions depend on the protein levels. However, understanding is very limited regarding how ICK protein levels are regulated. We fused Arabidopsis ICK sequences to the green fluorescent protein (GFP), and determined their effects on the fusion proteins in plants, yeast and E. coli. The N-terminal regions of ICKs drastically reduced GFP fusion protein levels in Arabidopsis plants. A number of short sequences of 10 - 20 residues were found to decrease GFP fusion protein levels, when fused at the N-terminus or C-terminus. Three of the four short sequences from ICK3 showed a similar function in yeast. Intriguingly, three short sequences from ICK1 and ICK3 caused the degradation of the fusion proteins in E. coli. In addition, computational analyses showed that ICK proteins were mostly disordered and unstructured except for the conserved C-terminal region, suggesting that ICKs are intrinsically disordered proteins. This study thus has identified a number of short protein-destabilizing sequences and evidence suggests that some of them may cause protein degradation through structural disorder and instability.