Minisymposium 7: Protein turnover

Abs # 17003: The chloroplast protease, ClpP4, is a substrate of AtCHIP, an E3 ubiquitin ligase involved in stress response in plants

Presenter:	Zhang, Hong , hong.zhang@ttu.edu
Authors	Shen, Guoxin  (A)   Yan, Juqiang  (A)   Luo, Ester  (A)   Clarke, Adrian K. (B)   Zhang, Hong  (A)
Affiliations:	(A): Texas Tech University (B): Goteborg University

The Arabidopsis gene AtCHIP encodes a U-box-containing E3 ubiquitin ligase that plays a critical role in temperature stress tolerance [Plant Physiol., 132, 861-869]. Overexpression of AtCHIP leads to increased sensitivity to both high and low temperatures in Arabidopsis. To understand the molecular mechanism of AtCHIP’s involvement in temperature stresses, proteins that physically interact with AtCHIP were identified from yeast two-hybrid screening using AtCHIP as bait. One AtCHIP-interacting protein is the nuclear-encoded chloroplast protease ClpP4, which belongs to a large family of ATP-dependent, Ser-type proteases found in chloroplast and bacteria. We expressed ClpP4 in bacterial cells and purified ClpP4 protein for ubiquitylation experiments. AtCHIP is able to add three ubiquitin molecules to ClpP4 in vitro, which not only confirms the protein-protein interaction data from yeast two-hybrid screening, but also suggests that ClpP4 might be AtCHIP’s substrate in vivo. Since ClpP4 is part of a major protein-degradation machinery in chloroplast stroma, its ubiquitylation by AtCHIP suggests that AtCHIP might regulate protein degradation in chloroplast. Our analysis of other AtCHIP-interacting proteins further support the possibility that AtCHIP plays important roles in protein quality control in chloroplast because it interacts with at least two more nuclear encoded proteases in chloroplast. AtCHIP orthologs in animals (i.e. CHIP proteins) interact with Hsp70, Hsp90, and proteasome, and they have been shown to be critical in the regulation of protein quality control in cytoplasm. In the light of our new findings, we now believe that AtCHIP and animal CHIP proteins regulate protein turnover metabolism in both cytoplasm and organelles.