Systematic mutagenesis of the leucine-rich repeat (LRR) domain of CCR4 reveals specific sites for binding to CAF1 and a separate critical role for the LRR in CCR4 deadenylase activity

被引:33
作者
Clark, LB [1 ]
Viswanathan, P [1 ]
Quigley, G [1 ]
Chiang, YC [1 ]
McMahon, JS [1 ]
Yao, G [1 ]
Chen, JJ [1 ]
Nelsbach, A [1 ]
Denis, CL [1 ]
机构
[1] Univ New Hampshire, Dept Biochem & Mol Biol, Durham, NH 03824 USA
关键词
D O I
10.1074/jbc.M313202200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
CCR4, a poly(A) deadenylase of the exonuclease III family, is a component of the multiprotein CCR4-NOT complex of Saccharomyces cerevisiae that is involved in mRNA degradation. CCR4, unlike all other exonuclease III family members, contains a leucine-rich repeat (LRR) motif through which it makes contact to CAF1 and other factors. The LRR residues important in contacting CAF1 were identified by constructing 29 CCR4 mutations encompassing a majority ( 47 of 81) of residues interstitial to the conserved structural residues. Two-hybrid and immunoprecipitation data revealed that physical contact between CAF1 and the LRR is blocked by mutation of just two alpha-helix/beta-helix strand loop residues linking the first and second repeats. In contrast, CAF16, a potential ligand of CCR4, was abrogated in its binding to the LRR by mutations in the N terminus of the second beta-strand. The LRR domain was also found to contact the deadenylase domain of CCR4, and deletion of the LRR region completely inhibited CCR4 enzymatic activity. Mutations throughout the beta-sheet surface of the LRR, including those that did not specifically interfere with contacts to CAF1 or CAF16, significantly reduced CCR4 deadenylase activity. These results indicate that the CCR4-LRR, in addition to binding to CAF1, plays an essential role in the CCR4 deadenylation of mRNA.
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页码:13616 / 13623
页数:8
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