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.
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.
Clark
Lisa B
LB
Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham, New Hampshire 03824, USA.
Viswanathan
Palaniswamy
P
Quigley
Gloria
G
Chiang
Yueh-Chin
YC
McMahon
Jeffrey S
JS
Yao
Gang
G
Chen
Junji
J
Nelsbach
Andreas
A
Denis
Clyde L
CL
D000595:Amino Acid Sequence; D004789:Enzyme Activation; D016350:Leucine Zippers / Q000502:physiology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000502:physiology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D011506:Proteins; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; C078657:CCR4 protein, S cerevisiae; D012260:Ribonucleases; C079009:POP2 protein, S cerevisiae