The CCR4±NOT complex from Saccharomyces cerevisiae is a general transcriptional regulatory complex. The proteins of this complex are involved in several aspects of mRNA metabolism, including transcription initiation and elongation and mRNA degradation. The evolutionarily conserved CCR4 protein, which is part of the cytoplasmic deadenylase, contains a C-terminal domain that displays homology to an Mg 2+ -dependent DNase/phosphatase family of proteins. We have analyzed the putative enzymatic properties of CCR4 and have found that it contains both RNA and singlestranded DNA 3¢±5¢ exonuclease activities. CCR4 displays a preference for RNA and for 3¢ poly(A) substrates, implicating it as the catalytic component of the cytoplasmic deadenylase. Mutations in the key, conserved catalytic residues in the CCR4 exonuclease domain abolished both its in vitro activities and its in vivo functions. Importantly, CCR4 was active as a monomer and remained active in the absence of CAF1, which links CCR4 to the remainder of the CCR4±NOT complex components. These results establish that CCR4 and most probably other members of a widely distributed CCR4-like family of proteins constitute a novel class of RNA±DNA exonucleases. The various regulatory effects of the CCR4±NOT complex on gene expression may be executed in part through these CCR4 exonuclease activities. Keywords: CCR4±NOT complex/deadenylase/ exonuclease/mRNA degradation/transcription IntroductionThe CCR4 protein from Saccharomyces cerevisiae is an evolutionarily conserved protein, and has orthologs in all higher eukaryotic organisms examined to date. In yeast, the CCR4 protein has been identi®ed in the CCR4±NOT protein complex, which exists in two forms in vivo: 1.0 and 1.9 MDa. The smaller core complex, which has been puri®ed Bai et al., 1999;Chen et al., 2001), contains the following proteins: CCR4, CAF1, ®ve NOT proteins (NOT1±NOT5), CAF40 and CAF130.The CCR4 gene was identi®ed initially by mutations that suppressed spt10-enhanced ADH2 expression and affected the derepression of ADH2 and other nonfermentative genes (Denis, 1984). However, it has become clear since then that CCR4, CAF1 and the NOT proteins affect diverse processes in yeast and do so both positively and negatively (Denis and Malvar, 1990;Sakai et al., 1992; Struhl, 1993, 1994;Liu et al., 1998Liu et al., , 2001Bai et al., 1999) Biochemical studies in our laboratory have shown that the CCR4 protein contains three major functional domains (Draper et al., 1994). The N-terminal region, which is rich in glutamines and asparagines, contains an activation domain that may interact with the transcriptional machinery. The central region of the protein (residues 350±473) contains ®ve tandem copies of a leucine-rich repeat (LRR) domain (Malvar et al., 1992). This domain has been shown to interact with CAF1 (Draper et al., 1995) and other putative components of the 1.9 MDa CCR4±NOT complex (Liu et al., 1997(Liu et al., , 2001 and to be the region of CCR4 that links CCR4 to the 1.0 MDa CCR4±NOT complex (Draper et al., 199...

The CCR4-NOT complex (1 mDa in size), consisting of the proteins CCR4, CAF1, and NOT1 to NOT5, regulates gene expression both positively and negatively and is distinct from other large transcriptional complexes in Saccharomyces cerevisiae such as SNF/SWI, TFIID, SAGA, and RNA polymerase II holoenzyme. The physical and genetic interactions between the components of the CCR4-NOT complex were investigated in order to gain insight into how this complex affects the expression of diverse genes and processes. The CAF1 protein was found to be absolutely required for CCR4 association with the NOT proteins, and CCR4 and CAF1, in turn, physically interacted with NOT1 through its central amino acid region from positions 667 to 1152. The NOT3, NOT4, and NOT5 proteins had no significant effect on the association of CCR4, CAF1, and NOT1 with each other. In contrast, the NOT2, NOT4, and NOT5 interacted with the C-terminal region (residues 1490 to 2108) of NOT1 in which NOT2 and NOT5 physically associated in the absence of CAF1, NOT3, and NOT4. These and other data indicate that the physical ordering of these proteins in the complex is CCR4-CAF1-NOT1-(NOT2, NOT5), with NOT4 and NOT3 more peripheral to NOT2 and NOT5. The physical separation of CCR4 and CAF1 from other components of the CCR4-NOT complex correlated with genetic analysis indicating partially separate functions for these two groups of proteins. ccr4 or caf1 deletion suppressed the increased 3-aminotriazole resistance phenotype conferred by not mutations, resulted in opposite effects on gene expression as compared to several not mutations, and resulted in a number of synthetic phenotypes in combination with not mutations. These results define the CCR4-NOT complex as consisting of at least two physically and functionally separated groups of proteins.

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The mouse CAF1 (mCAF1) is an ortholog of the yeast (y) CAF1 protein, which is a component of the CCR4-NOT complex, the major cytoplasmic deadenylase of Saccharomyces cerevisiae. Although CAF1 protein belongs to the DEDDh family of RNases, CCR4 appears to be the principle deadenylase of the CCR4-NOT complex. Here, we present evidence that mCAF1 is a processive, 3 -5 -RNase with a preference for poly(A) substrates. Like CCR4, increased length of RNA substrates converted mCAF1 into a processive enzyme. In contrast to two other DEDD family members, PAN2 and PARN, mCAF1 was not activated either by PAB1 or capped RNA substrates. The rate of deadenylation in vitro by yCCR4 and mCAF1 were both strongly influenced by secondary structures present in sequences adjacent to the poly(A) tail, suggesting that the ability of both enzymes to deadenylate might be affected by the context of the mRNA 3 -untranslated region sequences. The ability of mCAF1 to complement a ycaf1 deletion in yeast, however, did not require the RNase function of mCAF1. Importantly, yCAF1 mutations, which have been shown to block its RNase activity in vitro, did not inactivate yCAF1 in vivo, and mRNAs were deadenylated in vivo at nearly the same rate as found for wild type yCAF1. These results indicate that at least in yeast the CAF1 RNase activity is not required for its in vivo function.

A fundamental problem in proteomics is the identification of protein complexes and their components. We have used analytical ultracentrifugation with a fluorescence detection system (AU-FDS) to precisely and rapidly identify translation complexes in the yeast Saccharomyces cerevisiae. Following a one-step affinity purification of either poly(A)-binding protein (PAB1) or the large ribosomal subunit protein RPL25A in conjunction with GFP-tagged yeast proteins/RNAs, we have detected a 77S translation complex that contains the 80S ribosome, mRNA, and components of the closed-loop structure, eIF4E, eIF4G, and PAB1. This 77S structure, not readily observed previously, is consistent with the monosomal translation complex. The 77S complex abundance decreased with translational defects and following the stress of glucose deprivation that causes translational stoppage. By quantitating the abundance of the 77S complex in response to different stress conditions that block translation initiation, we observed that the stress of glucose deprivation affected translation initiation primarily by operating through a pathway involving the mRNA cap binding protein eIF4E whereas amino acid deprivation, as previously known, acted through the 43S complex. High salt conditions (1M KCl) and robust heat shock acted at other steps. The presumed sites of translational blockage caused by these stresses coincided with the types of stress granules, if any, which are subsequently formed.