Purification and characterization of the 1.0 MDa CCR4-NOT complex identifies two novel components of the complex.
The CCR4-NOT complex is an evolutionarily conserved, transcriptional regulatory complex that is involved in controlling mRNA initiation, elongation and degradation. The CCR4-NOT proteins from Saccharomyces cerevisiae exist in two complexes, 1.9x10(6) Da and 1.0x10(6) Da (1.0 MDa) in size, and individual components of these complexes display such disparate functions as binding to and restricting TFIID functions, contacting SAGA and contributing to mRNA deadenylation. As a first step in characterizing the functional roles of the 1.0 MDa complex, we have purified it to near homogeneity. Mass spectrometric analysis was subsequently used to identify all the components of the complex. The 1.0 MDa complex was found to contain CCR4, CAF1, NOT1-5 and two new proteins, CAF40 and CAF130. CAF130 and CAF40 are two unique yeast proteins, with CAF40 displaying extensive homology to proteins from other eukaryotes. Immunoprecipitation and gel filtration experiments confirmed that CAF130 and CAF40 are components of both of the 1.9 MDa and 1.0 MDa CCR4-NOT complexes. Biochemical analysis indicated that the CAF40 and CAF130 proteins bind to the NOT1 protein and exist in a location separate from the two other subsets of proteins in the complex: the CCR4 and CAF1 proteins, and the NOT2, NOT4 and NOT5 proteins. Moreover, CAF40 was able to interact with human NOT1, suggesting that human CAF40 would also be a component of the recently identified human CCR4-NOT complex. Analysis of caf40 and caf130 deletions indicated that they elicited phenotypes shared by defects in other CCR4-NOT genes. The distinct location of CAF40 and CAF130 and the evolutionary conservation of CAF40 implicate them in novel roles in the function of the CCR4-NOT complex.
Chen
J
J
Department of Biochemistry and Molecular Biology, Rudman Hall, University of New Hampshire, Durham, NH 03824, USA.
Rappsilber
J
J
Chiang
Y C
YC
Russell
P
P
Mann
M
M
Denis
C L
CL
D001665:Binding Sites; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D019143:Evolution, Molecular; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017353:Gene Deletion; D006801:Humans; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D008954:Models, Biological; D008970:Molecular Weight; D010641:Phenotype; D011233:Precipitin Tests; D011485:Protein Binding; D011506:Proteins; D012260:Ribonucleases; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017385:Sequence Homology; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
C446517:CAF130 protein, S cerevisiae; C446516:CAF40 protein, S cerevisiae; C079592:CDC39 protein, S cerevisiae; C489780:CNOT8 protein, human; D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D046911:Macromolecular Substances; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C078657:CCR4 protein, S cerevisiae; D012260:Ribonucleases; C079009:POP2 protein, S cerevisiae