Characterization of Mutations in NOT2 Indicates that it Plays an Important Role in Maintaining the Integrity of the CCR4–NOT Complex

The NOT2 protein is a component of the CCR4–NOT complex that plays multiple roles in the regulation of mRNA production in the yeast Saccharomyces cerevisiae. We have identified four novel not2 mutations and have characterized these and two previously described alleles as to the means by which they affect CCR4–NOT function. While two of the not2 alleles, not2-4 (carrying a G31R alteration) and not2∷L9P, resulted in severe growth defects and caused a not phenotype at the HIS3 locus, these phenotypes appear to arise from partially different effects. The not2∷L9P mutation resulted in complete loss of the 1.9×106 Da (1.9 MDa) CCR4–NOT complex, and the not2∷L9P protein displayed increased ability to associate with the NOT5 protein. In contrast, the not2-4 allele destabilized the CCR4–NOT complex to a lesser extent and had no effect on NOT5 association with NOT2. Instead, as previously reported, it displayed defective interactions with ADA2, a component of the SAGA complex. The not2∷R165G also abrogated NOT2 ability to interact with ADA2 but had little effect on the integrity of the CCR4–NOT complex. Alterations in NOT2 contacts to ADA2, therefore, do not necessarily result in effects on the CCR4–NOT complex nor result in severe growth defects. We also observed that the four NOT2 N-terminal mutations affected NOT5 association with the CCR4–NOT complexes, suggesting that it is the N terminus of NOT2 that contacts and stabilizes NOT5 interactions. These results indicate that it is the loss of the integrity of the CCR4–NOT complex which leads to severe not2 phenotypes and that the NOT2 contacts to ADA2 play a lesser role in NOT2 function.