Characterization of CAF4 and CAF16 Reveals a Functional Connection between the CCR4-NOT Complex and a Subset of SRB Proteins of the RNA Polymerase II Holoenzyme

Abstract

The CCR4-NOT transcriptional regulatory complex affects transcription both positively and negatively and consists of the following two complexes: a core 1 × 106 dalton (1 MDa) complex consisting of CCR4, CAF1, and the five NOT proteins and a larger, less defined 1.9-MDa complex. We report here the identification of two new factors that associate with the CCR4-NOT proteins as follows: CAF4, a WD40-containing protein, and CAF16, a putative ABC ATPase. Whereas neither CAF4 nor CAF16 was part of the core CCR4-NOT complex, both CAF16 and CAF4 appeared to be present in the 1.9-MDa complex. CAF4 also displayed physical interactions with multiple CCR4-NOT components and with DBF2, a likely component of the 1.9-MDa complex. In addition, both CAF4 and CAF16 were found to interact in a CCR4-dependent manner with SRB9, a component of the SRB complex that is part of the yeast RNA polymerase II holoenzyme. The three related SRB proteins, SRB9, SRB10, and SRB11, were found to interact with and to coimmunoprecipitate DBF2, CAF4, CCR4, NOT2, and NOT1. Defects in SRB9 and SRB10 also affected processes at the ADH2 locus known to be controlled by components of the CCR4-NOT complex; an srb9 mutation was shown to reduce ADH2 derepression and either an srb9 or srb10 allele suppressed spt10-enhanced expression of ADH2. In addition, srb9 and srb10 alleles increased ADRl C -dependent ADH2 expression; not4 and not5 deletions are the only other known defects that elicit this phenotype. These results suggest a close physical and functional association between components of the CCR4-NOT complexes and the SRB9, -10, and -11 components of the holoenzyme.