The CCR4 transcriptional regulatory complex consisting of CCR4, CAF1, DBF2 and other unidentified factors is one of several groups of proteins that affect gene expression. Using mass spectrometry, we have identified the 195, 185 and 116 kDa species which are part of the CCR4 complex. The 195 and 185 kDa proteins were found to be NOT1 and the 116 kDa species was identical to NOT3. NOT1, 2, 3 and 4 proteins are part of a regulatory complex that negatively affects transcription. All four NOT proteins were found to co-immunoprecipitate with CCR4 and CAF1, and NOT1 co-purified with CCR4 and CAF1 through three chromatographic steps in a complex estimated to be 1.2x10(6) Da in size. Mutations in the NOT genes affected many of the same genes and processes that are affected by defects in the CCR4 complex components, including reduction in ADH2 derepression, defective cell wall integrity and increased sensitivity to monoand divalent ions. Similarly, ccr4, caf1 and dbf2 alleles negatively regulated FUS1-lacZ expression, as do defects in the NOT genes. These results indicate that the NOT proteins are physically and functionally part of the CCR4 complex which forms a unique and novel complex that affects transcription both positively and negatively.
The CCR4-NOT transcriptional regulatory complex affects transcription both positively and negatively and consists of the following two complexes: a core 1 ؋ 10 6 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 ADR1 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.
cAMP-dependent protein kinase (cAPK) is implicated in the inactivation of the yeast transcriptional activator ADR1, which regulates glucose-repressible ADH2 gene expression. The interdependence of cAPK, SCH9 (a protein kinase that when overexpressed can functionally substitute for cAPK), and the CCR1 (SNF1) protein kinase that is required for ADH2 expression was studied. SCH9 was found to be required for ADH2 expression in contrast to the inhibitory role played by cAPK. CCR1 and SCH9 were observed to affect ADH2 expression independently of both ADR1 and cAPK. In contrast, cAPK was shown to exert its effects on ADH2 solely through ADR1. These results indicate that the SCH9 and CCR1 protein kinases are components of regulatory pathways separate from that utilized by cAPK to control ADR1 activity and ADH2 expression.
The rate ofADH2 transcription increases dramatically when Saccharomyces cerevisiae cells are shifted from glucose to ethanol growth conditions. Since ADH2 expression under glucose growth conditions is strictly dependent on the dosage of the transcriptional activator ADR1, we investigated the possibility that regulation of the rate of ADR1 protein synthesis plays a role in controlling ADR1 activation of ADH2 transcription. We found that the rate of ADRI protein synthesis increased 10-to 16-fold within 40 to 60 min after glucose depletion, coterminous with initiation ofADH2 transcription. Changes in ADRI mRNA levels contributed only a twofold effect on ADR1 protein synthetic differences. The 510-nt untranslated ADRI mRNA leader sequence was found to have no involvement in regulating the rate of ADR1 protein synthesis. In contrast, sequences internal to ADRI coding region were determined to be necessary for controlling ADRI translation. The ADRIC mutations which enhance ADR1 activity under glucose growth conditions did not affect ADR1 protein translation. ADR1 was also shown to be multiply phosphorylated in vivo under both ethanol and glucose growth conditions. Our results indicate that derepression of ADH2 occurs through multiple mechanisms involving the ADR1 regulatory protein.In the yeast Saccharomyces cerevisiae, glucose represses the transcription of numerous genes, including those required for ethanol and alternate sugar metabolism, the glyoxylate shunt, the tricarboxylic acid cycle, respiration, gluconeogenesis, and mitochondrial function. Several regulatory genes which, when mutated, either allow glucoseinsensitive transcription or inhibit derepression upon glucose removal have been identified (34). The mechanisms by which glucose repression occurs, however, remain largely obscure. We have previously implicated the glucose-induced adenyl cyclase signalling system in the inhibition of alcohol dehydrogenase II (ADH II; ADH2 gene) expression (7,13,16). This mechanism, though, would account for only a small portion of the total glucose repression of ADH2 (7,14). We show in the present study that glucose-dependent control of ADR1 protein synthesis contributes significantly to the overall glucose regulation of ADH2 expression.The ADR1 regulatory protein is a transcriptional activator of the ADH2 gene (14,33) and is required for the dramatic 500-fold increase in ADH II activity, which is initiated upon depletion or removal of glucose from the growth medium (8). Activation of ADH2 transcription requires the binding of ADR1 protein to a 22-bp dyad upstream activation sequence in the noncoding region of the ADH2 gene (35). Evidence
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