The CCR4 and CAF1 proteins of the CCR4-NOT complex are physically and functionally separated from NOT2, NOT4, and NOT5

Abstract

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.

FIG. 1

CCR4 requires CAF1 to immunoprecipitate NOT proteins. Immunoprecipitations with CCR4 antibody were conducted in caf1 (A792, pop2) and wild-type (A790) strains. Lanes 1 and 2, protein extracts from strains A790 and A792, respectively; lanes 3 and 4, immunoprecipitated (Ip) proteins analyzed by Western analysis using antibodies directed against NOT1 through NOT5 and CCR4. The NOT1 antibody used in these experiments could not detect NOT1 protein in crude (cr.) extracts (lanes 1 and 2), but other results indicate that NOT1 is present in both CAF1- and caf1-containing strains (20).

FIG. 2

CCR4 associates with CAF1 in the absence of NOT3, NOT4, NOT5, and the N-terminal 396 residues of NOT1. (A) Immunoprecipitations (Ip) were conducted with anti-CCR4 antibody. Western analysis was conducted with antibody directed against NOT1, NOT3, CCR4, NOT4, CAF1, NOT2, or NOT5 as indicated. An enhanced chemiluminescence-based system was used for NOT1, CCR4, and NOT5 Western blots for lanes 1 to 4, whereas an alkaline phosphatase-based system was used for the remainder of the results. Strains: wild type (wt), KY803; not1-2, MY8; not3, MY508; not4, MY537; not5, MY1735. (B) Yeast extracts from KY803 (wild type [wt]), 1393-4a (not2), and MY1735 (not5) were analyzed by gel filtration chromatography using a Superose 6 10/30 column. The protein extracts were precleared by centrifugation at 100,000 × g for 1 min, and 200 μl of sample was loaded onto the column. The flow rate was 0.2 ml/min, and a 0.5-ml volume was collected in each fraction; 100 μl from each fraction were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting using CCR4 and CAF1 antibodies. Molecular weight markers for the gel filtration experiment were blue dextran (2 × 10⁶ Da), thyroglobulin (0.67 × 10⁶ Da), and bovine serum albumin (6.6 × 10⁴ Da). (C) Immunoprecipitations were conducted in strain MY1737 [not1 pNOT1(396–2108)] and wild-type backgrounds with CAF1 antibody. Lanes 1 and 2 contain 1/10 of the crude extract (Cr. Ex.) protein input used for the immunoprecipitations (Ip) in lanes 3 and 4. Western analysis was conducted with anti-CCR4 and anti-NOT antibodies as indicated. NOT4 and NOT2 proteins in the crude extracts in lanes 1 and 2 were visible in the original Western blots and were in equal abundance for the two strains. IgG, immunoglobulin G.

FIG. 2

CCR4 associates with CAF1 in the absence of NOT3, NOT4, NOT5, and the N-terminal 396 residues of NOT1. (A) Immunoprecipitations (Ip) were conducted with anti-CCR4 antibody. Western analysis was conducted with antibody directed against NOT1, NOT3, CCR4, NOT4, CAF1, NOT2, or NOT5 as indicated. An enhanced chemiluminescence-based system was used for NOT1, CCR4, and NOT5 Western blots for lanes 1 to 4, whereas an alkaline phosphatase-based system was used for the remainder of the results. Strains: wild type (wt), KY803; not1-2, MY8; not3, MY508; not4, MY537; not5, MY1735. (B) Yeast extracts from KY803 (wild type [wt]), 1393-4a (not2), and MY1735 (not5) were analyzed by gel filtration chromatography using a Superose 6 10/30 column. The protein extracts were precleared by centrifugation at 100,000 × g for 1 min, and 200 μl of sample was loaded onto the column. The flow rate was 0.2 ml/min, and a 0.5-ml volume was collected in each fraction; 100 μl from each fraction were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting using CCR4 and CAF1 antibodies. Molecular weight markers for the gel filtration experiment were blue dextran (2 × 10⁶ Da), thyroglobulin (0.67 × 10⁶ Da), and bovine serum albumin (6.6 × 10⁴ Da). (C) Immunoprecipitations were conducted in strain MY1737 [not1 pNOT1(396–2108)] and wild-type backgrounds with CAF1 antibody. Lanes 1 and 2 contain 1/10 of the crude extract (Cr. Ex.) protein input used for the immunoprecipitations (Ip) in lanes 3 and 4. Western analysis was conducted with anti-CCR4 and anti-NOT antibodies as indicated. NOT4 and NOT2 proteins in the crude extracts in lanes 1 and 2 were visible in the original Western blots and were in equal abundance for the two strains. IgG, immunoglobulin G.

FIG. 2

CCR4 associates with CAF1 in the absence of NOT3, NOT4, NOT5, and the N-terminal 396 residues of NOT1. (A) Immunoprecipitations (Ip) were conducted with anti-CCR4 antibody. Western analysis was conducted with antibody directed against NOT1, NOT3, CCR4, NOT4, CAF1, NOT2, or NOT5 as indicated. An enhanced chemiluminescence-based system was used for NOT1, CCR4, and NOT5 Western blots for lanes 1 to 4, whereas an alkaline phosphatase-based system was used for the remainder of the results. Strains: wild type (wt), KY803; not1-2, MY8; not3, MY508; not4, MY537; not5, MY1735. (B) Yeast extracts from KY803 (wild type [wt]), 1393-4a (not2), and MY1735 (not5) were analyzed by gel filtration chromatography using a Superose 6 10/30 column. The protein extracts were precleared by centrifugation at 100,000 × g for 1 min, and 200 μl of sample was loaded onto the column. The flow rate was 0.2 ml/min, and a 0.5-ml volume was collected in each fraction; 100 μl from each fraction were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting using CCR4 and CAF1 antibodies. Molecular weight markers for the gel filtration experiment were blue dextran (2 × 10⁶ Da), thyroglobulin (0.67 × 10⁶ Da), and bovine serum albumin (6.6 × 10⁴ Da). (C) Immunoprecipitations were conducted in strain MY1737 [not1 pNOT1(396–2108)] and wild-type backgrounds with CAF1 antibody. Lanes 1 and 2 contain 1/10 of the crude extract (Cr. Ex.) protein input used for the immunoprecipitations (Ip) in lanes 3 and 4. Western analysis was conducted with anti-CCR4 and anti-NOT antibodies as indicated. NOT4 and NOT2 proteins in the crude extracts in lanes 1 and 2 were visible in the original Western blots and were in equal abundance for the two strains. IgG, immunoglobulin G.

FIG. 3

Localization of the NOT1 protein region that is sufficient for binding CCR4. (A) LexA-NOT1(667–1152) is sufficient for binding to CCR4 and CAF1. LexA-NOT1 fusions as indicated were expressed in strain EGY188, and immunoprecipitations were conducted with anti-CCR4 antibody. Western analysis was conducted with anti-LexA antibody. The crude protein extracts in lanes 1 to 5 contain 1/10 of the amount of extract used for the immunoprecipitations (Ip) displayed in lanes 6 to 10, respectively. IgG, immunoglobulin G. (B) The C terminus of NOT1 binds NOT2, NOT4, and NOT5. Strain EGY188 containing either LexA-NOT1(1–1152) or LexA-NOT1(1490–2108) was immunoprecipitated with LexA antibody. Western analysis using the antibodies as indicated was conducted as detailed in Fig. 1. Cr. Ex., crude extract.

FIG. 4

Gel filtration analysis of CCR4 in NOT1 mutant backgrounds. (A) Strain KY803 (wild type); (B) strain MY1738 [not1 pNOT1(1319–2108)]; (C) strain MY1737 [not1 pNOT1(396–2108)]; (D) strain MY8 (not1-2). Gel filtration chromatography was conducted as described for Fig. 2B. Anti-CCR4 antibody was used to detect the CCR4 protein.

FIG. 5

CCR4, CAF1, and NOT5 protein levels in not mutant backgrounds. All strains were grown to mid-log phase in YEP medium containing 5% glucose. Cells were harvested and lysed, and 40 μg of total protein was loaded in each lane. The RNA helicase homolog DHH1 was used as an internal control to demonstrate equivalent loading on the sodium dodecyl sulfate-polyacrylamide gel. Strains used: KY803 (wild type [wt]); MY8 (not1-2); MY1737 [not1 pNOT1(396–2108)]; MY1738 [not1 pNOT1(1319–2108)]; MY16 (not2-1); 1393-4a (not2); MY508 (not3); MY537 (not4); MY1735 (not5). Western blot analysis was conducted with antibodies directed against CCR4, CAF1, NOT5, and DHH1 as indicated.

FIG. 6

NOT5 coimmunoprecipitates with NOT2 in the absence of CAF1, NOT3, and NOT4. (A) LexA-NOT5 immunoprecipitates NOT2 in the absence of CAF1, NOT3, and NOT4. Immunoprecipitations (Ip) were conducted with anti-LexA antibody, and Western analysis used antibodies as indicated; LexA-NOT5 is full-length NOT5 fused to LexA(1–202). Lanes: 1, KY803 (wild type [wt]); 2, KY803-c1 (caf1); 3, MY508 (not3); 4, MY537 (not4). In the original Western blots, CCR4 was immunoprecipitated in lane 3. (B) NOT5 antibody immunoprecipitates NOT2. Antibody against CCR4 (lane 1) or NOT5 (lane 2) was used for immunoprecipitations from strain KY803. (C) NOT3 immunoprecipitates NOT1, NOT2, and NOT5 in the absence of CCR4, CAF1, and NOT4. Anti-NOT3 antibody was used to conduct the immunoprecipitations. WT (wild type), strain KY803; ccr4, KY803-1; caf1, KY803-c1; not4, MY537. Western analysis was conducted as detailed above.

FIG. 7

Model for protein contacts in the CCR4-NOT complex. Based on the results presented herein, CAF1 is presumed to bind to residues 667 to 1152 of NOT1, CCR4 binds to CAF1, and NOT2 and NOT5 interact with the C-terminal residues 1490 to 2108 of NOT1 in no particular order. NOT4 is placed on the periphery of NOT2 and NOT5, and it is presumed that NOT3 makes contacts with both NOT2, NOT5, or NOT4 and the N terminus of NOT1.