Article Figures & Data
Figures
- Fig. 1.
CCR4 requires CAF1 to immunoprecipitate NOT proteins. Immunoprecipitations with CCR4 antibody were conducted incaf1 (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- andcaf1-containing strains (20).
- Fig. 2.
CCR4 associates with CAF1 in the absence ofNOT3, 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 NOT1mutant 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 notmutant 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 [not1pNOT1(396–2108)]; MY1738 [not1pNOT1(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.
Tables
- Table 1.
Yeast strains used
Strain Genotype A790 MATα aro7 his3 leu2 ura3 A792 MATα aro7 his3 leu2 ura3 pop2-Δ3::LEU2 KY803 MAT a leu2-PET56 trp1-Δ1 ura3-52 gal2 gcn4-Δ1 KY803-1 Isogenic to KY803 exceptccr4::URA3 KY803-c1 Isogenic to KY803 except caf1::LEU2 MY8 Isogenic to KY803 except not1-2 MY8-1d Isogenic to MY8 exceptccr4::URA3 MY8-c1c Isogenic to MY8 except caf1::LEU2 MY1737 Isogenic to KY803 excepthis3::TRP1 not1::LEU2 pRS426-NOT1(396–2108) MY1738 Isogenic to KY803 except MATα not1::LEU2 pRS426-NOT1(1319–2108) MY16 Isogenic to KY803 except not2-1 MY16-1c Isogenic to MY16 exceptccr4::URA3 MY508 Isogenic to KY803 except not3::URA3 MY508-c1b Isogenic to MY508 exceptcaf1::LEU2 MY25 Isogenic to KY803 except not3-2 MY25-1b Isogenic to MY25 exceptccr4::URA3 MY537 Isogenic to KY803 except not4::URA3 MY537-1 Isogenic to MY537not4::ura3 MY537-1-1b Isogenic to MY537-1 ccr4::URA3 MY1735 Isogenic to KY803 exceptnot5::URA3 EGY188 MAT a ura3 his3 trp1 LexA-LEU2 EGY188-1 Isogenic to EGY188 exceptccr4::URA3 EGY188-c1 Isogenic to EGY188 except caf1::URA3 EGY188-1-1 Isogenic to EGY188 exceptccr4::ura3 EGY188-c1-1 Isogenic to EGY188 except caf1::ura3 787-6b MATα adh1-11 ADR1-5C-TRP1 ura3 his3 leu2 trp1 1076-2c-1 MATα ura3 his3 leu2 trp1 ccr4::ura3::TRP1 1278-5d MAT a ura3 his3 leu2 trp1 caf1::LEU2 1393-4a MATα his3/his2 leu2 ura3 not2::TRP1 1402-1a MATα ura3 his3 leu2 trp1 not4::URA3 1402-4a MATα ura3 his3 leu2 trp1 not4::URA3 1422-21 MATα adh1-11 ura3 his3 leu2 trp1 not1::LEU2 pRS426-NOT1(1490–2108) 1469-2-1c MAT a ura3 his3 leu2 trp1 ccr4::ura3::TRP1 1462-3c MAT a ura3 his3 leu2 trp1 not5::URA3 - Table 2.
Effects of not1 truncations onADH2 expressiona
NOT1 plasmid LexA plasmid ADH II activity (mU/mg; mean ± SEM)b NOT1(396–2108) None 2,900 ± 450 NOT1(1319–2108) None 910 ± 150 LexA-NOT1(1–2108) 2,300 ± 150 LexA-NOT1(1–1152) 1,800 ± 120 LexA-NOT1(1–667) 830 ± 100 None LexA-NOT1(1–2108) 2,200 ± 320 LexA-NOT1(1490–2108) 1,000 ± 230 ↵a ADH II assays were conducted after growth of strain 1422–21 on YEP medium containing 3% ethanol. ADH II activities in a strain carrying an integrated full-lengthNOT1 gene is generally 2,500 to 3,000 mU/mg. LexA plasmids contain NOT1 sequences fused to LexA-202(7). NOT1 plasmids were pRS426 derivatives.
↵b Average of at least three separate transformants.
- Table 3.
Synthetic lethalities between deletions inccr4 and caf1 and mutations in thenot genesa
Construct Growth wt ccr4 caf1 wt + + + not1-2 30°C + + + 34°C + − − pNOT1(1490–2108) not1 + Lethal ND not2 + Lethal Lethal not2-1 30°C + + ND 34°C + − ND not3 + + + not3-2 30°C + + ND 37°C + − ND not4 + + + not5 + Lethal Lethal ↵a Growth was determined at 30°C on YEP medium supplemented with 2% glucose unless otherwise indicated. +, growth; −, no growth; lethal, the gene pair resulted in cell death; ND, not done. Isogenic strains used: wild type (wt), KY803;not1-2, MY8; not1-2 ccr4, MY8-1d; not1-2 caf1, MY8-c1c; not3, MY508; not3 caf1, MY508-c1b; not2-1, MY16; not2-1 ccr4, MY16-1c;not3, MY508; not3 caf1, MY508-c1b;not3-2, MY25; not3-2 ccr4, MY25-1b;not4, MY537-1; not4 ccr4, MY537-1-1b. not3 ccr4 segregants were obtained from 1076-2c-1 × MY508 crosses, and not4 caf1 segregants were obtained from 1278-5d × 1402-1a and 1278-5d × 1402-4a crosses. pNOT1(1480–2108) not1 ccr4 lethality was confirmed by analysis of segregants of diploid 1471 [1422-21/pNOT1(1480–2108)/pLexA-NOT1 × 1469-2-1c], ccr4 not2 lethality was confirmed from strain 1441 (188-1/pLexA-NOT2 × 1393-4a), ccr4 not5 lethality was confirmed from strain 1469 (1076-2c-1/pLexANOT5 × 1462-3a),caf1 not2 lethality was confirmed from strain 1442 (188-c1/pLexA-CAF1 × 1393-4a), and caf1 not5 lethality was confirmed from strain 1469 (1076-2c-1/pLexA-NOT5 × 1462-3c).
- Table 4.
caf1 and ccr4 disruptions suppressnot-induced 3-AT phenotypesa
Relevant genotype Growth on 3-AT (mM) 0 5 10 Wild type + − − caf1 + − − ccr4 + − − not1-2 + + + not1-2 caf1 + w − not1-2 ccr4 + − − not3-2 + + + not3-2 ccr4 + − − not4 + + + not4 caf1 + − − not4 ccr4 + − − ↵a All strains are isogenic to KY803 except for the indicated allele. Growth was monitored on minimal medium lacking histidine and supplemented with 3-AT as indicated.
