Article Figures & Data
Figures
- Fig. 1.
Coimmunoprecipitation of LexA-MOB1 with B42-DBF2. Crude extracts (Cr. Ex.) from strain EGY188 containing either B42 and LexA-MOB1 (lanes 1, 4, and 7), B42-DBF2 and LexA (lanes 2, 5, and 8), or B42-DBF2 and LexA-MOB1 (lanes 3, 6, and 9) were incubated with either anti-LexA antibody (lanes 4 to 6) or anti-HA1 antibody (lanes 7 to 9), and the resulting immunoprecipitates (Ip) were subjected to electrophoresis on an SDS–10% polyacrylamide gel. Western analysis was conducted as described previously (8), and the blot was probed with HA1 antibody. Lanes 1 to 3 have crude extracts containing B42 (lane 1) or B42-DBF2 (lanes 2 and 3). The same extracts were immunoprecipitated and analyzed as described above. The blot was probed with LexA antibody. Lanes 1 and 3 contain LexA-MOB1 from crude extracts, and lane 2 has crude extract containing LexA. LexA-MOB1 was capable of being immunoprecipitated with anti-LexA antibody from a strain containing B42 and LexA-MOB1 (data is not shown). Molecular masses are as follows: B42, 10 kDa; B42-DBF2, 72 kDa; LexA, 22 kDa; and LexA-MOB1, 54 kDa.
- Fig. 2.
GST-MOB1 binds B42-DBF2 and B42-CAF1 from crude extracts. The GST and GST-MOB1 proteins expressed in E. coliwere bound to glutathione-agarose beads and then incubated with crude extracts (Cr. Ex.) from the EGY191 strain containing either B42 (lane 1), B42-DBF2 (lane 2), or B42-CAF1 (lane 3). The beads were then boiled with SDS sample buffer, the eluted protein was loaded on an SDS–10% polyacrylamide gel. HA1-containing proteins were detected by Western analysis as previously described (8). Lanes: 1 to 3, crude extracts; 4 to 6, GST incubated with crude extracts from EGY191/B42, EGY191/B42-DBF2, and EGY191/B42-CAF1, respectively; 7 to 9, same as 4 to 6, respectively, except crude extracts were incubated with GST-MOB1. The molecular mass of B42-CAF1 is 54 kDa.
- Fig. 3.
DBF2 binds to MOB1 at physiological concentrations. Extracts from strains S7-4A (wt) and S7-4A-c-myc were incubated with either preimmune MOB1 serum (pI) (lanes 3 and 4), anti-MOB1 antibody (lanes 5 and 6), or anti-c-myc antibody (lanes 7 and 8), and resulting immunoprecipitates (Ip) were subjected to electrophoresis on an SDS–8% polyacrylamide gel. Western analysis was conducted as described previously (8). The upper portion of the blot was probed with c-myc antibody (Ab), and the lower portion was probed with MOB1 antibody. Lanes 1 and 2, crude extracts (CE) containing DBF2-c-myc (lane 2) and/or MOB1 (lanes 1 and 2). DBF2-c-myc is 62 kDa and MOB1 is 34 kDa in size.
- Fig. 4.
Binding of MOB1 to DBF2. (A) Coomassie-stained GST, GST-MOB1, and GST-Vpu. GST fusions were induced as described elsewhere (9), bound to glutathione-agarose beads, eluted from the beads by boiling, and fractionated on an SDS–8% polyacrylamide gel. (B) T7 fusion proteins were translated in vitro with [35S]methionine as described in Materials and Methods. One milliliter of each radioactive protein was separated by SDS-polyacrylamide gel electrophoresis and identified following fluorography. Ten milliliters of each in vitro-translated protein was incubated with 50 mg of a GST fusion and, after washing, eluted by boiling. Molecular masses (in kilodaltons) are indicated on the left.
- Fig. 5.
MOB1 is expressed under cell cycle control. A culture of strain CG378 was synchronized by use of α-factor, and samples were taken for RNA hybridization analysis to determine the levels of the MOB1 (□) and DBF2 (▵) transcripts (12). Actin transcript levels were also determined as a control and used to normalize the MOB1 andDBF2 levels in the graph. The percentages of buds in the synchronized population are also shown as an indication of culture synchrony (○).
- Fig. 6.
Ability of B42-DBF2 and B42-MOB1 to suppress themob1 defect. Strain FLY59 mob1(pRS316-MOB1) was transformed with five different plasmids, expressing B42 alone, B42-DBF2, B42-MOB1(145–314), B42-MOB1(79–314), or B42-MOB1(9–314) as indicated. Transformants were grown overnight in medium lacking uracil, and about 15 × 104 cells of each strain, including the FLY59 strain without any B42 plasmids (−), were plated on medium containing fluoroorotic acid. The picture was taken after 72 h of incubation at 30°C.
- Fig. 7.
Coimmunoprecipitation of LexA-DBF20 with B42-MOB1 and B42-mob1 temperature-sensitive fusions. Extracts from diploid strain EGY188/EGY191 containing LexA-DBF20 (full length) and either B42-MOB1(79–314) (lane 1), B42–mob1-95 (lane 2), B42–mob1-77 (lane 3), or B42–mob1-55 (lane 4) were incubated with LexA antibody, and the resulting immunoprecipitates (Ip) were subjected to electrophoresis on an SDS–10% polyacrylamide gel. Western analysis was conducted as described previously (8), and the blot was probed with HA1 antibody. The positions of molecular mass markers (in kilodaltons) are indicated on the left.
Tables
- Table 1.
Yeast strains used in this study
Strain Genotype EGY188 MAT a ura3 his3 trp1 LexA-LEU2 EGY188-c1 Isogenic to EGY188 except caf1::URA3 EGY188-1 Isogenic to 188 except ccr4::URA3 EGY191 MATα ura3 his3 trp1 LexA-LEU2 991-1-1b MAT a ura3 his3 trp1 leu2 dbf2::URA3 1045-2b MATα ura3 his3 trp1 leu2 dbf20::TRP1 1300-1a MAT a ura3 his3 trp1 leu2 mob1-77 CG378 MAT a ura3 leu2 trp1 FLY30 MAT a ura3 his3 trp1 leu2 mob1-77 FLY59 MAT a ura3 his3 trp1 leu2 mob1::HIS3; contains plasmid pRS316-MOB1 (URA3) S7-4A MAT a dbf2::URA3 ura3 leu2 ade5 trp1 his7 S7-4A-c-myc Isogenic to S7-4A excepttrp1::c-myc-DBF2-TRP1 - Table 2.
Two-hybrid interaction of MOB1 with DBF2 and CAF1a
LexA fusion B42 fusion β-gal (U/mg)b LexA-DBF2(1–561) B42-MOB1(9–314) 260 LexA-DBF2(1–561) B42-MOB1(79–314) 530 LexA-DBF2(1–561) B42-MOB1(145–314) 66 LexA-DBF2(1–561) B42 <1 LexA B42-MOB1(9–314) <1 LexA-DBF2(1–220) B42-MOB1(9–314) 59 LexA-DBF2(1–220) B42-MOB1(145–314) 120 LexA-DBF2(1–220) B42 <1 LexA-MOB1(9–314) B42-DBF2(1–561) 6,700 LexA-MOB1(9–314) B42-DBF2(205–561) 350 LexA-MOB1(9–314) B42-CAF1 300 LexA-MOB1(9–314) B42 81 ↵a Strains were grown on minimal medium lacking uracil, histidine, and tryptophan and supplemented with 2% raffinose and 2% galactose as previously described (8). All assays were done in EGY188/EGY191 diploids containing the p34 reporter (eight LexA operators upstream of the GAL1-lacZ promoter). B42-DBF2(1–561) and B42-DBF2(205–561) were expressed to comparable levels as determined by Western analysis (data not shown).
↵b β-Galactosidase (β-Gal) activities represent the average of determinations for at least three separate transformants. The standard error of the mean was less than 20% in each case.
- Table 3.
Suppression analysis of MOB1 and DBF2
Genotype Suppression with plasmida: B42-MOB1 B42-DBF2 B42 37°C Caffeine 37°C Caffeine 37°C Caffeine mob1-77b + + + + − − dbf2c + + + + − − ↵a Growth was monitored on yeast extract-peptone agar plates supplemented with 2% each galactose and raffinose and incubated at 37°C and on the same plates additionally supplemented with 8 mM caffeine but incubated at 30°C. B42-MOB1 refers to MOB1(9–314), although B42-MOB1(79–314) complemented as well. Similar results were obtained for a mob1-95 allele or for a dbf2 temperature-sensitive allele. +, suppression evident; −, no suppression evident.
↵b Strain 1300-1a.
↵c Strain 991-1-1b.
- Table 4.
Two-hybrid interactions of wild-type and mutated forms of MOB1 with DBF2, MPS1, and DBF20
LexA fusiona B42 fusionb β-gal activity (U/mg)c LexA-DBF2 B42-MOB1 530 LexA-DBF2 B42–MOB1-55 (T85P, Q167R, Y183H) 70 LexA-DBF2 B42–MOB1-77 (E151K)d 40 LexA-DBF2 B42–MOB1-95 (L157P, A158I) 20 LexA-MPS1 B42-MOB1 100 LexA-MPS1 B42–MOB1-55 (T85P, Q167R, Y183H) 40 LexA-MPS1 B42–MOB1-77 (E151K)d 120 LexA-MPS1 B42–MOB1-99 (L157P, A158I) 120 LexA-DBF20 B42-MOB1 7.7 LexA-DBF20 B42–MOB1-55 (T85P, Q167R, Y183H) <1 LexA-DBF20 B42–MOB1-77 (E151K)d <1 LexA-DBF20 B42–MOB1-95 (L157P, A158I) <1 ↵a LexA-DBF2 contains full-length DBF2(1–561), LexA-MPS1 contains full-length MPS1(1–764), LexA-DBF20 contains full-length DBF20(1–564), and B42-MOB1 fusions contain residues 79 to 314 of MOB1.
↵b Specific mutations associated with the MOB moieties of the constructs are given in parentheses; e.g., T85P indicates a substitution of a proline for the threonine at position 85.
↵c β-Galactosidase (β-Gal) activities represent the average of three to six separate determinations. The standard error of the mean was less than 15% in each case. Assays were conducted as described in Table 2. All B42-MOB1 fusions were found to be expressed to comparable levels, as determined by Western analysis, and the LexA fusions were comparably expressed as well (data not shown). Assays were conducted in EGY188/EGY191 diploids containing the p34 reporter.
↵d The original mob1-77temperature-sensitive mutant contained an additional mutation, N65I, but the E151K alteration is sufficient to make MOB1 unable to fully complement a mob1 defect (data not shown).
- Table 5.
Transactivation effects of LexA-MOB1 variants
LexA fusiona β-Gal (U/mg)b LexA-MOB1 230 LexA–MOB1-55 (T85P, Q167R, Y183H) 100 LexA–MOB1-77 (E151K) 440 LexA–MOB1-95 (L157P, A158I) 36 ↵a Assays were conducted as described in Table 2. All LexA-MOB1 fusions were expressed to comparable levels as determined by Western analysis (data not shown). LexA-MOB1 fusions contain residues 79 to 314 of MOB1 and were expressed in EGY188 containing the p34 reporter.
↵b β-Galactosidase (β-Gal) activities represent the average of the determinations for at least three transformants. The standard error of the mean was less than 10% in each case except for LexA–MOB1-95, in which it was 21%.
