Characterization of Fus3 localization: active Fus3 localizes in complexes of varying size and specific activity - PubMed (original) (raw)

Characterization of Fus3 localization: active Fus3 localizes in complexes of varying size and specific activity

K Y Choi et al. Mol Biol Cell. 1999 May.

Free PMC article

Abstract

The MAP kinase Fus3 regulates many different signal transduction outputs that govern the ability of Saccharomyces cerevisiae haploid cells to mate. Here we characterize Fus3 localization and association with other proteins. By indirect immunofluorescence, Fus3 localizes in punctate spots throughout the cytoplasm and nucleus, with slightly enhanced nuclear localization after pheromone stimulation. This broad distribution is consistent with the critical role Fus3 plays in mating and contrasts that of Kss1, which concentrates in the nucleus and is not required for mating. The majority of Fus3 is soluble and not bound to any one protein; however, a fraction is stably bound to two proteins of approximately 60 and approximately 70 kDa. Based on fractionation and gradient density centrifugation properties, Fus3 exists in a number of complexes, with its activity critically dependent upon association with other proteins. In the presence of alpha factor, nearly all of the active Fus3 localizes in complexes of varying size and specific activity, whereas monomeric Fus3 has little activity. Fus3 has highest specific activity within a 350- to 500-kDa complex previously shown to contain Ste5, Ste11, and Ste7. Ste5 is required for Fus3 to exist in this complex. Upon alpha factor withdrawal, a pool of Fus3 retains activity for more than one cell cycle. Collectively, these results support Ste5's role as a tether and suggest that association of Fus3 in complexes in the presence of pheromone may prevent inactivation in addition to enhancing activation.

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Figures

Figure 1

Figure 1

Effect of α factor addition and withdrawal on Fus3-HA kinase activity. Strain EY960 (EY940 + pYEE121, FUS3-HA CEN URA3) was induced with α factor for 2 h, and then the α factor was washed out and the G1-arrested cells were allowed to recover for 3 h. Duplicate samples were taken at the indicated time intervals during the α factor addition and withdrawal; one sample was pelleted and frozen for cell extracts, and the other was fixed with formaldehyde for microscopic analysis. A total of 18 time points (0–17, 120 min in the presence of α factor, 180 min after α factor washout) were analyzed for protein levels, tyrosine phosphorylation, and kinase activity, and a total of 17 time points were analyzed for cell morphology (0–16, 120 min in the presence of α factor, 165 min after α factor washout). Fus3-HA was detected with 12CA5 antibody, and Fus3-HA tyrosine phosphorylation was detected with an anti-phosphotyrosine antibody as previously described (Elion et al., 1993). Kinase assays were performed as described (see MATERIALS AND METHODS). Top panel, Abundance of Fus3-HA by immunoblot analysis of 25 μg of whole-cell extract. Second panel, Tyrosine phosphorylation of Fus3-HA immunoprecipitated from 200 μg of whole-cell extract. Third panel, Cell morphology. Fourth panel, Fus3-HA kinase assay of associated substrates immunoprecipitated from 200 μg of whole-cell extract. Samples were separated on a 10% (38:2) acrylamide:bis-acrylamide SDS gel. Cell percentages are averages of three fields of 100 cells each.

Figure 2

Figure 2

Immunoprecipitation of Fus3-HA from 35S-labeled cells. Strains EY960 and EY1124 (EY940 with FUS3 [pYEE114] or FUS3-HA [pYEE121]) was labeled with 35S (see MATERIALS AND METHODS), and cells were harvested either without inducing with α factor, or after 20 and 60 min of α factor induction. Whole-cell extracts were prepared as described (see MATERIALS AND METHODS) and 200 μg of total protein was immunoprecipitated with 12CA5 for analysis. (A) Effect of salt in the immunoprecipitation. Samples were separated on a 10% (38:3 acrylamide:bis-acrylamide) SDS gel. Shown is an 18-h exposure of the autoradiogram. (B) Effect of detergents in the immunoprecipitation. Samples were separated on a 7.5% (30:0.8 acrylamide:bis-acrylamide) SDS gel. Shown is a 90-h exposure of the autoradiogram. For both panels A and B, extracts were prepared as described in MATERIALS AND METHODS and were then immunoprecipitated under the conditions described in the figure (note that in panel B, Set 1 also contained 1% bovine serum albumin and Set 2 also contained 1% ovalbumin). After immunoprecipitation, samples were washed with modified H-buffer (Elion et al., 1993). Fus3-HA is indicated by the arrow. ±HA indicates whether the strain contains Fus3-HA (+HA) or Fus3 (−HA). The dots indicate the positions of the 60- and 70-kDa proteins in panel B. DOC is deoxycholate. Extracts in panels A and B are from separate experiments.

Figure 3

Figure 3

Immunoprecipitation of Fus3-HA from 32P04-labeled cells. Strain EY940 containing FUS3 or FUS3-HA on a CEN plasmid (pYEE114 or pYEE121) was labeled with 32P-orthophosphate (see MATERIALS AND METHODS), and cells were harvested either in the absence of α factor induction or after 20 and 60 min of induction. Whole-cell extracts were prepared as described (see MATERIALS AND METHODS), and 200 μg of total protein were immunoprecipitated with 12CA5 antibody. Samples were separated on 7.5% (30:0.8 acrylamide:bis-acrylamide) SDS gels. (A) Immunoprecipitation of Fus3-HA in RIPA or modified H buffer, with or without final incubation with 4 μg RNAse for 5 min on ice. Shown is a 4.5-d exposure of the autoradiogram. ±HA indicates whether the strain contains Fus3-HA (+HA) or Fus3 (−HA). (B) Samples were immunopreciptated in modified H buffer and then treated with RNAse and DNAse as indicated. Shown is a 6.5-d exposure of the autoradiogram. Arrows indicate the position of Fus3-HA.

Figure 4

Figure 4

Localization of Fus3-HA by indirect immunofluorescence. (A) FUS3-HA, vegetative growth; (B) FUS3-HA, 60-min α factor induction; (C) FUS3, 90-min α factor induction; (EY940 containing either FUS3 or FUS3-HA on a CEN plasmid [pYEE121]) grown either in the absence or presence of α factor, was prepared as described in MATERIALS AND METHODS and stained with 12CA5 antibody and affinity-purified donkey-anti-mouse IgG antibody conjugated to rhodamine-like Cy3. Micrographs shown are Cy3 fluorescence, DAPI fluorescence, and Nomarski differential interference contrast, as labeled. Cells were photographed with Fuji Super HGII-100 film using comparable exposure times.

Figure 5

Figure 5

Comparison of short- and long-term exposure to α factor on Fus3-HA localization. (A) FUS3-HA, vegetative growth; (B) FUS3-HA, 10-min α factor induction; (C) FUS3-HA; 3-h α factor induction. Cells were prepared as in Figure 4 and photographed with comparable exposure times. Micrographs shown are Cy3 fluorescence and DAPI fluorescence.

Figure 6

Figure 6

High-speed fractionation of Fus3-HA. Strain EY960 was either uninduced (lanes 1 and 2) or induced with α factor for 1 h (lanes 3 and 4) and whole-cell extracts prepared as described in MATERIALS AND METHODS, including the clarification step of 10 min at 13,000 rpm. Total protein (1 mg) in 500 μl of modified H buffer was centrifuged at 100,000 × g (61,000 rpm in a microcentrifuge) for 30 min at 4°C. The 500-μl supernatant (s) was removed, diluted with 500 μl of 2× SDS sample buffer, and boiled. The pellet (p) was resuspended by boiling in 1 ml of 1× SDS sample buffer. Equal amounts (30 μl) of each sample were separated on a SDS polyacrylamide gel and immunoblotted with 12CA5 and tubulin (4A1) antibodies. Alternate lanes were used to ensure that signals are not from spillover from an adjacent lane.

Figure 7

Figure 7

Fus3 abundance and kinase activity across a 10–30% glycerol density gradient. (A) Fus3-HA kinase activity across the gradient. Fus3-HA from each fraction was immunoprecipitated with 12CA5 antibody and assayed for Fus3 activity using exogenous casein as described (see MATERIALS AND METHODS). Shown is an 8-h exposure of the autoradiogram. (B) Distribution of Fus3-HA protein across a 10–30% glycerol density gradient, performed as in Figure 8. A portion of each fraction (50 μl) was used for immunoblot analysis to detect Fus3-HA with 12CA5 antibody. (C) Graphic representation of Fus3-HA kinase activity, abundance, and specific activity across the glycerol gradient (see MATERIALS AND METHODS). Open circle, Fus3-HA kinase activity; triangles, Fus3-HA protein; solid circles, Fus3-HA-specific activity. Strain EY940 containing FUS3-HA on a CEN plasmid (pYEE121) was used for extract preparation. Cells were grown and induced with 50 nM α factor for 1 h. The asterisk indicates a ∼55 kDa protein that cross-reacts with 12CA5.

Figure 8

Figure 8

Fus3R42 abundance and kinase activity across a 10–30% glycerol density gradient. (A) Fus3R42-HA kinase activity across the gradient assayed as described in Figure 6. (B) Distribution of Fus3R42-HA protein across the 10–30% glycerol density gradient, performed as in Figure 8. A portion of each fraction (50 μl) was used for immunoblot analysis to detect Fus3R42-HA with 12CA5 antibody. Strain EY940 containing FUS3R42-HA on a CEN plasmid was used for extract preparation. Cells were grown as in Figure 6. A similar exposure time is shown as in Figure 7B. (C) Longer exposure of the immunoblot in panel B showing Fus3R42-HA at the bottom of the gradient. The asterisk indicates a ∼55 kDa protein that cross-reacts with 12CA5.

Figure 9

Figure 9

Distribution of Fus3, Ste11M, and Ste7M across a 10–30% glycerol density gradient in the absence of Ste5. Whole-cell extracts were prepared from a ste5Δ ste11Δ strain (EY1881) harboring either pKC11 (pGAL1-STE11M) and pKCS5 (pGAL1-STE7M) or pKC11R444 (pGAL1-STE11R444M) and pKCS5R220 (pGAL1-STE7R220M) and separated across a 10–30% glycerol gradient as described (see MATERIALS AND METHODS). (A and C) Immunoblots of the distribution of Fus3, Ste11M and Ste7M, Ste11R444M, and Ste7R220M across the gradients. A portion of each fraction (50 μl) was used for immunoblot analysis to detect Ste11M and Ste7M with the 9E10 antibody, and 120 μl of each fraction was used to detect Fus3 with a rabbit polyclonal antibody to Fus3 (see MATERIALS AND METHODS; Choi et al., 1994). (B and D) Plots of the relative amount of each protein. Protein levels were quantitated by densitometric scans of exposures of the immunoblots as in Figure 7C. The highest value for each protein was standardized as 100%. Arrows above the figure represent the positions of native-state-size markers separated in a separate glycerol gradient (albumin, 68 kDa; aldolase, 158 kDa; catalase, 232 kDa; throglobulin, 669 kDa). The asterisks indicate proteins that cross-react with the anti-Fus3 antiserum.

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