The pheromone-induced nuclear accumulation of the Fus3 MAPK in yeast depends on its phosphorylation state and on Dig1 and Dig2

Abstract

Background: Like mammalian MAP kinases, the mating-specific Fus3 MAPK of yeast accumulates in the nuclei of stimulated cells. Because Fus3 does not appear to be subjected to active nucleo-cytoplasmic transport, it is not clear how its activation by mating pheromone effects the observed change in its localization. One possibility is that the activation of Fus3 changes its affinity for nuclear and cytoplasmic tethers.

Results: Dig1, Dig2, and Ste12 are nuclear proteins that interact with Fus3. We found that the pheromone-induced nuclear accumulation of a Fus3-GFP reporter is reduced in cells lacking Dig1 or Dig2, whereas Fus3T180AY182A-GFP localization was unaffected by the absence of these proteins. This suggests that Dig1 and Dig2 contribute to the retention of phosphorylated Fus3 in the nucleus. Moreover, overexpression of Ste12 caused the hyper-accumulation of Fus3-GFP (but not Fus3T180AY182A-GFP) in the nuclei of pheromone-treated cells, suggesting that Ste12 also plays a role in the nuclear retention of phosphorylated Fus3, either by directly interacting with it or by transcribing genes whose protein products are Fus3 tethers. We have previously reported that overexpression of the Msg5 phosphatase inhibits the nuclear localization of Fus3. Here we show that this effect depends on the phosphatase activity of Msg5, and provide evidence that both nuclear and cytoplasmic Msg5 can affect the localization of Fus3.

Conclusion: Our data are consistent with a model in which the pheromone-induced phosphorylation of Fus3 increases its affinity for nuclear tethers, which contributes to its nuclear accumulation and is antagonized by Msg5.

Figure 1

Dig1 and Dig2 are required for the pheromone-induced nuclear accumulation of Fus3. Strains of the indicated genotype were transformed with Fus3-GFP and grown to mid-log phase. The cultures were then split and grown with or without the addition of 12 nM pheromone. Images were acquired from the treated and untreated cells three hours later. The RNCF values were determined as described in the Materials and Methods, and their distributions represented in histograms. The number of cells (y axis) is plotted as a function of the RNCF values (x axis). Mean RNCF values are indicated ± s.d. In each panel, the untreated and treated cultures are represented by the left and right histograms, respectively. Arrows indicate the mean RNCF value for each culture. Representative micrographs from which the quantitative data were derived are shown beneath each histogram. (A)Wild type cells; (B) dig1Δ cells; (C)dig2Δ cells.

Figure 2

Ste12 overexpression causes hyper-accumulation of Fus3 in the nuclei of pheromone-treated cells. Wild type cells were transformed with either the Fus3-GFP reporter or the Fus3^T180AY182A-GFP reporter, and either the GAL1-STE12 plasmid or an empty vector. Strains were grown to mid-log phase in sucrose medium and galactose was added to a concentration of 2% two hours before pheromone treatment (GAL1 promoter on). The cultures were then split and grown with or without the addition of 12 nM pheromone. Images were acquired from the untreated and treated cultures in 2 hour intervals. The mean RNCF ± s.d. values were determined as described in the Materials and Methods, and are represented in histograms as follows: wild type cells expressing Fus3-GFP–untreated (white bars) and treated (dark gray bars); wild type cells expressing Fus3-GFP and overexpressing Ste12–untreated (light gray bars) and treated (black bars); wild type cells expressing Fus3^T180AY182A-GFP and overexpressing Ste12–untreated (stippled bars) and treated (hatched bars). Asterisks indicate statistically significant differences between strains with ρ = 0.003. See additional file 2 for images representative of each time point sampled for Figure 2.

Figure 3

Localization of the wild type, NLS-tagged, and NES-tagged forms of Msg5. A set of 15Dau transformants carrying various centromeric reporter plasmids–PTP2-GFP, PTP3-GFP, MSG5-GFP, NLS-MSG5^M45A-GFP, and NES-MSG5^M45A-GFP–were grown to mid-log phase, stained with 20 μg/ml DAPI, and examined using fluorescent and differential interference contrast (DIC) microscopy. In each panel, representative images are displayed as follows: FITC (left), DAPI (middle), and DIC (right). RNCF values for Msg5-GFP, NLS-MSG5^M45A-GFP, and NES-MSG5^M45A-GFP were quantified in the same manner as for Fus3-GFP, and are represented as histograms. RNCF values are indicated as the mean ± s.d. (A)PTP2-GFP cells (top); PTP3-GFP cells(bottom); (B)MSG5-GFP cells; (C)NLS-MSG5^M45A-GFP cells; (D)NES-MSG5^M45A-GFP cells.

Figure 4

Deficient Msg5 phosphatase activity in either the nucleus or cytoplasm confers increased sensitivity to pheromone. Halo tests were performed as described in [83]. (A)Wildtype cells; (B) GAL-MSG5 cells; (C) NES-MSG5^M45A/NLS-MSG5^M45A cells; (D)NES-MSG5^M45A/NLS-MSG5^M45AC319A; (E) NES-MSG5^M45AC319A/NLS-MSG5^M45A cells.