Pheromone-induced polarization is dependent on the Fus3p MAPK acting through the formin Bni1p - PubMed (original) (raw)

Pheromone-induced polarization is dependent on the Fus3p MAPK acting through the formin Bni1p

Dina Matheos et al. J Cell Biol. 2004 Apr.

Abstract

During mating, budding yeast cells reorient growth toward the highest concentration of pheromone. Bni1p, a formin homologue, is required for this polarized growth by facilitating cortical actin cable assembly. Fus3p, a pheromone-activated MAP kinase, is required for pheromone signaling and cell fusion. We show that Fus3p phosphorylates Bni1p in vitro, and phosphorylation of Bni1p in vivo during the pheromone response is dependent on Fus3p. fus3 mutants exhibited multiple phenotypes similar to bni1 mutants, including defects in actin and cell polarization, as well as Kar9p and cytoplasmic microtubule localization. Disruption of the interaction between Fus3p and the receptor-associated Galpha subunit caused similar mutant phenotypes. After pheromone treatment, Bni1p-GFP and Spa2p failed to localize to the cortex of fus3 mutants, and cell wall growth became completely unpolarized. Bni1p overexpression suppressed the actin assembly, cell polarization, and cell fusion defects. These data suggest a model wherein activated Fus3p is recruited back to the cortex, where it activates Bni1p to promote polarization and cell fusion.

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Figures

Figure 1.

Figure 1.

Fus3p phosphorylates Bni1p both in vitro and in vivo. (A) Wild-type FLAG-Fus3p and analogue-sensitive FLAG-Fus3pQ93G were immunoprecipitated from mitotic and pheromone-induced extracts, and in vitro kinase assays were performed as described in the Materials and methods. Nonradioactive ATP was added to the indicated concentrations as a competitive inhibitor of endogenous kinases. The inhibitor analogue 1-Na PP1 was added to 5 μM in indicated lanes. The Fus3p panel and the top Fus3pQ93G panel were exposed to x-ray film for equivalent times; the bottom Fus3pQ93G panel shows a shorter exposure to allow visualization of individual protein bands. (B) Wild-type FLAG-Fus3p or inhibitor-sensitive FLAG-Fus3pQ93G was immunoprecipitated from pheromone-induced extracts, and kinase assays were performed as described for A. Myelin basic protein (MBP) was added as an exogenous substrate. 1-Na PP1 was added to 10 μM in indicated lanes. (C) In vitro phosphorylation of Bni1p by Fus3p. Top: HA-Bni1p was immunoprecipitated out of yeast and added to FLAG-Fus3pQ93G in the in vitro kinase assay. Bottom: Western blots were performed using anti-HA antibody (12CA5 at 1:2,500 dilution) to identify HA-Bni1p. In both cases, a strain containing no HA tagged proteins (EY699) was used as negative control. (D) In vivo phosphorylation of Bni1p is partially dependent on Fus3p. Top: wild-type (MY8195) and _fus3_Δ (MY8196) overexpressing HA-BNI1 were induced with α-factor pheromone and labeled with [32P]orthophosphate before immunoprecipitation as described in the Materials and methods. Bottom: cells treated under the same conditions were processed for Western blot analysis to determine total amount of protein immunoprecipitated from each strain. EY699 was used as the negative control.

Figure 2.

Figure 2.

fus3 mutants have mislocalized actin and fail to polarize in response to pheromone. (A) Cells were treated for 2.5 h with α-factor, fixed, and stained with rhodamine-phalloidin to examine actin localization by fluorescence microscopy as described in the Materials and methods. (B) WT (EY699), _bni1_Δ (MY8188), _fus3_Δ (EY700), and gpa1 K21E R22E (MY8193) were scored for their ability to form shmoos after 1.5 h of exposure to pheromone (n > 100). (C) Cells were scored for their actin localization phenotype by fluorescence microscopy (n > 100).

Figure 3.

Figure 3.

Both fus3 mutants and _gpa1_K21E R22E mutants mislocalize Kar9p. Cells containing p_GAL-GFP_-KAR9 were pregrown in raffinose, induced for 2 h by addition of galactose, and then pheromone was added for another 3 h. After brief fixation, WT (MY8189), _bni1_Δ (MY), fus3-Q93G (MY7494), and gpa1 K21E R22E (MY8194) cells were scored for GFP-Kar9p localization (n > 100). To inactivate Fus3pQ93G in vivo, 10 μM 1-Na PP1 was added after 60 min of pheromone induction. An equivalent amount of DMSO was added to a separate culture as a control. Cells were scored as having (from left to right): a single cortical dot, a line of localization, multiple dots near the shmoo tip, and dispersed dots elsewhere in the cell body, possibly on misoriented microtubules.

Figure 4.

Figure 4.

Both fus3 and _gpa1_K21E R22E mutants have misaligned microtubules. WT (EY699), _bni1_Δ (MY8188), fus3_Δ (EY700), gpa1 K21E R22E (MY8193), and fus3 p_GAL-BNI1 (MY8196) cells were induced with pheromone for 3 h and processed for immunofluorescence. Microtubules were detected with YOL1/34 as the primary antibody and the nucleus was stained with DAPI (n > 100). Cells were scored as having (from left to right): a single bundle of cytoplasmic microtubules going to the shmoo tip, a single bundle of cytoplasmic microtubules going to the shmoo tip plus other additional bundles oriented away from the shmoo tip, a spray of microtubules going to the shmoo tip, no cytoplasmic microtubules going to the shmoo tip, and long nuclear microtubules with short or nonexistent cytoplasmic microtubules.

Figure 5.

Figure 5.

Bni1p is not localized properly in shmoos in a fus3 mutant. (A) WT (MY8195), _fus3_Δ (MY8196), and gpa1 K21E R22E (MY) strains containing pGAL-BNI1-GFP were grown in the presence of galactose for 2 h and then split into two cultures either with or without the addition of pheromone for an additional 2 h. Cells were fixed briefly with formaldehyde and examined by fluorescence microscopy. For fus3 mutants, a brightfield image of the cells is also shown to indicate the outline of the cell. (B) Quantification of Bni1p localization in the strains (n > 200). (C) Total Bni1p was monitored by Western blots on extracts from cultures treated in the same way as for the microscopy. 12CA5 anti-HA antibody was used to detect Bni1p.

Figure 6.

Figure 6.

Spa2p does not localize to the shmoo tips in fus3 and bni1 mutants. (A) WT (EY699), _bni1_Δ (MY8188), and _fus3_Δ (EY700) cells transformed with p426S2G (expressing Spa2p-GFP) were induced with pheromone for 2 h, fixed with formaldehyde, and processed for immunofluorescence. Rabbit polyclonal anti-GFP antibody (from CLONTECH Laboratories, Inc.) was used at 1:25 dilution in PBS-BSA. (B) Cells were scored for GFP-Spa2p localization at the shmoo tip (n > 100).

Figure 7.

Figure 7.

Overexpression of BNI1 suppresses the actin polarization and shmooing defect of a fus3 mutant. (A) BNI1 expression was induced for 2 h with galactose and then pheromone was added for another 2 h to either WT (EY699), _fus3_Δ (EY700), or _fus3_Δ p_GAL_-BNI1 (MY8196). Cells were fixed and examined by differential interference contrast or brightfield microscopy to determine whether they had formed shmoos. (B) The same strains as in A were stained with rhodamine-phalloidin and examined by fluorescence microscopy to determine the localization of actin (n > 200). (C) Representative cells in which overexpression of Bni1p has restored actin localization. Examples of cells with aberrant multiple shmoo projections are shown in the right-most panels.

Figure 8.

Figure 8.

Overexpression of BNI1 suppresses the cell fusion defect of a fus3 mutant. WT (EY699), _fus3_Δ (EY700), and _fus3_Δ p_GAL_-BNI1 (MY8196) cells were mated to a _MAT_α fus1fus2 (MY1814) double mutant for 6 h as described in the Materials and methods. Cells were then fixed with methanol/acetic acid, stained with DAPI, and scored for the appearance of a septum (by differential interference contrast microscopy) and for the number and location of the nuclei (by fluorescence microscopy). Zygotes were scored as having a cell fusion defect based on the presence of a septum separating two unfused nuclei (n > 100).

Figure 9.

Figure 9.

New cell wall growth is not polarized in a fus3 mutant. WT (EY699), _cln3_Δ (MY5802), _fus3_Δ_cln3_Δ (MY5801), and _bni1_Δ (MY8188) were labeled for old growth (green) and new growth (red) after pheromone treatment using FITC- and TRITC-labeled Con A. Cells were visualized and deconvolved using a DeltaVision microscope.

Figure 10.

Figure 10.

A model of Fus3p-dependent Bni1p activation. Upon pheromone binding to its receptor, a heterotrimeric G protein dissociates. The Gβγ subunit, comprised of Ste4p and Ste18p, respectively, is required for activation of the MAPK module and for recruitment of Far1p, Cdc42p, and Cdc24p, which establish the site for the future shmoo projection. Free Gpa1p recruits phosphorylated active Fus3p back to the site of receptor activation, facilitating the local Fus3p-dependent phosphorylation of Bni1p and leading to the stable localization of Bni1p at the incipient shmoo site. Bni1p then nucleates actin assembly and other functions required for polarization of the cell toward the pheromone gradient.

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