Gβ phosphorylation is critical for efficient chemotropism in yeast

Abstract

SummaryMating yeast cells interpret complex pheromone gradients and polarize their growth in the direction of the closest partner. Chemotropic growth depends on both the pheromone receptor and its associated G-protein. Upon activation by the receptor, Ga dissociates from Gbc and Gb is subsequently phosphorylated. Free Gbc signals to the nucleus via a MAPK cascade and recruits Far1-Cdc24 to the incipient growth site. It is not clear how the cell establishes and stabilizes the axis of polarity, but this process is thought to require local signal amplification via the Gbc-Far1-Cdc24 chemotropic complex, as well as communication between this complex and the activated receptor. Here we show that a mutant form of Gb that cannot be phosphorylated confers defects in directional sensing and chemotropic growth. Our data suggest that phosphorylation of Gb plays a role in localized signal amplification and in the dynamic communication between the receptor and the chemotropic complex, which underlie growth site selection and maintenance.

Gβ promotes pheromone receptor polarization and yeast chemotropism by inhibiting receptor phosphorylation

“…7A). Previously published data suggest that Gα recruits the activated MAPK Fus3 to phosphorylate cortical Gβ (32, 33) and demonstrate roles for the Gα-Fus3 interaction in directional sensing and chemotropic shmooing (21, 33, 34). Given the data reported here, phosphorylation of Gβ by a MAPK would be expected to enhance its interaction with Yck1/2.…”

“…Because Gβ adopts a polarized localization before pheromone-induced morphogenesis occurs (21), its direct interaction with Yck1 raises the possibility that Gβγ promotes receptor polarization by locally inhibiting receptor phosphorylation. Consistent with this, a region of the plasma membrane enriched in hypo-phosphorylated receptor arises in response to isotropic pheromone treatment (6, 22).…”

“…In contrast, cells defective in gradient sensing or chemotropism, or wild-type cells confused by exogenous pheromone (fig. S3), did not orient directly toward their partners, and ultimately formed angled zygotes (21). …”

“…Strain AIY109 was generated by integrating ste4 T320A S335A into strain YDB111 (22) in situ with Msc1-cut YIplac128/ste4 T320A S335A NΔ112 (pAIB130). Strain AIY197 was generated by integrating Hpa1-cut LHP1921 (53) into RDY114 (21). Strain AIY301 was generated by integrating Apa1-digested YIplac211-GIC2-PBD-RFP (54) into DMY169 (6).…”

“…The Sal I– and Hind III–digested PCR product was inserted into YCplac22/ GAL1 (55). YIplac128/ste4 T320A S335A NΔ112 was constructed by PCR-amplifying ste4 T320A S335A , minus the first 112 bases of the coding region, from RDY120 (21) with the oligomers 5'-CGCGAATTCTGCGCTTCCACAGAACTAATG-3' and 5'-CGCGGATCCAAATAGAGGCCGCCAGACAAG-3'. The PCR fragment digested with Eco RI and Bam HI was subcloned into YIplac128 (55) to generate AIB130.…”