Figure 4. Phosphorylation of serine 185 interferes with membrane recruitment of Ste5 and prevents Gβγ binding. (A) The localization of 3xGFP-tagged WT Ste5 (upper panel), Ste5S185A (middle panel), or Ste5S185D (lower panel) expressed in WT cells exposed to α-factor was analyzed by light microscopy. The percentage of cells with accumulated GFP-signal at shmoo tips was quantified by counting ≥100 cells for each strain. (B) Recruitment of 3xGFP-tagged WT Ste5, Ste5S185A or Ste5S185D was quantified in single cells at the indicated times upon addition of α-factor (t = 0). The membrane-to-cytoplasmic ratio was calculated using YeastQuant (Pelet et al., 2012) using TMD-mCherry to segment the plasma membrane. The solid line represents the median of the single-cell traces and the shaded area the 25th–75th percentiles. (C) FIG1-qV reporter expression was measured by FACS in ste5Δ cells expressing from the estradiol-inducible GAL promoter WT Ste5 (diamond), nonphosphorylatable Ste5S185A (square), or phospho-mimicking Ste5S185D (triangle) fused to a TMD and plotted in arbitrary units for different estradiol concentrations. For control, WT Ste5 (x) and Ste5S185D (*) were also expressed without TMD fusion. (D) GST-Ste4–Ste18 purified from yeast was immobilized on glutathione-Sepharose beads and incubated in vitro with either 6His-tagged Ste5 WT (Ste5149–238) or S185D (Ste5149–238 S185D) RING domains expressed in E. coli. Bound and flow-through fractions were collected and analyzed by Western blotting with the indicated antibodies. +, protein added; −, protein omitted. An aliquot of the input fraction controls for the presence of the specified proteins. GST-Ste4–Ste18 preferentially binds to the nonphosphorylated Ste5-RING domain. (E) Overlay of [1H, 15N] correlation spectra of WT (left panel), S185A (middle panel), and S185D (right panel) Ste5 RING-H2 domain in free form (black contours) and in complex with Gβγ subunits (red). Ste5 adopts a better-defined fold upon binding Gβγ binding with larger peak dispersion. In contrast to WT and the S185A mutant, the S185D RING-H2 domain fails to interact with Gβγ heterodimers.
Figure 4.

Phosphorylation of serine 185 interferes with membrane recruitment of Ste5 and prevents Gβγ binding. (A) The localization of 3xGFP-tagged WT Ste5 (upper panel), Ste5S185A (middle panel), or Ste5S185D (lower panel) expressed in WT cells exposed to α-factor was analyzed by light microscopy. The percentage of cells with accumulated GFP-signal at shmoo tips was quantified by counting ≥100 cells for each strain. (B) Recruitment of 3xGFP-tagged WT Ste5, Ste5S185A or Ste5S185D was quantified in single cells at the indicated times upon addition of α-factor (t = 0). The membrane-to-cytoplasmic ratio was calculated using YeastQuant (Pelet et al., 2012) using TMD-mCherry to segment the plasma membrane. The solid line represents the median of the single-cell traces and the shaded area the 25th–75th percentiles. (C) FIG1-qV reporter expression was measured by FACS in ste5Δ cells expressing from the estradiol-inducible GAL promoter WT Ste5 (diamond), nonphosphorylatable Ste5S185A (square), or phospho-mimicking Ste5S185D (triangle) fused to a TMD and plotted in arbitrary units for different estradiol concentrations. For control, WT Ste5 (x) and Ste5S185D (*) were also expressed without TMD fusion. (D) GST-Ste4–Ste18 purified from yeast was immobilized on glutathione-Sepharose beads and incubated in vitro with either 6His-tagged Ste5 WT (Ste5149–238) or S185D (Ste5149–238 S185D) RING domains expressed in E. coli. Bound and flow-through fractions were collected and analyzed by Western blotting with the indicated antibodies. +, protein added; −, protein omitted. An aliquot of the input fraction controls for the presence of the specified proteins. GST-Ste4–Ste18 preferentially binds to the nonphosphorylated Ste5-RING domain. (E) Overlay of [1H, 15N] correlation spectra of WT (left panel), S185A (middle panel), and S185D (right panel) Ste5 RING-H2 domain in free form (black contours) and in complex with Gβγ subunits (red). Ste5 adopts a better-defined fold upon binding Gβγ binding with larger peak dispersion. In contrast to WT and the S185A mutant, the S185D RING-H2 domain fails to interact with Gβγ heterodimers.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal