Function of the MAPK scaffold protein, Ste5, requires a cryptic PH domain

Abstract

Ste5, the prototypic mitogen-activated protein kinase (MAPK) scaffold protein, associates with plasma membrane-tethered Gbetagamma freed upon pheromone receptor occupancy, thereby initiating downstream signaling. We demonstrate that this interaction and membrane binding of an N-terminal amphipathic alpha-helix (PM motif) are not sufficient for Ste5 action. Rather, Ste5 contains a pleckstrin-homology (PH) domain (residues 388-518) that is essential for its membrane recruitment and function. Altering residues (R407S K411S) equivalent to those that mediate phosphoinositide binding in other PH domains abolishes Ste5 function. The isolated PH domain, but not a R407S K411S derivative, binds phosphoinositides in vitro. Ste5(R407S K411S) is expressed normally, retains Gbetagamma and Ste11 binding, and oligomerizes, yet is not recruited to the membrane in response to pheromone. Artificial membrane tethering of Ste5(R407S K411S) restores signaling. R407S K411S loss-of-function mutations abrogate the constitutive activity of gain-of-function Ste5 alleles, including one (P44L) that increases membrane affinity of the PM motif. Thus, the PH domain is essential for stable membrane recruitment of Ste5, and this association is critical for initiation of downstream signaling because it allows Ste5-bound Ste11 (MAPKKK) to be activated by membrane-bound Ste20 (MAPKKKK).

Figure 1.

Sequence alignment and analysis of the predicted S. cerevisiae Ste5 PH domain. (A) Alignment of the S. cerevisiae Ste5 PH domain (residues 388–518) with corresponding sequences in orthologs from Saccharomyces bayanus, Saccharomyces mikatae, Saccharomyces paradoxus, Ashbya gossypii, and Kluyveromyces lactis. (B) Alignment of the S. cerevisiae Ste5 PH domain with selected mammalian PH domains. Secondary structure elements (green arrows, β-strands; blue cylinder, α-helix) depict those in the crystal structure of the PLCδ1 PH domain (Lemmon and Keleti 2005). Basic residues in the β1–β2 loop and the conserved Trp in the C-terminal helix are highlighted (bold). (C) Secondary structure elements in the Ste5 PH domain predicted by the indicated algorithms: 3D-PSSM, SSPro, YAPSIN, PSIPRED, and SYMPRED. (Green/E) β-strand; (blue/H) α-helix; (C) random coil.

Figure 2.

The PH domain is essential for Ste5 function in vivo. (A) Ste5 primary structure. (White boxes) Regions implicated in binding the indicated proteins; (dotted line) location of the PM motif; (solid line) location of the predicted PH domain. Positions of previously characterized hyperactive alleles (P44L, C226Y, S770N) and basic residues mutated in this study (R407S K411S R416S R420S) are indicated. (B) Strain BYB69 (ste5Δ) was transformed with either an empty CEN vector or the same vector expressing wild-type Ste5, Ste5(R407S K411S), Ste5(K416S R420S), or Ste5(R407S K411S K416S R420S) from either the native STE5 promoter (left) or from the GAL1 promoter (right), and patch-mating assays were carried out as described in Materials and Methods. (C) Extracts of the same cultures as in B were prepared, resolved by SDS-PAGE, and analyzed by immunoblotting with either an α-Ste5 antiserum (gift of K. Benjamin) (left) or affinity-purified α-Ste5 IgG (Hasson et al. 1994) (right).

Figure 3.

The Ste5 PH domain binds PtdIns(4,5)P₂. (A) Overlay on immobilized lipids. The indicated purified GST fusions (1 μM) were incubated with commercial filter strips (three right-most panels) on which the indicated phospholipids and related compounds were spotted in the pattern shown (left). (B) Binding to liposomes. Samples (1 μM) of the indicated purified GST fusions (input) were incubated with synthetic vesicles of the following compositions: PC/PE (77% DOPC, 22% DOPE); PI (52% DOPC, 22% DOPE, 10% DOPS, 5% DOPA, 10% PI); PI(4)P; and PIP2 [same as PI vesicles, except 5% PI and 5% PI(4)P or 5% PI(4,5)P₂, respectively], each marked with 1% Texas Red-PE. (C) Summary of liposome-binding assays. Values, obtained as in B, represent the amount of vesicle-bound protein (expressed as percent of input) averaged over three to four independent trials. Bars indicate standard error. (D) Effect of vesicle content of PtdIns(4,5)P₂. Binding assays were performed with GST–Ste5 PH domain or GST alone, as a control, as in B with liposomes containing the indicated fraction of PI(4,5)P₂ (when PIP2 exceeded 10%, DOPC was decreased correspondingly).

Figure 4.

The PH domain is required for pheromone-induced membrane recruitment of Ste5. Strain BYB84 (ste5Δ) (A) or strain YAS1 (ste5Δ ste11Δ) (B) transformed with CEN plasmids expressing from the GAL1 promoter wild-type Ste5-GFP (pCJ80), Ste5(R407S K411S)-GFP (pLG35), or Ste5(K416S R420S)-GFP (pLG36) were grown, induced with galactose, treated with pheromone for 1 h, and viewed by deconvolution microscopy, as indicated in Materials and Methods. Panels depict a collage of representative images; arrowheads indicate Ste5-GFP accumulated at the cell cortex.

Figure 5.

Forced membrane anchoring rescues the Ste5 PH domain mutant. (A) Membrane targeting elements. CTM (cylinder), C-terminal α-helical transmembrane segment of Snc2; S-palmitoylated and S-farnesylated (zig-zags) C-terminal segment of wild-type (CCAAX) and unmodified mutant (SSAAX) Ras2. (B) Mating proficiency of BYB69 (ste5Δ) cells expressing either empty vector, wild-type Ste5, or the indicated Ste5 mutants, not fused to any membrane targeting domain (no MTD) or fused to the elements shown in A, was analyzed using a patch-mating assay as in Figure 2B.

Figure 7.

The Ste5 PH domain acts separately from, but synergistically with, Gβγ. (A) The Ste5 PH domain mutant retains Gβγ binding. Strain BYB84 (ste5Δ)—expressing from the GAL1 promoter either a vector control (−) or the c-Myc epitope-tagged Ste5 derivatives described in Figure 5A and coexpressing STE4 and STE18 under the control of the divergent GAL1–GAL10—was lysed, subjected to immunoprecipitation, and analyzed by immunoblotting as in Figure 6A using α-Ste5 antisera and α-Ste4 antisera. A trace of Ste4 associates with antibody-coated beads nonspecifically in the absence of Ste5, as we have observed previously (Inouye et al. 1997a). (B) Loss of PH domain function abrogates the constitutive activity of hyperactive Ste5 alleles. Strains BYB69 (ste5Δ) or BYB88 (ste4Δ ste5Δ) were transformed with empty 2μm-DNA vector or the same vector expressing from the GAL1 promoter either wild-type Ste5 or Ste5(R407S K411S), Ste5(P44L) or Ste5(P44L R407S K411S), Ste5(S770N) or Ste5(R407S K411S S770N), or Ste5(C226Y) or Ste5(C226Y R407S K411S), as indicated, and patch-mating assays were done as in Figure 2B. (C) Loss of PH domain function still abrogates the enhanced activity of the PM motif mutant, Ste5(P44L)–GST. Strain BYB88 (ste4Δ ste5Δ) was transformed with a CEN vector expressing from the GAL1 promoter either Ste5(P44L)–GST or Ste5(P44L R407S K411S)–GST, and patch-mating assays were performed.