1. Volume 20, Issue 1, Pages 21-32 (October 2005)
A Membrane Binding Domain in the Ste5 Scaffold Synergizes with Gβγ Binding to Control Localization and Signaling in Pheromone Response 
Matthew J. Winters, Rachel E. Lamson, Hideki Nakanishi, Aaron M. Neiman, Peter M. Pryciak 
Molecular Cell 
Volume 20, Issue 1, Pages (October 2005)
DOI: /j.molcel
Copyright © 2005 Elsevier Inc. Terms and Conditions

2. Figure 1 A Cortical Localization Domain in the Ste5 N Terminus
See Table S2 for plasmids.
(A) Behavior of the Ste5-NT fragment. Ste5 and Ste5ΔN are from Pryciak and Huntress (1998).
(B) Top, cortical localization of GFP-Ste5-NT fusions in ste4Δ ste5Δ cells (PPY886). Neither GFP alone nor GFP-Ste5-NT-ΔNLS are excluded from the nucleus, but they lack the strong nuclear enrichment of GFP-Ste5-NT. Bottom, dominant negativity. Wild-type (wt) cells (PPY1368) harboring plasmids as above were plated on –His/Raff/Gal plates and overlaid with filter disks containing α factor (10 or 2 nmol).
(C) Mutant Ste5-NT fragments were tested for localization to nucleus (nuc) and cortex (cort), for dominant negativity (dom neg), and for binding to Ste4 (Gβ). Mutations were also moved to full-length Ste5 and tested for α factor response. Results are summarized; see Figure S1 and Figure 4 for supporting data. #, localization results with C180A were ambiguous.
(D) Ste5 residues 37–76 are sufficient for plasma membrane localization in PPY886. Top, GFP fusions to Ste5 residues 1–214, 1–125, or two copies of residues 1–125 expressed in tandem ([1–125]x2). Bottom, GST is fused at the N terminus of GFP, yielding GST-GFP-Ste5 fusions of Ste5 residues 1–125, 37–125, or 37–76.
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3. Figure 2 Membrane Binding by the Ste5 PM/NLS Domain
(A) Bacterially expressed GST-Ste5(37–76) was copelleted with liposomes containing 100% PC or 80% PC plus 20% supplemental phospholipid. Top, protein in supernatant (S) and pellet (P) fractions. Bottom, the fraction of input protein recovered in the pellet (percentage bound); the mean ± SD is from two to five trials, depending on the lipid.
(B) Liposome binding is not observed for GST alone.
(C) Binding of GST-Ste5(37–76) to PC liposomes containing increasing molar percentage of PA, PI(4)P, or PIP2 (mean ± SD, n = 3–5).
(D) Localization of GFP-Ste5-NT (pPP1750) in cells bearing temperature-sensitive alleles of STT4, PIK1, or MSS4. Localization was normal at 23°C (data not shown); cells shown were incubated at 37°C for 60 min.
(E) Effect of increased PA synthesis. GST-GFP-Ste5(1–125) was expressed in PPY886 with a vector control (left) or a plasmid expressing diacylglycerol (DAG) kinase (right). Arrowheads show recruitment of the Ste5 fragment to intracellular lamellae.
(F) Disruption of nuclear transport does not block membrane localization of the Ste5 N terminus. Strain PPY1649 harboring plasmid pPP1978 was incubated at 23°C or was shifted to 37°C for 60 min.
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4. Figure 3
Separation of Nuclear-Targeting and Membrane-Targeting Signals in the Ste5 PM/NLS Domain
(A) A potential amphipathic α helix, shown by helical wheel projection of residues 45–65 (underlined). Black circles, charged residues; gray circles, uncharged polar residues; and white squares, nonpolar residues. At left are two basic residues at the C-terminal end, K66 and R67; their inclusion in the helix would disrupt amphipathicity, and so they could either be uninvolved or terminate the α helix with a different secondary structure.
(B) Summary of mutations and their effects on localization of N-terminal Ste5 fragments.
(C) Top, effects of mutations on binding of GST-Ste5(37–76) to PC + 7% PIP2 liposomes. Bottom, quantitation of multiple trials (mean ± SD; n = 5–8).
(D) Signaling role of the Ste5 PM/NLS domain correlates with membrane-targeting activity, not nuclear-targeting activity. (i) Localization of GST-GFP-Ste5(1–125) fragments in ste4Δ ste5Δ strain PPY886. (ii) Dominant negativity of Ste5-NT(1–214) fragments in wt strain PPY1368. (iii) Basal localization of full-length Ste5-GFPx3 expressed from the STE5 promoter in ste5Δ strain PPY858. (iv) Pheromone response of full-length Ste5-myc13 expressed from the STE5 promoter in PPY858. Note that the vector control expresses GFP in column (i), but not (iii).
(E) Anti-myc immunoblot showing Ste5-myc13 levels in PPY858.
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5. Figure 4
A Membrane-Targeted Ste11 Derivative Reveals a Functional Role for the Ste5 PM/NLS Domain Independent of Gβγ
(A) Membrane-targeted Ste11 causes constitutive mating pathway signaling that requires Ste5, but not Ste4 (Gβ). The indicated strains harbored PGAL1-driven forms of Ste11 fused to the membrane-targeting prenylation/palmitoylation domain from Ras2 (Cpr) or a control sequence (Cpr-SS) (Pryciak and Huntress, 1998). Growth arrest was assessed by spotting onto −His/Glu (GLU) or −His/Raff/Gal (GAL) plates. Transcriptional activation was measured by FUS1-lacZ assay (mean ± SD; n = 6).
(B) Ste5 mutants were compared for their ability to mediate signaling initiated by α factor, Ste11-Cpr, or Ste11-4 in strain PPY858 (left) or PPY886 (middle, right). Bars, FUS1-lacZ activation (mean ± SD; n = 6).
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6. Figure 5
Functional Replacement of the Ste5 PM/NLS Domain by Heterologous Membrane Binding Domains
(A) Schematic description of chimeric proteins.
(B) Anti-GFP immunoblot showing levels of Ste5-GFPx3 chimeras.
(C) Restoration of pheromone response. FUS1-lacZ levels were measured (mean ± SD; n = 4) with and without α factor (αF).
(D) Subcellular localization of chimeric Ste5-GFPx3 fusions before and after α factor treatment.
In all panels, strain PPY858 (ste5Δ) harbored plasmids described in Table S2.
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7. Figure 6 Increased Membrane Binding by Hyperactive Ste5 Mutants
(A) Sites of mutations.
(B) Constitutive signaling by Ste5 mutants, expressed from the GAL1 promoter with or without a C-terminal GST tag, in PPY886 (ste4Δ ste5Δ). Bars, mean ± SD (n = 4). See Figure S4 for additional data.
(C) Liposome binding by wt, P44L, and T52M forms of GST-Ste5(37–76). Top, representative result using 5% PIP2. Bottom, results of multiple trials using 5% or 7% PIP2 were normalized to wt binding from each trial and combined (mean ± SD, n = 5–8).
(D) The P44L and T52M mutations enhance membrane localization of GFP-Ste5(1–125), expressed with or without an N-terminal GST tag in PPY886.
(E) Relationship between increased membrane binding and reduced nuclear localization. (i and ii) T52M increases membrane localization of Ste5(37–76), which shows minimal nuclear enrichment; strain, PPY886. (iii and iv) T52M increases membrane localization of Ste5(1–125) even when nuclear localization is disrupted by incubation of gsp1-1ts cells at 37°C; strain, PPY1649. (v and vi) T52M does not cause nuclear depletion when membrane binding is disrupted by the NLSb mutation; strain, PPY886.
(F) The P44L and T52M mutations cause constitutive membrane localization of full-length Ste5. GFP-Ste5 or GFP-Ste5-GST derivatives were expressed in PPY886 (ste4Δ ste5Δ) or PPY1215 (ste4Δ ste7Δ).
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8. Figure 7 Creation of New Hyperactive Ste5 Mutants
(A) The P44L and T52M mutations add hydrophobic side chains to the predicted nonpolar face of the amphipathic α helix. Pro44 was not shown on the helical wheel in Figure 3A because of its helix-breaking nature, but P44L is predicted to lie on the nonpolar face.
(B) Alignment of PM/NLS sequences in Ste5 orthologs from S. cerevisiae, S. bayanus, S. castelli, and S. kluyveri. Thr52 and Gln59 (arrowheads) are polar residues that are predicted to lie on the nonpolar face of the helix (see Figure 3A) and thus impede membrane binding.
(C) New mutations created in the PM/NLS domain.
(D) Constitutive signaling by the new Ste5 mutants in PPY886 (ste4Δ ste5Δ). Also tested in the same vectors were the ΔNLS, NLSo, NLSb, and NLSm mutants. Bars, mean ± SD (n = 4). Numerical values are shown where signaling was significantly greater than wt Ste5 but was not evident in the bar graphs.
(E) Localization of some GFP-Ste5 mutants (without GST) in PPY886 or in ste4Δ ste7Δ strain PPY1215 (bottom right).
(F) Increased Ste5-membrane affinity compensates for decreased Ste5-Gβγ affinity. Ste5 variants were expressed from the STE5 promoter in PPY657 (ste4Δ ste5Δ) along with wt Ste4 (Gβ) or Ste4 mutants (K55E and D62G) with moderate Ste5 binding defects (Figure S5C). Bars, mean ± SD (n = 4).
(G) Overexpression partially rescues Ste5 mutants lacking the PM/NLS domain. Ste5 derivatives expressed from a CEN ARS plasmid (low copy) or a 2 μm plasmid (high copy) were tested for mating and FUS1-lacZ induction (mean ± SD; n = 3) by α factor (in PPY858).
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9. Figure 8
Model for the Role of Membrane Binding in Ste5 Localization and Signaling
For clarity, some pathway components are omitted from panels (B)–(D) in order to emphasize factors governing Ste5 localization.
(A) Original model for Ste5 membrane recruitment triggered by Gβγ, and its role in promoting the Ste20→Ste11 step.
(B) Revised model, in which membrane recruitment of Ste5 requires cooperativity between a weak Ste5-Gβγ interaction and a weak Ste5-membrane interaction. The cylinder denotes a putative amphipathic α helix formed by the PM/NLS domain.
(C) The Ste5-membrane interaction can also synergize with the Ste5-Ste11 interaction in cells expressing membrane-tethered Ste11 (Ste11-Cpr).
(D) Hyperactive Ste5 mutants have PM/NLS domains with increased membrane affinity, causing membrane localization and signaling in the absence of Gβγ.
Molecular Cell  , 21-32DOI: ( /j.molcel )
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