Membrane recruitment of the kinase cascade scaffold protein Ste5 by the Gbetagamma complex underlies activation of the yeast pheromone response pathway - PubMed (original) (raw)

Membrane recruitment of the kinase cascade scaffold protein Ste5 by the Gbetagamma complex underlies activation of the yeast pheromone response pathway

P M Pryciak et al. Genes Dev. 1998.

Abstract

In the Saccharomyces cerevisiae pheromone response pathway, the Gbetagamma complex activates downstream responses by an unknown mechanism involving a MAP kinase cascade, the PAK-like kinase Ste20, and a Rho family GTPase, Cdc42. Here we show that Gbetagamma must remain membrane-associated after release from Galpha to activate the downstream pathway. We also show that pheromone stimulates translocation of the kinase cascade scaffold protein Ste5 to the cell surface. This recruitment requires Gbetagamma function and the Gbetagamma-binding domain of Ste5, but not the kinases downstream of Gbetagamma, suggesting that it is mediated by Gbetagamma itself. Furthermore, this event has functional significance, as artificial targeting of Ste5 to the plasma membrane, but not intracellular membranes, activates the pathway in the absence of pheromone or Gbetagamma. Remarkably, although independent of Gbetagamma, activation by membrane-targeted Ste5 requires Ste20, Cdc42, and Cdc24, indicating that their participation in this pathway does not require them to be activated by Gbetagamma. Thus, membrane recruitment of Ste5 defines a molecular activity for Gbetagamma. Moreover, our results suggest that this event promotes kinase cascade activation by delivering the Ste5-associated kinases to the cell surface kinase Ste20, whose function may depend on Cdc42 and Cdc24.

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Figures

Figure 1

Figure 1

Rescue of Ste18ΔC signaling defect by fusion with heterologous membrane targeting domains. (A) Transcriptional induction. Fus1–LacZ activation is shown, after galactose induction of Ste18 derivatives for 4 or 18 hr (left) or for 4 hr ± α-factor (αF; right). Bars, average of four measurements of two transformants (left) or mean ±

s.d.

for three transformants (right). Strains: PPY 885, PPY 865. Plasmids, from top to bottom: pPP449, pGS18–WT, pGS18ΔC, pGS18ΔC–NTM, pGS18ΔC–Nmyr, pGS18ΔC–CTM, pGS18ΔC–Cpr. (B) Mating of strains in A, right. Partner: PPY 262. Bars, mean ±

s.d.

for three transformants. (C) Growth arrest. Lawns of transformants (as in A, right) were exposed for 4 days at 30°C on −TRP + RAFF + GAL to filter disks containing 25 μl of 500 μ

m

α-factor. (D) Rescue by fusion of prenylation/palmitoylation sequence to Ste4. Patch mating tests of Ste4–MTD fusions for complementation of ste4Δ (PPY 794; left), or ability to rescue mating in a ste18Δ strain (PPY 832) that also expressed a Ste18ΔC allele from pBH21–Q98ter (right). Plasmids, from top to bottom: pPP449, pGS4, pGS4–Nmyr, pGS4–NTM, pGS4–Cpr, pGS4–CTM. (E) Signaling by Ste4–Cpr still requires the Ste18 amino terminus. Patch mating of a ste18Δ strain (PPY832) expressing the indicated plasmid-borne Ste4 and Ste18 alleles: pGS4 (Ste4), pGS4–Cpr (Ste4–Cpr), pBH21–WT (WT), pBH21–Q98ter (Ste18ΔC), pRS425 (vector).

Figure 2

Figure 2

Activation of the pheromone response pathway by membrane targeting of Ste5. (A) Schematic description of Ste5 and Ste5ΔN fusions. (Top) Regions of Ste5 that bind Gβγ (Whiteway et al. 1995) or the kinases Fus3, Kss1, Ste11, and Ste7 (Choi et al. 1994), or facilitate oligomerization (Yablonski et al. 1996; Inouye et al. 1997). (Right) Carboxy-terminal MTDs. Zigzag lines: palmitoylated and farnesylated Cys Cys (CC) residues in the Cpr sequence. (B) Effects of Ste5 and Ste5ΔN MTD fusions on the pheromone response pathway. Fus1–LacZ induction ±α-factor (αF; left), and mating (right; partner: PPY 198). Bars, mean ± range for two transformants. PPY 858 (ste5Δ) harbored plasmids, from top to bottom: pPP449, pGS5, pGS5–CTM, pGS5–Cpr, pGS5–Cpr-SS, pGS5ΔN, pGS5ΔN–CTM, pGS5ΔN–Cpr, pGS5ΔN–Cpr–SS. (C) Growth arrest activated by carboxy-terminal MTD fusions to Ste5ΔN. Transformants (as in B) were streaked on −TRP + RAFF + GAL plates and incubated for 4 days at 30°C. Analogous fusions to full-length Ste5 gave similar results (not shown). (D) Membrane-targeted Ste5 derivatives rescue mating in cells lacking pheromone receptors. Strains: PPY 858, PPY 409. Plasmids, as in B. (E) Membrane-targeted Ste5 derivatives activate the pathway even when expression levels are reduced by glucose. Strains: PPY 640, PPY 858. Plasmids: pRS413, pH–GS5, pH–GS5–CTM, pH–GS5ΔN–CTM. Bars, mean ±

s.d.

for four transformants.

Figure 3

Figure 3

Localization of GFP–Ste5 fusions. (A) Cell surface recruitment of GFP–Ste5 in response to pheromone. PPY 858 harboring pGFP–GS5 was examined in the absence or presence of α-factor (αF; 2-hr treatment). Representative fields are shown of both DIC and fluorescence (GFP) images. (B) GFP–Ste5 localization at different times after addition of pheromone. Cells were as in A. (C) GFP–Ste5ΔN cannot translocate to the cell surface. PPY 858 harboring pGFP–GS5ΔN and either vector (pRS413; left) or a Ste5 plasmid (pH–GS5; right) is shown after treatment with α-factor. (D) Plasma membrane localization of membrane-targeted Ste5. A GFP–Ste5–CTM fusion (pGFP–GS5–CTM) was visualized without pheromone treatment in both a ste5Δ strain (PPY 858, left), where signaling was activated, and a ste11Δ strain (PPY 890, right), where signaling was blocked. GFP–Ste5–Cpr (pGFP–GS5–Cpr) gave similar results (not shown).

Figure 4

Figure 4

Cell surface recruitment of GFP–Ste5 is a function of the free Gβγ complex that does not require kinase cascade activity. (A) Requirement for heterotrimeric G protein but not kinases. Mutant strains expressed GFP–Ste5 (pH–GFP–GS5). For examination in the presence of α-factor (αF), a galactose-inducible Ste4 construct (pL19) was also included (except in the ste4Δ strain, as it would complement the ste4Δ mutation); although not required (not shown), this enhanced pheromone-induced cell surface localization of GFP–Ste5 in ste20Δ, ste11Δ, and ste7Δ strains. Strains, from top to bottom: PPY 889, PPY 858, PPY 860, PPY 890, PPY 891. (B) Activated Gβγ, but not activated Ste5 or Ste11, can cause translocation of GFP–Ste5 to the cell surface. GFP–Ste5 (pH–GFP–GS5) or GFP–Ste20 (pRL116) fusion proteins were visualized in projection-containing cells induced without pheromone by using galactose-inducible constructs, from left to right: pL19 (Gal–Ste4), pGS5–CTM (Ste5–CTM), pRD–STE11–H3 (Ste11ΔN), pH–GS5–CTM (Ste5–CTM). Cells were analyzed 2–8 hr after galactose induction. Representative examples are shown; GFP–Ste5 appeared at projection tips consistently when induced by Gal–Ste4, but not when induced by Ste5–CTM or Ste11ΔN.

Figure 5

Figure 5

Pathway activation by Ste5ΔN fusions requires delivery to the plasma membrane. Localization (left) and Fus1–LacZ induction (right) for carboxy-terminal MTD fusions to GFP–Ste5ΔN. Snc2D, Sso1B, and Cpr MTD fusions show peripheral rim localization indicative of plasma membrane. Intracellular circular localization observed with Sed5 and Sec22 MTD fusions are reminiscent of endoplasmic reticulum, and punctate spots seen with Sso1B, Sed5, and Sec22 are reminiscent of Golgi localization. With the Sec22 MTD fusion, faint plasma membrane localization was also detectable (arrowheads). All images show cells untreated with pheromone. Strain: PPY 858. Plasmids, from top to bottom: pGFP–GS5ΔN, pGFP–GS5ΔN–Snc2D, pGFP–GS5ΔN–Sso1B, pGFP–GS5ΔN–Sed5, pGFP–GS5ΔN–Sec22, pGFP–GS5ΔN–Cpr, pGFP–GS5ΔN–Cpr–SS. Bars, mean ±

s.d.

for four transformants.

Figure 6

Figure 6

Critical role of Ste20, Cdc42, and Cdc24 in pathway activation by membrane-targeted Ste5 derivatives. (A) Analysis of requirement for Ste4 and Ste20. Fus1–LacZ induction in mutant strains expressing galactose-inducible Ste5, Ste11, or Ste12 derivatives. Strains: PPY 858, PPY 886, PPY 860. Plasmids, from top to bottom: pH–GS5, pH–GS5–CTM, pH–GS5ΔN–CTM, pRD–STE11–H3, pNC252. Bars, mean ±

s.d.

for four transformants. (B) Growth arrest. Streaked transformants (as in A) grown for 5 days at 30°C on a −HIS + RAFF + GAL plate are shown. (C) Dependence on Cdc24 and Cdc42. Fus1–LacZ induction in wild-type vs. cdc mutant strains was compared after pathway activation by pheromone-dependent or -independent methods. (Left) ste5Δ CDC24 (PPY 655) or ste5Δ cdc24-1 (data combined for PPY 697 and PPY 698) strains harbored, from top to bottom: pL–GS5, pL–GS5–CTM, pL–GS5ΔN–CTM, pRD–STE11–H3, pNC252. Bars, mean ±

s.d.

for four transformants. (Right): A bar1 cdc42-1 strain (PPY 911) harbored either pRS314–CDC42–WT (CDC42) or pRS314 (cdc42-1) plus, from top to bottom: pRS315, pL–GS5–CTM, pL–GS5ΔN–CTM, pNC252. Results using the Ste11ΔN allele in this strain are absent because they gave highly variable results, for unknown reasons, and therefore were inconclusive. Bars, mean ±

s.d.

for four transformants.

Figure 6

Figure 6

Critical role of Ste20, Cdc42, and Cdc24 in pathway activation by membrane-targeted Ste5 derivatives. (A) Analysis of requirement for Ste4 and Ste20. Fus1–LacZ induction in mutant strains expressing galactose-inducible Ste5, Ste11, or Ste12 derivatives. Strains: PPY 858, PPY 886, PPY 860. Plasmids, from top to bottom: pH–GS5, pH–GS5–CTM, pH–GS5ΔN–CTM, pRD–STE11–H3, pNC252. Bars, mean ±

s.d.

for four transformants. (B) Growth arrest. Streaked transformants (as in A) grown for 5 days at 30°C on a −HIS + RAFF + GAL plate are shown. (C) Dependence on Cdc24 and Cdc42. Fus1–LacZ induction in wild-type vs. cdc mutant strains was compared after pathway activation by pheromone-dependent or -independent methods. (Left) ste5Δ CDC24 (PPY 655) or ste5Δ cdc24-1 (data combined for PPY 697 and PPY 698) strains harbored, from top to bottom: pL–GS5, pL–GS5–CTM, pL–GS5ΔN–CTM, pRD–STE11–H3, pNC252. Bars, mean ±

s.d.

for four transformants. (Right): A bar1 cdc42-1 strain (PPY 911) harbored either pRS314–CDC42–WT (CDC42) or pRS314 (cdc42-1) plus, from top to bottom: pRS315, pL–GS5–CTM, pL–GS5ΔN–CTM, pNC252. Results using the Ste11ΔN allele in this strain are absent because they gave highly variable results, for unknown reasons, and therefore were inconclusive. Bars, mean ±

s.d.

for four transformants.

Figure 7

Figure 7

Model for pheromone response pathway activation by Gβγ. (A) General models. Cdc24, Cdc42, and Ste20 are required for Gβγ to activate the downstream kinase cascade (Ste11, Ste7, and Fus3, shown associated with the scaffold protein Ste5). A priori, Gβγ could either activate these proteins (left) or promote the action of already active proteins on the kinase cascade (right). Our observations favor the scheme on the right. Whether Cdc24/Cdc42 act by way of Ste20 is still controversial (see text). (B) Detailed model for molecular activity of Gβγ. Asterisks indicate active proteins, and Cdc24, Cdc42, and Ste20 are suggested to be active before exposure to pheromone. The model proposes that on exposure of cells to pheromone, liberated Gβγ recruits Ste5 and its associated kinases to the membrane, and thus into proximity of active Cdc24, Cdc42, and Ste20, resulting in activation of the kinase cascade by Ste20. The model also incorporates the recently described interaction of Gβγ with Ste20 (Leeuw et al. 1998), which is shown as contributing to the formation of a Ste5–Gβγ–Ste20 complex. Possible subsequent Ste5 fates are indicated below. For simplicity, we show all three kinases—Ste11, Ste7, and Fus3—accompanying Ste5 to the cell surface, but it is possible that only a subset do so.

Figure 7

Figure 7

Model for pheromone response pathway activation by Gβγ. (A) General models. Cdc24, Cdc42, and Ste20 are required for Gβγ to activate the downstream kinase cascade (Ste11, Ste7, and Fus3, shown associated with the scaffold protein Ste5). A priori, Gβγ could either activate these proteins (left) or promote the action of already active proteins on the kinase cascade (right). Our observations favor the scheme on the right. Whether Cdc24/Cdc42 act by way of Ste20 is still controversial (see text). (B) Detailed model for molecular activity of Gβγ. Asterisks indicate active proteins, and Cdc24, Cdc42, and Ste20 are suggested to be active before exposure to pheromone. The model proposes that on exposure of cells to pheromone, liberated Gβγ recruits Ste5 and its associated kinases to the membrane, and thus into proximity of active Cdc24, Cdc42, and Ste20, resulting in activation of the kinase cascade by Ste20. The model also incorporates the recently described interaction of Gβγ with Ste20 (Leeuw et al. 1998), which is shown as contributing to the formation of a Ste5–Gβγ–Ste20 complex. Possible subsequent Ste5 fates are indicated below. For simplicity, we show all three kinases—Ste11, Ste7, and Fus3—accompanying Ste5 to the cell surface, but it is possible that only a subset do so.

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References

    1. Adams AE, Johnson DI, Longnecker RM, Sloat BF, Pringle JR. CDC42 and CDC43, two additional genes involved in budding and the establishment of cell polarity in the yeast Saccharomyces cerevisiae. J Cell Biol. 1990;111:131–142. - PMC - PubMed
    1. Akada R, Kallal L, Johnson DI, Kurjan J. Genetic relationships between the G protein βγcomplex, Ste5p, Ste20p and Cdc42p: Investigation of effector roles in the yeast pheromone response pathway. Genetics. 1996;143:103–117. - PMC - PubMed
    1. Carraway KL, Carraway CA. Signaling, mitogenesis and the cytoskeleton: Where the action is. Bioessays. 1995;17:171–175. - PubMed
    1. Chant J, Stowers L. GTPase cascades choreographing cellular behavior: Movement, morphogenesis, and more. Cell. 1995;81:1–4. - PubMed
    1. Choi KY, Satterberg B, Lyons DM, Elion EA. Ste5 tethers multiple protein kinases in the MAP kinase cascade required for mating in S. cerevisiae. Cell. 1994;78:499–512. - PubMed

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