The RA Domain of Ste50 Adaptor Protein Is Required for Delivery of Ste11 to the Plasma Membrane in the Filamentous Growth Signaling Pathway of the Yeast Saccharomyces cerevisiae

Abstract

In Saccharomyces cerevisiae, pheromone response requires Ste5 scaffold protein, which ensures efficient G-protein-dependent recruitment of mitogen-activated protein kinase (MAPK) cascade components Ste11 (MAPK kinase kinase), Ste7 (MAPK kinase), and Fus3 (MAPK) to the plasma membrane for activation by Ste20 protein kinase. Ste20, which phosphorylates Ste11 to initiate signaling, is activated by binding to Cdc42 GTPase (membrane anchored via its C-terminal geranylgeranylation). Less clear is how activated and membrane-localized Ste20 contacts Ste11 to trigger invasive growth signaling, which also requires Ste7 and the MAPK Kss1, but not Ste5. Ste50 protein associates constitutively via an N-terminal sterile-alpha motif domain with Ste11, and this interaction is required for optimal invasive growth and hyperosmotic stress (highosmolarity glycerol [HOG]) signaling but has a lesser role in pheromone response. We show that a conserved C-terminal, so-called "Ras association" (RA) domain in Ste50 is also essential for invasive growth and HOG signaling in vivo. In vitro the Ste50 RA domain is not able to associate with Ras2, but it does associate with Cdc42 and binds to a different face than does Ste20. RA domain function can be replaced by the nine C-terminal, plasma membrane-targeting residues (KKSKKCAIL) of Cdc42, and membrane-targeted Ste50 also suppresses the signaling deficiency of cdc42 alleles specifically defective in invasive growth. Thus, Ste50 serves as an adaptor to tether Ste11 to the plasma membrane and can do so via association with Cdc42, thereby permitting the encounter of Ste11 with activated Ste20.

Binding the Atypical RA Domain of Ste50p to the Unfolded Opy2p Cytoplasmic Tail Is Essential for the High-Osmolarity Glycerol Pathway

“…Further studies will be required to identify the partner involved in the pheromone response pathway and its mode of interaction with the Ste50p-RA domain. Our findings demonstrate that the Ste50p-RA domain interacts not only with Rho GTPases (Cdc42p) as previously shown (Truckses et al, 2006) but also with two regions within an intrinsically disordered C-terminal cytoplasmic tail of Opy2p. Thus it appears that RA domains have a binding capacity not limited to the Ras family members as implied by their name.…”

“…Ste50p has also been shown to be involved in the activation of the pseudohyphal growth through the interaction of the Ste50p-RA domain with the Rho GTPase Cdc42p (Truckses et al, 2006). Several Ste50p-RA domain mutations affecting pseudohyphal growth have been previously identified, namely K254A, I267A/L268A, and R274A/ N276A, which implicated these residues in binding Cdc42p (Truckses et al, 2006).…”

“…The Ste50p-RA domain has been previously shown to interact with the small GTPase Cdc42p, a member of Rho type of the Ras superfamily. This interaction is implicated as a critical part of the activation of the Ste11p/Ste7p/Kss1pMAP kinase cascade controlling filamentous growth (Truckses et al, 2006) The interaction of the Ste50p-RA domain with Opy2p activating the HOG pathway is likely to be qualitatively different from that seen for Cdc42p since, in contrast to the GTPase, the associating region of Opy2p is predicted to be largely unstructured.…”

“…This region contains a predicted domain (aa 235-327; Schultz et al, 1998;Kiel and Serrano, 2006;Letunic et al, 2009). This Ste50p-RA domain has been shown by in vitro binding assays to interact with specific Rho-type small GTPases rather than the classically expected Ras family members (Truckses et al, 2006;Annan et al, 2008). The initial construct corresponding to the predicted Ste50p-RA domain showed a well-dispersed 1 H-15 N HSQC spectrum indicative of a folded protein (data not shown), with the exception of the N-terminal region that was unfolded.…”

“…The main function of Ste50p in the activation of each of these MAP kinase pathways is in plasma membrane localization of the Ste11p MAPKKK (Posas et al, 1998b;Wu et al, 1999Wu et al, , 2006Jansen et al, 2001). The Ste50p adaptor achieves this membrane association through the interaction of its RA (Ras association) domain with Opy2p (Wu et al, 2006) or with other membrane-anchored proteins, such as the small GTPase Cdc42p (Tatebayashi et al, 2006;Truckses et al, 2006). In each case it appears this membrane localization positions Ste11p for its activation by the PAK kinase member Ste20p, itself membrane-associated through interaction with Cdc42p (Peter et al, 1996;Leberer et al, 1997;Lamson et al, 2002;Ash et al, 2003).…”