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

Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, California 94720, USA

Ste5, the prototypic mitogen-activated protein kinase (MAPK) scaffold protein, associates with plasma membrane-tethered G freed upon pheromone receptor occupancy, thereby initiating downstream signaling. We demonstrate that this interaction and membrane binding of an N-terminal amphipathic -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 G 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).

[Keywords: Pheromone response; plasma membrane; mutants; yeast; Saccharomyces cerevisiae; Ste20]

Received January 26, 2006; revised version accepted May 8, 2006.

E-MAIL jthorner{at}berkeley.edu; FAX (510) 642-6420.

Supplemental material is available at http://www.genesdev.org.

Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1413706

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