Nuclear export and plasma membrane recruitment of the Ste5 scaffold are coordinated with oligomerization and association with signal transduction components.
The Ste5 scaffold activates an associated mitogen-activated protein kinase cascade by binding through its RING-H2 domain to a Gbetagamma dimer (Ste4/Ste18) at the plasma membrane in a recruitment event that requires prior nuclear shuttling of Ste5. Genetic evidence suggests that Ste5 must oligomerize to function, but its impact on Ste5 function and localization is unknown. Herein, we show that oligomerization affects Ste5 activity and localization. The majority of Ste5 is monomeric, suggesting that oligomerization is tightly regulated. Increasing the pool of Ste5 oligomers increases association with Ste11. Remarkably, Ste5 oligomers are also more efficiently exported from the nucleus, retained in the cytoplasm by Ste11 and better recruited to the plasma membrane, resulting in constitutive activation of the mating mitogen-activated protein kinase cascade. Coprecipitation tests show that the RING-H2 domain is the key determinant of oligomerization. Mutational analysis suggests that the leucine-rich domain limits the accessibility of the RING-H2 domain and inhibits export and recruitment in addition to promoting Ste11 association and activation. Our results suggest that the major form of Ste5 is an inactive monomer with an inaccessible RING-H2 domain and Ste11 binding site, whereas the active form is an oligomer that is more efficiently exported and recruited and has a more accessible RING-H2 domain and Ste11 binding site.
Wang
Yunmei
Y
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Elion
Elaine A
EA
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism; D028884:Karyopherins; D007930:Leucine / Q000235:genetics / Q000378:metabolism; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D009154:Mutation; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D044387:GTP-Binding Protein beta Subunits; D028884:Karyopherins; C401306:MSN5 protein, S cerevisiae; C080321:STE5 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C067576:Ste4 protein, S cerevisiae; D020930:MAP Kinase Kinase Kinases; C067018:Ste11 protein, S cerevisiae; D007930:Leucine