MUTATIONAL ANALYSIS OF STE5 IN THE YEAST SACCHAROMYCES-CEREVISIAE - APPLICATION OF A DIFFERENTIAL INTERACTION TRAP ASSAY FOR EXAMINING PROTEIN-PROTEIN INTERACTIONS
C. Inouye et al., MUTATIONAL ANALYSIS OF STE5 IN THE YEAST SACCHAROMYCES-CEREVISIAE - APPLICATION OF A DIFFERENTIAL INTERACTION TRAP ASSAY FOR EXAMINING PROTEIN-PROTEIN INTERACTIONS, Genetics, 147(2), 1997, pp. 479-492
Ste5 is essential for the yeast mating pheromone response pathway and
is thought to function as a scaffold that organizes the components of
the mitogen-activated protein kinase (MAPK) cascade. A new method was
developed to isolate missense mutations in Ste5 that differentially af
fect the ability of Ste5 to interact with either of two MAPK cascade c
onstituents, the MEKK (Ste11) and the MEK (Ste7). Mutations that affec
t association with Ste7 or with Ste11 delineate discrete regions of St
e5 that are critical for each interaction. Co-immunoprecipitation anal
ysis, examining the binding in vitro of Ste5 to Ste11, Ste7, Ste4 (G p
rotein beta subunit), and Fus3 (MAPK), confirmed that each mutation sp
ecifically affects the interaction of Ste5 with only one protein. When
expressed in a ste5 Delta cell, mutant Ste5 proteins that are defecti
ve in their ability to interact with either Ste11 or Ste7 result in a
markedly reduced mating proficiency. One mutation that clearly weakene
d (but did not eliminate) interaction of Ste5 with Ste7 permitted mati
ng at wild-type efficiency, indicating that an efficacious signal is g
enerated even when Ste5 associates with only a small fraction of (or o
nly transiently with) Ste7. Ste5 mutants defective in association with
Ste11 or Ste7 showed strong interallelic complementation when co-expr
essed, suggesting that the functional form of Ste5 in vivo is an oligo
mer.