Scaffold-mediated
symmetry breaking by Cdc42p.
Javier Irazoqui,
Daniel Lew
Pharmacology and Cancer Bgy., Duke University Medical Center, PO Box 3813,
Durham, NC 27710, USA (jei@duke.edu)
Yeast polarize
F-actin towards a cortical site before budding. The bud site selection system
including the GTPase Rsr1p, its regulators Bud2p and Bud5p, and cortical
landmarks, is essential for the spatial pattern of budding. Signals from this
system polarize the GTPase Cdc42p to the proper site, leading to polarized
actin and secretion. We hypothesize that Cdc42p polarization may be the result
of symmetry breaking. Recruitment of Cdc24p by GTP-Rsr1p may bias symmetry
breaking to the proper site in wild type cells. Assuming that genes involved in
symmetry breaking are essential in the absence of spatial cues, we tested for
interactions between rsr1Δ and polarity-related mutations. We found that bem1Δ is synthetic lethal
with rsr1Δ,
bud2Δ
and bud5Δ;
thus, Bem1p may be involved in symmetry breaking. We isolated novel BEM1 temperature-sensitive
alleles; at restrictive temperature, rsr1Δ bem1ts cells cannot
polarize Cdc42p, indicating that their inviability is likely due to the
inability to polarize. Site-directed mutagenesis of highly conserved residues
yielded both inactive and hyperactive Bem1p mutants. Immunolocalization of
these mutants defined three classes: a) inactive and delocalized, b) inactive
and localized, and c) hyperactive and localized. Our data suggest that Bem1p
functions in symmetry breaking by Cdc42p as a scaffold in a multiprotein
complex that establishes an axis of polarization for budding.