Mol Cell Biol, July 1998, p. 4053-4069, Vol. 18, No. 7
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Department of Biology, Yale University, New Haven, Connecticut 06520-8103
Received 4 November 1997/Returned for modification 22 December 1997/Accepted 7 April 1998
The yeast protein Spa2p localizes to growth sites and is important
for polarized morphogenesis during budding, mating, and pseudohyphal
growth. To better understand the role of Spa2p in polarized growth, we
analyzed regions of the protein important for its function and proteins
that interact with Spa2p. Spa2p interacts with Pea2p and Bud6p (Aip3p)
as determined by the two-hybrid system; all of these proteins exhibit
similar localization patterns, and spa2
,
pea2
, and bud6
mutants display similar
phenotypes, suggesting that these three proteins are involved in the
same biological processes. Coimmunoprecipitation experiments
demonstrate that Spa2p and Pea2p are tightly associated with each other
in vivo. Velocity sedimentation experiments suggest that a significant portion of Spa2p, Pea2p, and Bud6p cosediment, raising the possibility that these proteins form a large, 12S multiprotein complex. Bud6p has
been shown previously to interact with actin, suggesting that the 12S
complex functions to regulate the actin cytoskeleton. Deletion analysis
revealed that multiple regions of Spa2p are involved in its
localization to growth sites. One of the regions involved in Spa2p
stability and localization interacts with Pea2p; this region contains a
conserved domain, SHD-II. Although a portion of Spa2p is sufficient for
localization of itself and Pea2p to growth sites, only the full-length
protein is capable of complementing spa2 mutant defects,
suggesting that other regions are required for Spa2p function. By using
the two-hybrid system, Spa2p and Bud6p were also found to interact with
components of two mitogen-activated protein kinase (MAPK) pathways
important for polarized cell growth. Spa2p interacts with Ste11p (MAPK
kinase [MEK] kinase) and Ste7p (MEK) of the mating signaling pathway
as well as with the MEKs Mkk1p and Mkk2p of the Slt2p (Mpk1p) MAPK
pathway; for both Mkk1p and Ste7p, the Spa2p-interacting region was
mapped to the N-terminal putative regulatory domain. Bud6p interacts
with Ste11p. The MEK-interacting region of Spa2p corresponds to the
highly conserved SHD-I domain, which is shown to be important for
mating and MAPK signaling. spa2 mutants exhibit reduced
levels of pheromone signaling and an elevated level of Slt2p kinase
activity. We thus propose that Spa2p, Pea2p, and Bud6p function
together, perhaps as a complex, to promote polarized morphogenesis
through regulation of the actin cytoskeleton and signaling pathways.
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