Molecular Cell, Vol. 20, 21–32, October 7, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.molcel.2005.08.020
A Membrane Binding Domain in the Ste5 Scaffold
Synergizes with G?? Binding to Control
Localization and Signaling in Pheromone Response
Matthew J. Winters,1Rachel E. Lamson,1
Hideki Nakanishi,2Aaron M. Neiman,2
and Peter M. Pryciak1,*
1Department of Molecular Genetics and Microbiology
University of Massachusetts Medical School
Worcester, Massachusetts 01605
2Department of Biochemistry and Cell Biology
The State University of New York at Stony Brook
Stony Brook, New York 11794
Activation of mitogen-activated protein (MAP) kinase
cascade signaling by yeast mating pheromones in-
volves recruitment of the Ste5 scaffold protein to the
plasma membrane by the receptor-activated G?? di-
mer. Here, we identify a putative amphipathic ?-heli-
cal domain in Ste5 that binds directly to phospholipid
membranes and is required for membrane recruitment
by G??. Thus, Ste5 signaling requires synergistic Ste5-
G?? and Ste5-membrane interactions, with neither alone
being sufficient. Remarkably, the Ste5 membrane bind-
ing domain is a dual-function motif that also mediates
nuclear import. Separation-of-function mutations show
that signaling requires the membrane-targeting activity
of this domain, not its nuclear-targeting activity, and
heterologous lipid binding domains can substitute for
its function. This domain also contains imperfections
that reduce membrane affinity, and their elimination
results in constitutive signaling, explaining some pre-
vious hyperactive Ste5 mutants. Therefore, weak mem-
brane affinity is advantageous, ensuring a normal level
of signaling quiescence in the absence of stimulus and
imposing a requirement for G?? binding.
Eukaryotic cells respond to a wide variety of external
signals that influence decisions regarding division,
gene expression, and behavior. Cellular responses are
often initiated at the plasma membrane when stim-
ulated receptors create membrane binding sites for cy-
toplasmic proteins, leading to their activation. The reg-
ulated recruitment of proteins to the plasma membrane
can involve interactions with either membrane proteins
or membrane lipids (Pawson and Scott, 1997; Hunter,
2000; Teruel and Meyer, 2000). Subsequent disassem-
bly of these membrane-based signaling complexes can
allow for dissemination of the signal to other parts of
the cell or termination of signaling.
The mating pathway of the yeast Saccharomyces
cerevisiae (Dohlman and Thorner, 2001) provides a model
for the regulated assembly of signaling complexes at
the plasma membrane. Mating of two haploid cells is
triggered by pheromones, termed a factor and α factor,
which activate a signal transduction pathway con-
sisting of two main modules: (1) a seven transmem-
brane domain receptor coupled to a heterotrimeric G
protein (Gαβγ) and (2) a cascade of protein kinases re-
lated to mammalian MAP kinase cascades. Another
protein, Ste5, is critical for communication between
these two modules. Ste5 is termed a “scaffold” protein
for its ability to bind all three kinases in the MAP kinase
cascade, which promotes their mutual interactions and
thereby facilitates signal transmission (Elion, 2001).
Ste5 also serves an adaptor function, in that it binds to
the Gβγ dimer and thereby connects the MAP kinase
cascade to the stimulus-sensing receptor/G protein
module. Analogs of Ste5 act as scaffolds for other MAP
kinase cascades in both yeast and mammalian cells
(Whitmarsh and Davis, 1998; Morrison and Davis, 2003),
and thus, Ste5 is a prototype for understanding the role
of scaffolding in signal transduction.
Mating pheromones trigger the recruitment of Ste5 to
the plasma membrane (Pryciak and Huntress, 1998),
and similar translocations occur for other yeast and
mammalian scaffold proteins (Reiser et al., 2000; Muller
et al., 2001). This event clearly has functional signifi-
cance, because artificially targeting Ste5 to the mem-
brane causes constitutive signaling (Pryciak and Hunt-
ress, 1998). Membrane recruitment of Ste5 is thought
to initiate activation of the Ste5-associated kinase
cascade by allowing the MAP kinase kinase kinase
(MAPKKK) Ste11 to become phosphorylated by its
membrane-localized activator, the PAK family kinase
Ste20 (Pryciak and Huntress, 1998; van Drogen et al.,
2000). The kinases downstream of Ste11, namely Ste7
and Fus3, are also brought to the membrane by Ste5,
which thereby serves as an activation platform for the
entire MAP kinase cascade (van Drogen et al., 2001).
Photobleaching studies suggest that the activated
MAPK Fus3 dissociates from Ste5 and then transits to
the nucleus to regulate cell cycle arrest and transcrip-
tional induction (van Drogen et al., 2001).
It is clear that membrane recruitment of Ste5 is pro-
moted by interaction with the receptor-activated Gβγ
dimer (Pryciak and Huntress, 1998; Mahanty et al.,
1999). Nevertheless, several aspects of Ste5 regulation
remain poorly understood. For example, Ste5 also has
an affinity for the nucleus, and one study suggested
that shuttling through the nucleus allows Ste5 to be-
come competent for membrane recruitment, though by
an undefined mechanism (Mahanty et al., 1999). It has
also been suggested that Gβγ may induce changes in
the conformation or oligomerization state of Ste5 as
part of the signaling activation mechanism (Yablonski
et al., 1996; Inouye et al., 1997; Sette et al., 2000; Wang
and Elion, 2003). These notions are based in part on
the existence of hyperactive mutants of Ste5 that have
been hypothesized to mimic an active conformation,
but whether such conformational changes actually oc-
cur and whether they are induced by Gβγ is unknown.
In this study, we identify a domain in Ste5 that binds
directly to phospholipid membranes. This domain is re-
quired for pheromone response in a manner that sug-
gests it acts cooperatively with the Gβγ binding do-
Figure 1. A Cortical Localization Domain in the Ste5 N Terminus
See Table S2 for plasmids.
(A) Behavior of the Ste5-NT fragment. Ste5 and Ste5?N are from
Pryciak and Huntress (1998).
(B) Top, cortical localization of GFP-Ste5-NT fusions in ste4D ste5D
cells (PPY886). Neither GFP alone nor GFP-Ste5-NT-?NLS are ex-
cluded from the nucleus, but they lack the strong nuclear enrich-
ment of GFP-Ste5-NT. Bottom, dominant negativity. Wild-type (wt)
cells (PPY1368) harboring plasmids as above were plated on –His/
Raff/Gal plates and overlaid with filter disks containing α factor (10
or 2 nmol).
(C) Mutant Ste5-NT fragments were tested for localization to nu-
cleus (nuc) and cortex (cort), for dominant negativity (dom neg),
and for binding to Ste4 (Gβ). Mutations were also moved to full-
length Ste5 and tested for α factor response. Results are summa-
rized; see Figure S1 and Figure 4 for supporting data. #, localization
results with C180A were ambiguous.
(D) Ste5 residues 37–76 are sufficient for plasma membrane local-
ization in PPY886. Top, GFP fusions to Ste5 residues 1–214, 1–125,
main, with neither interaction alone being sufficiently
strong to allow functional recruitment of Ste5. Remark-
ably, the Ste5 membrane binding domain is identical to
a previously characterized nuclear localization se-
quence, suggesting a revised view of the role of nuclear
shuttling in Ste5 function. Moreover, we find that the
normal level of signaling quiescence in the absence of
Gβγ activation depends on the imperfect nature of the
Ste5 membrane binding domain and that at least some
previous hyperactive Ste5 mutants are explained by an
increased affinity for membranes.
A G??-Independent Membrane Localization
Domain Maps to the Ste5 NLS
Previous work showed that the pheromone-activated
Gβγ dimer recruits Ste5 to the plasma membrane (Pry-
ciak and Huntress, 1998). Unexpectedly, we later found
that an amino-terminal fragment of Ste5 (Ste5-NT; resi-
dues 1–214) could localize to the cell cortex even with-
out pheromone treatment (often asymmetrically; see
Discussion) when overexpressed from the GAL1 pro-
moter (Figures 1A and 1B). Although this fragment con-
tains the binding site for Gβγ, its cortical localization
was still observed in cells lacking the Ste4 (Gβ) subunit,
such as ste4D ste5D cells (Figure 1B), as well as in cells
lacking any of the other mating pathway components
(data not shown).
To identify the responsible sequences, we made a
series of small deletions (Figure 1C). A deletion referred
to as ?NLS (?48–67), which removes a previously de-
fined nuclear localization sequence (NLS) (Mahanty et
al., 1999), eliminated cortical localization of the Ste5-
NT fragment as well as its strong nuclear enrichment
(Figures 1B and 1C). Previously, the NLS was found to
be required for membrane recruitment of full-length
Ste5 in response to pheromone, leading to the proposal
that nuclear shuttling allows Ste5 to become compe-
tent for recruitment (Mahanty et al., 1999). Our findings
show that the NLS governs cortical localization in a way
that is inherent to the Ste5-NT fragment and, hence,
can be studied in the absence of Gβγ, pathway signal-
ing, and other Ste5 sequences.
Though it did not affect binding to Ste4 (Gβ), the
?NLS mutation disrupted the ability of the Ste5-NT
fragment to display dominant-negative activity (Figures
1B and 1C), which is thought to reflect inhibition of Gβγ
(Whiteway et al., 1995; Feng et al., 1998). Therefore, al-
though not part of the Gβγ binding site, the NLS is
nonetheless required for functional interaction of the
Ste5 N terminus with Gβγ. In contrast, interaction with
Gβγ in both assays was disrupted, but cortical localiza-
tion was unperturbed by two mutations flanking the
RING-H2 domain, ?152–173 and ?169–171, which re-
semble the RING-H2 mutation C180A (Inouye et al.,
1997; Feng et al., 1998) but have the advantage of dis-
rupting only Gβγ binding and not other interactions
or two copies of residues 1–125 expressed in tandem ([1–125]x2).
Bottom, GST is fused at the N terminus of GFP, yielding GST-GFP-
Ste5 fusions of Ste5 residues 1–125, 37–125, or 37–76.
Cooperative Interactions Control Ste5 Localization
not nuclear targeting, best explains the role of the PM/
NLS domain in Ste5 function. These include: (1) direct
membrane binding by the PM/NLS domain, (2) separa-
tion-of-function mutations in the PM/NLS domain, and
(3) functional replacement of the PM/NLS domain with
lipid binding, but not nuclear-targeting, domains.
Is there a benefit to having the PM domain also pos-
sess NLS activity? Nuclear targeting could help mini-
mize promiscuous membrane contact by reducing
cytoplasmic concentrations of Ste5, and several obser-
vations suggest that nuclear sequestration of Ste5 is
inhibitory (Mahanty et al., 1999; Kunzler et al., 2001).
Competition between membrane and nuclear targeting
could also sharpen the transition between on and off
states (Ferrell, 1998). Notably, nuclear localization of
Ste5 is not required to propagate signal into the nu-
cleus, because trapping Ste5 at the plasma membrane
does not disrupt signaling (Pryciak and Huntress,
1998). Instead, Fus3 likely carries signal into the nu-
cleus after activation on membrane-localized Ste5 (van
Drogen et al., 2001).
Interestingly, dual-function targeting motifs are not
unique to Ste5, as two related examples were reported
recently: one in the S. cerevisiae transcription factor
Opi1 (Loewen et al., 2004) and another in the S. pombe
cytokinesis factor Mid1 (Celton-Morizur et al., 2004).
Given the diverse functions of these proteins, this strat-
egy may ultimately prove to be widespread.
Strains and Plasmids
Yeast strains are listed in Table S1. Plasmids are listed in Table S2;
DNA regions amplified by PCR or subjected to mutagenesis were
sequenced to ensure the absence of spurious mutations.
GFP fusions to full-length Ste5 or Ste5 fragments were visualized
without fixation. Results representative of multiple repeated experi-
ments are shown. Fusions expressed from the GAL1 promoter were
induced with 2% galactose for 60–180 min., depending on expres-
sion strength. Nuclear localization was verified in some experi-
ments by DAPI staining (data not shown). Ste5 expressed at native
levels was visualized as a Ste5-GFPx3fusion to increase signal
levels; cells were grown in selective medium either with or without
α factor (10 ?M for 60 min).
Assays of growth arrest, mating, and FUS1-lacZ induction were
performed as described previously (Pryciak and Huntress, 1998;
Lamson et al., 2002). Halo assays of growth arrest used 10 nmol
and 2 nmol α factor (Figure 1B) or 10 nmol α factor (Figure 3D). To
assay growth arrest by galactose-inducible constructs (Figure 4A),
cell suspensions were spotted onto selective GLU (2% glucose) or
GAL (2% raffinose + 2% galactose) plates. Patch mating assays
used strain PT2α as the mating partner. To measure induction of
FUS1-lacZ by pheromone, cells were incubated with and without 5
?M α factor for 2 hr. FUS1-lacZ in response to galactose-inducible
constructs was measured 3 hr after addition of 2% galactose to
cultures in 2% raffinose medium. Levels of FUS1-lacZ activity were
quantified by β-galactosidase assay as described previously (Pry-
ciak and Huntress, 1998; Lamson et al., 2002).
Interactions were measured by using the two-hybrid reporter strain
PPY760 and were quantified by liquid β-galactosidase assays
(Lamson et al., 2002).
Wt and mutant derivatives of GST-Ste5(37–76) were expressed
from pGEX-6P-1 and purified from E. coli strain BL21-RIPL (Strata-
gene) by using glutathione Sepharose as described previously (Na-
kanishi et al., 2004); solutions included 0.2 mM PMSF and 0.1 mM
benzamidine to inhibit proteolysis. Purified GST fusions (or GST
alone) were pelleted with sucrose-laden liposomes in 20 mM
NaPO4(pH 7.5), 100 mM NaCl. After electrophoresis and Coomas-
sie blue staining, the fraction of input protein recovered in the pellet
(percentage bound) was quantified (Nakanishi et al., 2004). Lipo-
somes contained phosphatidylcholine (PC) alone or mixed with
other lipids; to maximize sensitivity to changes in binding affinity,
tests of Ste5 mutants used 5% or 7% PIP2, based on the half-
maximal binding point in Figure 2C.
Yeast cell extracts were prepared by glass bead lysis as described
previously (Lamson et al., 2002). Epitope-tagged Ste5 was immu-
noprecipitated with mouse anti-myc (9E10; Santa Cruz Biotech) or
mouse anti-GFP (clones 7.1 and 13.1; Roche) antibodies and de-
tected by immunoblotting with rabbit anti-myc (A-14; Santa Cruz
Biotech) or mouse anti-GFP (B34; Covance) antibodies using meth-
ods described previously (Lamson et al., 2002).
Supplemental Data include five figures and two tables and are
available with this article online at http://www.molecule.org/cgi/
We thank the following for gifts of plasmids and yeast strains: A.
Audhya and S. Emr, N. Edgington and B. Futcher, D. Jenness, D.
Lambright, M. Peter, P. Silver, and J. Thorner. We also thank D.
McCollum and C. Goutte for comments on the manuscript and J.
Thorner for communicating unpublished results. This work was
supported by grants from the National Institutes of Health to P.M.P
(GM57769) and A.M.N. (GM62184).
Received: April 4, 2005
Revised: July 27, 2005
Accepted: August 17, 2005
Published: October 6, 2005
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