The EMBO Journal (2004) 23, 3747–3757
www.embojournal.org
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2004 European Molecular Biology Organization | All Rights Reserved 0261-4189/04
THE
EMBO
JOURNAL
Genome-wide lethality screen identifies
new PI4,5P2 effectors that regulate
the actin cytoskeleton
Anjon Audhya1,7, Robbie Loewith2, Ainslie
B Parsons3,4, Lu Gao5,6, Mitsuaki Tabuchi1,
Huilin Zhou5,6, Charles Boone3,4,
Michael N Hall2 and Scott D Emr1,*
1
Department of Cellular and Molecular Medicine, The Howard Hughes
Medical Institute, University of California, San Diego School of
Medicine, La Jolla, CA, USA, 2Division of Biochemistry, Biozentrum,
University of Basel, Basel, Switzerland, 3Banting and Best Department
of Medical Research, University of Toronto, Toronto, Ontario, Canada,
4
Department of Molecular and Medical Genetics, University of Toronto,
Toronto, Ontario, Canada, 5Ludwig Institute for Cancer Research, La
Jolla, CA, USA and 6Department of Cellular and Molecular Medicine,
University of California, San Diego, La Jolla, CA, USA
To further understand the roles played by the essential
phosphoinositide PI4,5P2, we have used a synthetic lethal
analysis, which systematically combined the mss4ts mutation, partially defective in PI4P 5-kinase activity, with
each of approximately 4700 deletion mutations. This genomic screening technique uncovered numerous new candidate effectors and regulators of PI4,5P2 in yeast. In
particular, we identified Slm1 (Yil105c), a previously uncharacterized PI4,5P2 binding protein. Like Mss4, Slm1
and its homolog Slm2 (Ynl047c) were required for actin
cytoskeleton polarization and viability. Co-immunoprecipitation experiments revealed that Slm1 interacts with a
component of TORC2, a Tor2 kinase-containing complex,
which also regulates the actin cytoskeleton. Consistent
with these findings, phosphorylation of Slm1 and Slm2
was dependent on TORC2 protein kinase activity, both in
vivo and in vitro, and Slm1 localization required both
PI4,5P2 and functional TORC2. Together, these data suggest
that Slm1 and Slm2 function downstream of PI4,5P2 and
the TORC2 kinase pathway to control actin cytoskeleton
organization.
The EMBO Journal (2004) 23, 3747–3757. doi:10.1038/
sj.emboj.7600384; Published online 16 September 2004
Subject Categories: cell & tissue architecture; signal
transduction
Keywords: actin; phosphoinositide; polarity; Rho GTPase;
TOR
*Corresponding author. Division of Cellular and Molecular Medicine,
HHMI, UCSD School of Medicine, Cellular and Molecular Medicine Bldg,
Rm 318, 9500 Gilman Drive, 3rd Floor, La Jolla, CA 92093-0668, USA.
Tel.: þ 1 858 534 6462; Fax: þ 1 858 534 6414; E-mail: semr@ucsd.edu
7
Present address: Ludwig Institute for Cancer Research, La Jolla, CA
92093, USA
Received: 11 May 2004; accepted: 5 August 2004; published online:
16 September 2004
& 2004 European Molecular Biology Organization
Introduction
Over the past decade, derivatives of the lipid phosphatidylinositol, collectively known as phosphoinositides, have
emerged as essential regulatory molecules involved in a
diverse spectrum of cellular processes (Fruman et al, 1998;
Takenawa and Itoh, 2001). Multiple characterized effectors
of phosphoinositides harbor specific domains important
for lipid recognition. Pleckstrin homology (PH) domains
from several proteins have been shown to interact with
phosphoinositides, sometimes showing a clear preference
for a single lipid (Lemmon and Ferguson, 2000). The PH
domain from phospholipase C-d1 (PLCd1) specifically
binds PI4,5P2 and its soluble head group IP3 in vitro
(Cifuentes et al, 1993), and when fused to GFP, can be
used as an in vivo probe for this lipid (Stauffer et al, 1998).
These data suggest that proteins containing PH domains or
other phosphoinositide interacting motifs (i.e. FYVE, PX,
ENTH, etc.) are recruited and/or activated by binding
phosphoinositides on specific membrane compartments.
Through the action of a set of well-conserved and tightly
regulated phosphoinositide kinases, phosphatases, and
lipases, phosphoinositides can efficiently modulate downstream events mediated by their effectors in response to
a variety of stimuli. The yeast Saccharomyces cerevisiae
has proven to be a useful model system for the study of
phosphoinositides, showing essential roles for PI3P in endosomal membrane trafficking, PI4P in Golgi secretion, and
PI4,5P2 in endocytosis and actin cytoskeleton organization
(Odorizzi et al, 2000).
Generation of PI4,5P2 is catalyzed by a single phosphoinositide kinase in yeast, Mss4. Using temperature-sensitive
alleles of MSS4, multiple groups have demonstrated that
PI4,5P2 generated at the plasma membrane is required for
actin cytoskeleton organization in yeast (Desrivières et al,
1998; Homma et al, 1998). Correspondingly, studies using
several different biological systems indicate the existence of
numerous PI4,5P2 effector proteins involved in cytoskeletal
dynamics, including gelsolin, cofilin, the Arp2/3 activator
WASP, ERM proteins, and exchange factors for small Rhotype GTPases (Takenawa and Itoh, 2001). In particular, Mss4generated PI4,5P2 has been shown to recruit/activate the
Rho1 GTPase exchange factor Rom2, which is required for
Rho1 activation and maintenance of actin polarization
(Audhya and Emr, 2002). However, the mechanism(s) behind
how PI4,5P2 coordinates the recruitment/activation of its
many effectors remains unclear.
In addition to Mss4, multiple other factors have been
implicated in the regulation of actin cytoskeleton organization, several of which affect Rho1 activation. Previous studies
have shown that Tor2 also functions upstream of Rho1,
and tor2ts mutant cells exhibit a defect in actin organization similar to mss4ts cells (Schmidt et al, 1997). TOR, the
target of rapamycin, is a conserved phosphatidylinositol
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A Audhya et al
kinase-related protein kinase that controls cell growth in
response to nutrient conditions. Yeast cells express two TOR
homologs, Tor1 and Tor2, which can participate in two
distinct complexes, TORC1 and TORC2 (Loewith et al,
2002). TORC1 is rapamycin sensitive, contains either Tor1
or Tor2, and controls translation initiation, ribosome biogenesis, transcription, and other growth-related readouts. In
contrast, TORC2 is rapamycin insensitive, contains Tor2 but
not Tor1, and controls polarization of the actin cytoskeleton
(Loewith et al, 2002). In addition to Tor2, TORC2 also
contains five additional subunits: Avo1, Avo2, Avo3, Bit61,
and Lst8 (Loewith et al, 2002; Wedaman et al, 2003; Reinke
et al, 2004). Interestingly, tor2ts mutations can be suppressed
by overexpression of Mss4, suggesting an interaction between the Tor2 protein kinase and the Mss4 lipid kinase
(Helliwell et al, 1998).
To initiate an unbiased investigation of the physiological
roles of PI4,5P2, we conducted a genome-wide synthetic
lethal screen using a strain expressing a functionally
impaired, temperature-sensitive allele of MSS4. We identified two previously uncharacterized proteins, Slm1 and
Slm2, which are downstream effectors of both Mss4 and
TORC2 and are required for organization of the actin
cytoskeleton. These findings suggest an integration of
lipid kinase function with TORC2 protein kinase regulation,
both necessary for spatial and temporal regulation of actin
cytoskeleton dynamics.
Results
Synthetic genetic array analysis reveals an Mss4
network of genetic interactions
MSS4 encodes an essential PI4P 5-kinase in yeast. To extend
our understanding of Mss4 regulation and activity, we utilized a method, termed synthetic genetic array (SGA) analysis, for systematic construction and characterization of
double-mutant strains. An mss4ts mutation was combined
with each of approximately 4700 deletion mutations at the
permissive growth temperature for mss4ts cells. Under these
conditions, mss4ts cells generate approximately 40% reduced
levels of PI4,5P2 as compared to wild-type cells (Stefan et al,
2002), slightly compromising but not inactivating PI4,5P2dependent cellular functions. We reasoned that inviable or
slow-growing double-mutant progeny could identify a functional interaction between Mss4 and the product of the wildtype version of the deleted gene. Such proteins could represent regulators of the PI4P 5-kinase or targets of its product,
PI4,5P2. Previous work indicated that mutations in MSS4 are
lethal in combination with mutations that affect activation of
the Rho1 GTPase (Audhya and Emr, 2002). In agreement with
these studies, the SGA analysis identified Rom2, the exchange
factor for Rho1. Since Rom2 requires PI4,5P2 synthesis for its
appropriate localization (Audhya and Emr, 2002), the SGA
analysis should identify potential novel PI4,5P2 effectors.
In total, almost 80 double-deletion combinations were
initially isolated in the screen that resulted in a synthetic
growth defect. Of these, approximately half were confirmed
using an alternative method (see Supplementary Table I).
Multiple genes that were Synthetic Lethal with Mss4 (SLM)
encoded proteins that have been implicated in actin cytoskeleton organization and/or cell polarity including Cap1, Cap2,
Avo2, Spa2, Bem4, Boi2, Myo5, Cyk3, Ilm1, Van1, and Api2.
Additionally, multiple components of the GimC complex, a
chaperone believed to be important for normal folding of
actin monomers (Siegers et al, 1999), were isolated in this
screen. The identification of genes encoding proteins involved in a number of other processes including cellular
stress response, ribosomal biosynthesis, protein/lipid modification, vesicle trafficking, and mitochondrial function suggests that Mss4 is involved in multiple biological pathways.
Taken together, the results from the SGA analysis confirmed
an essential function for Mss4 in cytoskeletal organization,
but also indicated that Mss4/PI4,5P2 likely plays other roles
in maintaining cell structure and viability (Figure 1A).
Figure 1 SGA analysis using the mss4ts mutation indicates diverse roles for PI4,5P2. (A) The genes that are required for normal growth of
mss4ts cells are represented as nodes. Each node is color coded based on its functional classification as defined by published literature. (B)
Cartoon showing the domains of Slm1 and Slm2. Both proteins contain PH domains that interact with phosphoinositides. Additionally, Slm1
and Slm2 share a highly conserved domain, the Slm domain, which has been highlighted.
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Of particular interest is the SGA analysis that revealed a
genetic interaction between MSS4 and YIL105c, a gene encoding a protein that contains a PH domain (Figure 1B),
which has been shown to interact weakly with phosphoinositides in vitro (Yu et al, 2004). Moreover, plasma membrane
localization of the PH domain from Yil105c requires Mss4dependent PI4,5P2 production (Yu et al, 2004). Consistent
with studies of the Yil105c PH domain, full-length Yil105c
also interacted with multiple phosphoinositides in liposome
binding assays (Supplementary Figure 1).
The genetic interaction between the mss4ts mutation and
yil105cD was confirmed in the SEY6210 background, where
mss4tsyil105cD double-mutant cells exhibited a potent growth
defect at 31.51C in contrast to the near-normal growth of both
single-mutant strains at the same temperature (Figure 2A).
Analysis of phosphoinositide levels in an mss4ts mutant and
in mss4tsyil105cD double-mutant cells at 31.51C revealed
comparable levels of phosphoinositides (data not shown),
suggesting that Yil105c is not upstream of Mss4 and is
consistent with the idea that PI4,5P2 may play a role in the
recruitment/activation of Yil105c. Given its genetic interaction with the mss4ts mutation, we have named this gene
SLM1 for Synthetic Lethal with Mss4.
Since cells deleted for SLM1 alone failed to display any
significant phenotype, we performed a database search to
detect potential homologs that may serve a redundant function. This analysis led to the identification of YNL047c, which
encodes a protein that is 53% identical to Slm1. Due to this
strong similarity, we have named this gene SLM2 (Figure 1B).
Slm2 also contains a PH domain that binds phosphoinositides, with a preference for PI4,5P2 when expressed as a GFP
fusion in cells (Yu et al, 2004). Deletion of SLM2 did not yield
any noticeable effects on cell growth, but in combination
with deletion of SLM1, cells were no longer viable (Figure 2B).
Together, these data indicate that Slm1 and Slm2 serve an
essential function in yeast, likely downstream of Mss4/
PI4,5P2.
Although Slm1 and Slm2 appear to be redundant in function, only SLM1 was uncovered in the SGA analysis using
the mss4ts mutation. Consistent with this observation,
mss4tsslm2D double-mutant cells did not exhibit a significant
synthetic growth defect in contrast to mss4tsslm1D doublemutant cells. These data suggest that Slm1 may play a more
dominant role in cells as compared to Slm2. To test directly
the expression levels of Slm1 and Slm2, we measured the
cellular levels of each protein by first constructing strains that
expressed chromosomally GFP-tagged forms of each gene.
Both fusion proteins were found to be functional, as determined by the normal growth of slm1DSLM2-GFP and SLM1GFPslm2D cells. Western blot analysis revealed that levels of
Slm1 protein were approximately 10-fold higher than those of
Slm2 (Figure 2C), confirming that Slm1 is more abundant and
likely plays a more significant role than Slm2 in cells.
Consistent with these data, Slm1-GFP was clearly visible at
endogenous levels, while Slm2-GFP fluorescence was more
difficult to detect (see below).
Slm1 and Slm2 are new PI4,5P2 effectors
In contrast to the confluent plasma membrane localization of
the PH domains from both Slm1 and Slm2 (Yu et al, 2004),
localization of each full-length protein showed they were
enriched in punctate structures distributed at the cell periphery (Figure 3A and Supplementary Figure 2). The Slm1
patches were not polarized, as is the case with cortical
actin patches. Time-lapse video microscopy indicated only
Figure 2 Slm1 and Slm2 perform an essential function downstream
of Mss4. (A) slm1D, mss4ts and slm1Dmss4ts cells were grown at 26
or 31.51C for 60 h. (B) slm1D and slm2D cells were mated, and the
resulting diploids were sporulated and dissected. Tetrads (1–6) are
shown, yielding four viable spores (indicative of two slm1D colonies and two slm2D colonies), three viable spores (indicative of one
wild-type colony, one slm1D colony, one slm2D colony, and one
inviable slm1Dslm2D colony), or two viable spores (indicative of
two wild-type colonies and two inviable slm1Dslm2D colonies). The
arrows highlight inviable slm1Dslm2D colonies. (C) TCA-precipitated extracts from cells expressing SLM1-GFP or SLM2-GFP were
analyzed by SDS–PAGE followed by Western blot using a-GFP
antibodies to detect Slm1 and Slm2 or a-G6PDH antibodies as a
loading control.
& 2004 European Molecular Biology Organization
Figure 3 Localization of Slm1 is dependent on PI4,5P2 but not Factin. (A) Cells expressing SLM1-GFP and ABP1-RFP were visualized
by fluorescence microscopy in the presence or absence of latrunculin A. (B) mss4ts cells expressing SLM1-GFP were visualized by
fluorescence microscopy following a 60 min shift to 371C. (C)
slm1Dslm2D cells expressing slm1KA-GFP were visualized by fluorescence microscopy at 261C.
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occasional movement of Slm1 on the mother cell cortex,
while Slm1 patches were more dynamic on the daughter
cell cortex (Supplementary Movie 1). Additionally, Slm1
patches were not affected by latrunculin A, a drug that causes
actin depolymerization, indicating that Slm1 localization was
independent of F-actin (Figure 3A). Since the PH domains
from both proteins showed localizations dependent on Mss4
activity, localization of Slm1-GFP was analyzed in mss4ts
cells. Following shift to the nonpermissive temperature,
Slm1 appeared more soluble in mss4ts cells as compared to
wild-type cells treated identically, although a portion of Slm1
could still associate with cortical punctate structures
(Figure 3B). These data suggest that Slm1 is at least in part
recruited to the plasma membrane by PI4,5P2 and then
potentially stabilized by other protein–protein or protein–
lipid interactions.
To further investigate a role for PI4,5P2 in the recruitment
of Slm1 to the plasma membrane, we generated a mutant
form of Slm1 (slm1KA), which harbors point mutations in
critical lysine resides (K483A, K487A) in the PH domain that
were previously shown to be required for phosphoinositide
binding (Yu et al, 2004). slm1Dslm2D double-mutant cells
expressing slm1KA were viable but temperature sensitive for
growth, indicating an essential requirement for Slm1 phosphoinositide binding under conditions of stress. Strikingly,
even at permissive temperatures, slm1KA-GFP was largely
mislocalized, accumulating mostly in the cytoplasm, with
only a weak ability to associate with the plasma membrane,
similar to mss4ts cells expressing wild-type Slm1-GFP
(Figure 3C). Western blot analysis revealed that this form of
Slm1 was stable, both at permissive and nonpermissive
temperatures, indicating that cytoplasmic accumulation of
GFP fluorescence was not due to clipping of the GFP fluorophore. Together, these data suggest a role for Mss4-generated PI4,5P2 in the recruitment of Slm1 to the plasma
membrane, and also indicate the existence of other factor(s)
that contribute to Slm1 localization.
Slm1 and Slm2 are essential for normal actin
cytoskeleton organization
To gain further insight into the function of Slm1 and Slm2, we
constructed temperature-sensitive forms of SLM1 using errorprone PCR and plasmid shuffle techniques. Western blot
analysis indicated that each form of Slm1 was stable, but
three different patterns of Slm1 localization emerged from the
screen (Table I). One group of temperature-sensitive mutants
localized similarly to wild-type Slm1 at both permissive and
nonpermissive temperatures (class A). In contrast, a second
group of mutants showed normal localization at 261C, but
accumulated in the cytoplasm following shift to nonpermissive temperature (class B). Finally, a third group of mutants
showed strong cytoplasmic fluorescence independent of temperature, similar to slm1KA described above (class C).
Representative members from each class of mutants were
chosen for sequencing. Both class A and B mutants harbored
mutations between amino acids 192 and 436, a region we
refer to as the Slm domain, due to its high conservation
between Slm1 and Slm2 (Figure 1B). However, only class C
mutants contained mutations in the PH domain (amino acids
469–581). Although mutations in the Slm domain could affect
the localization of Slm1 (class B), its association with PI4,5P2
as determined by liposome binding assays was not significantly perturbed (Supplementary Figure 1). These data
further indicate the importance of the Slm1 PH domain for
localization, and also highlight an additional region of Slm1
as being important for both function and maintenance of
Slm1 at the plasma membrane.
Previous studies have indicated a role for PI4,5P2 in the
maintenance of actin cytoskeleton organization and cell wall
integrity, at least in part through activation of the Rho1–Pkc1
pathway. To determine if Slm1 and Slm2 also function as
downstream effectors of Mss4 in the maintenance of actin
polarization, we examined actin localization in all three
classes of slm1tsslm2D cells. At the permissive temperature,
both wild-type and slm1tsslm2D cells with relatively small
buds showed normal polarization of the actin cytoskeleton,
with cortical actin patches restricted to daughter cells and
actin cables extending through mother cells. In contrast
to wild-type cells, at the nonpermissive temperature
slm1tsslm2D cells exhibited a complete depolarization of the
actin cytoskeleton, with actin patches randomly distributed
throughout both mother and daughter cells and an apparent
loss of actin cables (Figure 4A). Additionally, extended
incubation of slm1tsslm2D cells at 371C (greater than 3 h)
resulted in cell lysis. These phenotypes were identical to
those observed in mss4ts cells placed at the nonpermissive
temperature.
Since actin cables were rather poorly preserved following
fixation, we directly examined actin cable organization in
living cells by visualizing a GFP fusion to the actin cable
binding protein Abp140 (Yang and Pon, 2002). Although long
actin cables oriented along the mother–bud axis were clearly
Table I Three classes of slm1ts alleles each show different localizations
Class
Strain
Mutation(s)
Growth
Localization
261C
371C
261C
371C
A
AAY1622
F236S, K254R, N436S
++
—
PM
PM
B
AAY1623
AAY1624
I207N, N371T, V392A
L192P
+++
++
—
—
PM
PM
C
C
C
AAY1625
AAY1626
N134S, D351A, K471T
K480Q, H498R
+++
++
—
—
C
C
C
C
Cells were grown at 26 or 371C for 2 days on synthetic growth media. For localization studies, cells were shifted to 371C for 1 h. Observations
were based on visualization of 4100 cells/condition.
PM: plasma membrane; C: cytoplasm.
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Figure 4 Slm1 and Slm2 control actin cytoskeleton organization
and polarized secretion. (A) Wild-type and slm1tsslm2D cells expressing either an activated allele of PKC1 or an empty vector were
shifted to 371C for 2 h, fixed and stained with rhodamine–phalloidin, and visualized by fluorescence microscopy. (B) Wild-type and
slm1tsslm2D cells expressing ABP140-GFP were shifted to 371C for
1 h and visualized by fluorescence microscopy. The arrows indicate
long actin cables found in wild-type cells. (C) slm1Dslm2D doublemutant and slm1Dslm2Dsac7D triple-mutant cells carrying
pRS416SLM1 were grown on 5-FOA for 4 days at 261C. (D) Wildtype and slm1tsslm2D cells expressing GFP-SEC4 were shifted to
371C for 1 h and visualized by fluorescence microscopy. Arrows
indicate Sec4 localization to the bud or mislocalization to the
mother cell cortex. (E) Wild-type and slm1tsslm2D cells expressing
GFP-SNC1 were shifted to 371C for 1 h and visualized by fluorescence microscopy.
visible in slm1tsslm2D cells at 261C, they were absent following a 60-min shift to the nonpermissive temperature
(Figure 4B). Under identical conditions, wild-type cells maintained actin cable orientation independent of temperature.
Together, these data indicate a requirement for Slm1 and
Slm2 in both actin patch polarization and actin cable stability/orientation.
Slm1 and Slm2 are important for polarization
of the secretory pathway
To further investigate the functions of Slm1 and Slm2, we
performed a gene dosage-dependent suppression screen
using class A slm1tsslm2D cells to identify proteins that
when overexpressed could suppress the temperature sensitivity of the mutant. Analysis of 40 000 transformants allowed
the isolation of five genes, each of which could reproducibly
suppress the growth defect of slm1tsslm2D cells: SLM1,
SLM2, PKC1, MSS4, and SEC4, a Rab-type GTPase essential
for protein secretion. Moreover, overexpression of these
genes also suppressed the actin defects characteristic of
slm1tsslm2D cells at 371C, suggesting that increased signaling
through the Rho1–Pkc1 cell integrity pathway could bypass
the requirement for Slm1 and Slm2 (Figure 4A and Table II).
Consistent with this idea, expression of PKC1R398P, encoding
a constitutively activated form of Pkc1, allowed growth of
cells completely lacking SLM1 and SLM2 (slm1Dslm2D) at
261C. However, at elevated temperatures, slm1Dslm2D cells
harboring PKC1R398P were no longer viable, indicating the
existence of other factors necessary for bypass of Slm1 and
Slm2. In contrast to the rescue of slm1Dslm2D cells by
PKC1R398P, expression of constitutively activated alleles of
BCK1 or MKK1, both downstream effectors of Pkc1, failed
to suppress the phenotypes of slm1tsslm2D or slm1Dslm2D
cells (Table II).
Since Pkc1 is an effector of Rho1, we tested whether
elevated Rho1-GTP levels were sufficient to bypass the requirement for Slm1 and Slm2 for cell viability at elevated
temperature. Deletion of SAC7, a gene that encodes a Rho1
guanine nucleotide activating protein, has been shown to
elevate levels of cellular Rho1-GTP (Schmidt et al, 1997).
Strikingly, in contrast to slm1Dslm2D double-mutant cells,
slm1Dslm2Dsac7D triple-mutant cells were viable both at 26
and 381C, indicating that Slm1 and Slm2 are dispensable
under conditions where Rho1 is independently activated
(Figure 4C).
Previous work has demonstrated a requirement for the
Sec4-dependent secretory pathway in transport of Rho1 (Abe
et al, 2003). Interestingly, inactivation of Sec4 causes a defect
in actin cytoskeleton organization (Mulholland et al, 1997),
possibly due to a defect in transport of Rho1 (or other
machinery necessary for actin organization) to sites of polarized growth. Since overexpression of Sec4 suppressed the
growth defect of slm1tsslm2D cells, we examined whether
Sec4 localization was perturbed in the absence of Slm1 and
Table II Genetic interactions in slm1tsslm2D cells
Plasmid or mutation in slm1tsslm2D cells
None
2m PKC1
2m MSS4
2m SEC4
PKC1R398P
BCK1-20
2m MKK1S386P
sac7D
sec4-8
Growth
Actin polarization
Snc1 polarization
281C
371C
261C
371C
261C
371C
+++
+++
+++
+++
+++
+++
+++
+++
—
—
++
++
++
++
—
—
+++
—
+++
+++
+++
+++
+++
+++
+++
+++
ND
—
++
++
++
++
—
—
+++
ND
+++
+++
+++
+++
+++
+++
+++
+++
ND
—
++
++
++
++
—
—
+++
ND
For growth studies, cells were grown at 28 or 371C for 2 days on synthetic growth media. For actin and Snc1 polarization studies, cells were
shifted to 371C for 2 h. Observations were based on visualization of 4100 small budded cells/condition. ND: not determined.
& 2004 European Molecular Biology Organization
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Slm2 activity. At 261C, Sec4 localized to sites of polarized
growth in slm1tsslm2D cells, similar to that observed in wildtype cells. However, following a 60-min shift to the nonpermissive temperature, Sec4 was mislocalized in greater than
75% of slm1tsslm2D cells, accumulating in the cytoplasm as
well as in unpolarized patches decorating the entire cell
periphery (Figure 4D). Since Sec4 transport requires polarized actin cables (Pruyne et al, 1998), these data are
consistent with a primary defect in actin cytoskeleton organization when Slm1 and Slm2 function is compromised.
Similar to the defect in Sec4 localization, examination of
another secretory vesicle marker, the v-SNARE Snc1, showed
that it also became unpolarized in slm1tsslm2D cells at
elevated temperature (Figure 4E). Increased signaling
through the Rho1–Pkc1 pathway or overexpression of Sec4,
which may elevate Rho1–Pkc1 signaling through enhanced
delivery of Rho1 to the cortex, returned Snc1 to sites of
polarized growth in slm1tsslm2D cells at elevated temperature
(Table II). Together, these data indicate that appropriate Sec4
localization to sites of polarized growth becomes limiting in
the absence of Slm1 and Slm2 activity, likely due to a primary
defect in actin organization. Furthermore, Sec4 mislocalization potentially exacerbates the defect in actin polarization
observed in slm1tsslm2D cells by virtue of its role in actin
organization. Consistent with this idea, combining the temperature-sensitive sec4-8 mutation with the slm1tsslm2D mutations resulted in synthetic lethality at 281C, a normally
permissive temperature for sec4-8 or slm1tsslm2D cells individually (Table II).
Phosphorylation of Slm1 and Slm2 requires TORC2
protein kinase activity
High-throughput two-hybrid analysis has previously suggested that Slm1 and Slm2 have a common set of interactors
(Uetz et al, 2000; Ito et al, 2001). To test directly whether
Slm1 functions as part of a complex, we used a tandem
affinity purification (TAP) method to isolate Slm1 and any
tightly associated proteins. From this analysis, we found
only one other copurifying protein by mass spectrometry
following stringent washing conditions, Slm2 (Figure 5A).
Interestingly, we identified several electrophoretic species of
both Slm1 and Slm2 in our purification. Western blot analysis
directed against the S-tag epitope fused to Slm1 (and used
during its purification) revealed that only two of the four
bands identified as being Slm1 by mass spectrometry contained the S tag. These data indicate that Slm1 can selfassociate in vivo. The existence of multiple forms of Slm1 and
Slm2 suggested that both proteins undergo post-translational
modification. To test this directly, we immunoprecipitated
newly synthesized Slm1-GFP and Slm2-GFP after briefly
labeling cells with 35S and chasing with unlabeled cysteine
and methionine. After 10 min of labeling, we detected single
bands corresponding to Slm1 and Slm2, but following a brief
period of chase, we observed a shift in the molecular weight
of Slm1, which migrated as two distinct, higher molecular
weight species, and Slm2, which migrated as a single, higher
molecular weight species (Figure 5B). Due to its higher level
of expression, we focused on Slm1 to determine if the gel shift
corresponded to phosphorylation.
Cells expressing Slm1-GFP were labeled with 32P, and
extracts were immunoprecipitated with GFP antibodies.
This analysis indicated that Slm1 was phosphorylated on at
3752 The EMBO Journal VOL 23 | NO 19 | 2004
Figure 5 Slm1 and Slm2 are phosphoproteins that form a tightly
associated complex in vivo. (A) Silver-stained SDS–PAGE gel showing the Slm1/Slm2 complex following TAP. Slm1-S refers to Slm1
fused to the S tag used during purification. Slm1-S* is a breakdown
product of Slm1-S as detected by Western blot analysis using
antibodies directed against the S tag. (B, D) Cells expressing
SLM1-GFP or SLM2-GFP were metabolically labeled for 10 min
with 35S-labeled cysteine and methionine and chased for the
indicated time. Extracts were immunoprecipitated with anti-GFP
antibodies, incubated in the presence or absence of shrimp alkaline
phosphatase (SAP), and subjected to SDS–PAGE analysis followed
by autoradiography. The asterisks indicate the phosphorylated
forms of Slm1. (C) Cells expressing SLM1-GFP were metabolically
labeled for the indicated time with 32P-labeled orthophosphate.
Extracts were immunoprecipitated with anti-GFP antibodies, followed by SDS–PAGE analysis and autoradiography.
least two sites in vivo (Figure 5C). Additionally, treatment of
S-labeled cell extracts with phosphatase eliminated the gel
mobility shift, confirming that the shift in Slm1 gel mobility
was specifically due to phosphorylation (Figure 5D). Similar
studies with Slm2 confirmed that it is also phosphorylated
(data not shown).
Since the Rho1–Pkc1 pathway responds to heat stress by
first causing depolarization of the actin cytoskeleton followed
by a period of repolarization (Delley and Hall, 1999), we also
analyzed the effect of elevated temperature on the phosphorylation of Slm1. Heat shock significantly reduced the
phosphorylation of Slm1 normally observed at low temperature. However, Slm1 phosphorylation recovered at later
time points (Figure 6A), which correlated with the timing
of actin cytoskeleton repolarization, again suggesting a
link between Slm1 activity and organization of the actin
cytoskeleton.
To determine which kinase(s) phosphorylated Slm1, we
first analyzed results from high-throughput two-hybrid analyses, which suggested that both Slm1 and Slm2 may interact
with Avo2, a component of the Tor2 protein kinase-containing TORC2 complex, required for proper actin cytoskeleton
organization (Loewith et al, 2002). Although TAP of Slm1
failed to indicate an interaction with Avo2, immunoprecipitation of Slm1-GFP from cells expressing Avo2-Myc showed
that Avo2 could associate with Slm1. This interaction was
extremely sensitive to salt conditions used during TAP, suggesting that Slm1 and Avo2 only weakly associate (Figure 6B).
While mutations in the Slm domain of Slm1 failed to perturb
35
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PI4,5P2 and TORC2 regulate actin organization
A Audhya et al
defect in Slm1 phosphorylation (Figure 6C and D).
Moreover, tor2ts cells failed to show recovery of Slm1 phosphorylation at the nonpermissive temperature, indicating that
Tor2 function is required for this process (Figure 6E). Since
PI4,5P2 also plays a role in Slm1 recruitment/activation, we
determined the effect of Mss4 inactivation on Slm1 phosphorylation. As in tor2ts cells, Slm1 phosphorylation failed to
recover in the absence of Mss4 activity, suggesting that
PI4,5P2 and Tor2 are both required for localization and
phosphorylation of Slm1 (Figure 6F). Consistent with these
data, the mutant form of Slm1 harboring mutations in its PH
domain was not normally phosphorylated, likely due to its
inability to target efficiently to the plasma membrane
(Figure 6G).
Tor2 has previously been demonstrated to function in two
different protein complexes, one required for the response to
nutrient availability (TORC1), while the other regulates actin
cytoskeleton organization (TORC2). Studies have also indicated that TORC1 is uniquely sensitive to the effects of the
drug rapamycin (Loewith et al, 2002). To determine if Slm1
phosphorylation requires TORC1, we treated cells with rapamycin and examined phosphorylation of Slm1. Following a
30 min pretreatment with rapamycin, we found that Slm1
phosphorylation was unperturbed, suggesting that TORC2,
but not TORC1, multiply phosphorylates Slm1 (Figure 6H).
Consistent with these data, cells specifically repressed for
TOR2 expression showed a defect in Slm1 phosphorylation
(Figure 6H).
Figure 6 Slm1 associates with the TORC2 component Avo2 and
depends on TORC2 for appropriate phosphorylation. (A) Wild-type
cells expressing SLM1-GFP were metabolically labeled for 10 min
with 35S-labeled cysteine and methionine, chased for 30 min and
shifted to 371C for the indicated time. Extracts were immunoprecipitated with anti-GFP antibodies and subjected to SDS–PAGE
analysis followed by autoradiography. (B) Extracts from cells expressing either AVO2-MYC alone or AVO2-MYC and SLM1-GFP were
immunoprecipitated using a-GFP antibodies under varying salt
conditions. Total Avo2 protein in the extract is shown below. (C)
Wild-type or avo2D cells expressing SLM1-GFP were metabolically
labeled as in Figure 5A. Below each gel is the percentage of dually
phosphorylated Slm1-GFP as determined by densitometry. (D)
Wild-type or avo2D cells expressing SLM1-GFP were metabolically
labeled as in Figure 5C. (E, F) tor2ts and mss4ts cells expressing
SLM1-GFP were metabolically labeled as in (A). (G) slm1Dslm2D
cells expressing SLM1-GFP or slm1KA-GFP were treated as in
Figure 5B. (H) Wild-type cells or cells grown for 16 h in glucosecontaining media and expressing TOR2 under the control of an
integrated GAL1-10 promoter (to repress expression of genomic
TOR2) were metabolically labeled for 10 min with 35S-labeled
cysteine and methionine and chased for the indicated time in the
presence or absence of rapamycin (200 ng/ml). Extracts were
treated as in Figure 5A.
this interaction, mutations in the PH domain of Slm1 resulted
in a reduced association between Slm1KA and Avo2
(Supplementary Figure 3). However, the weakened interaction may be an indirect consequence of Slm1KA mislocalization. Additionally, SGA analysis of the mss4ts mutation
revealed a genetic interaction with AVO2, which was confirmed in the SEY6210 background (Figure 1A and
Supplementary Table I). Consistent with a role for TORC2 in
phosphorylation of Slm1, avo2D cells exhibited a kinetic
& 2004 European Molecular Biology Organization
TORC2 phosphorylates Slm1 and Slm2 in vitro
and is partially required for Slm1 localization
To investigate further a role for TORC2 in phosphorylation of
Slm1 and Slm2, recombinant forms of Slm1 and Slm2 were
purified from bacteria and subjected to an in vitro kinase
assay. In the presence of purified TORC2, both GST-Slm1 and
GST-Slm2, but not GST alone, were phosphorylated, similar to
a previously characterized mammalian TOR substrate, 4EBP1
(Figure 7A). However, purified TORC2 containing a kinasedead form of Tor2 failed to phosphorylate Slm1 or Slm2,
suggesting that a contaminant from the purification was not
responsible for the observed kinase activity.
Finally, to investigate a function for Slm1 phosphorylation
by TORC2, we tested whether the localization of Slm1 depends on Tor2 activity. Since our studies have shown a partial
role for PI4,5P2 in the localization of Slm1, we examined
whether the remaining plasma membrane association of
Slm1 was dependent on Tor2. Therefore, wild-type and
tor2ts cells expressing a mutant form of Slm1 that cannot
interact with PI4,5P2 (slm1KA-GFP) were visualized following
a 90 min shift to 371C. In contrast to wild-type cells, tor2ts
cells failed to exhibit any localization of slm1KA-GFP to the
cell periphery. Cells lacking other plasma membraneassociated protein kinase activities, including pkc1ts, bck1D,
slt2D, tpk1Dtpk2tstpk3D, ypk1tsypk2D, and pkh1tspkh2D
cells, did not affect slm1KA-GFP localization (Figure 7B and
data not shown). Together, these data suggest that Slm1 and
Slm2 function downstream of TORC2 in signaling to the actin
cytoskeleton, and represent the first substrates of TORC2
protein kinase activity.
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PI4,5P2 and TORC2 regulate actin organization
A Audhya et al
regulation of actin cytoskeleton organization. Furthermore,
our screen showed that deletion of AVO2, which encodes a
nonessential component of the Tor2 protein kinase-containing TOR complex 2, is lethal in combination with the mss4ts
mutation, suggesting that TORC2 and Mss4 function together
in regulating actin organization. Consistent with this hypothesis, overexpression of MSS4 suppresses a tor2 mutation
(Helliwell et al, 1998), and both Slm1 and Slm2 are substrates
of the TORC2 kinase. Together, our data suggest the existence
of multiple PI4,5P2 effectors in yeast, whose actions can be
coordinated through additional signaling events to regulate
polarity of the actin cytoskeleton. Importantly, Slm1 and
Slm2 are the first effectors identified that require both
TORC2-dependent phosphorylation and Mss4-dependent recruitment to control the actin cytoskeleton, possibly through
activation of Rho1. We speculate that Mss4 functions as
a sensor for cell stress, such as heat shock, generating
variable levels of PI4,5P2 to alter actin polarization and
cell wall integrity, while other inputs, such as TORC2
kinase regulation, further modulate the magnitude/duration
of these changes.
Multiple roles for PI4,5P2 revealed by synthetic genetic
array analysis
Previous studies have established that PI4,5P2 plays essential
roles in both actin cytoskeleton organization and endocytosis, and several effectors of PI4,5P2 have been identified that
are important for these processes (Takenawa and Itoh, 2001).
Using an unbiased, genome-wide-based approach in yeast,
we have identified several new genes that may be involved in
regulating PI4,5P2 synthesis or that function downstream of
PI4,5P2 (Figure 8). When combined with the mss4ts mutation,
several components of two protein chaperone complexes, the
Hsp70–Hsp90 complex and the GIMC complex (Siegers et al,
Figure 7 Slm1 phosphorylation is TORC2 dependent in vitro. (A)
SW80-1D cells expressing TOR2 under the control of an integrated
GAL1-10 promoter and TAP-tagged AVO2 were transformed with
either vector, (HA)3TOR2 (TOR2) or (HA)3TOR2D2998E (TOR2KD).
After growth for 16 h in glucose-containing media (to repress
expression of genomic TOR2), TORC2 (via Avo2-TAP) was precipitated with IgG-Sepharose beads and tested for Slm1, Slm2, and
4EBP1 kinase activity. Shown are autoradiographs (Phos-Slm1,
Phos-Slm2, Phos-4EBP1) and the corresponding Coomassie bluestained region of the SDS–PAGE gel (Slm1, Slm2, 4EBP1). The
bottom panel is a Western blot showing the amount of HA-tagged
TOR2/TOR2KD added to the respective kinase assays. (B) Wild-type
or tor2ts cells expressing slm1KA-GFP were visualized by fluorescence microscopy following a 90 min shift to 371C.
Discussion
We and others have previously shown that regulation of
PI4,5P2 synthesis at the plasma membrane is critical for
regulation of the actin cytoskeleton, at least in part through
activation of the Rho1 GTPase (Desrivières et al 1998;
Homma et al, 1998; Audhya and Emr, 2002). Using an
unbiased approach to identify additional factors involved in
PI4,5P2 signaling, we have identified two new PI4,5P2 effectors, Slm1 and Slm2, which also appear to be critical for
3754 The EMBO Journal VOL 23 | NO 19 | 2004
Figure 8 Regulation of actin cytoskeleton organization by PI4,5P2.
Genetic interactions identified in the Mss4 SGA analysis indicate
multiple roles for Mss4-mediated PI4,5P2 production in actin
organization. In particular, PI4,5P2 recruitment/activation of the
Slm1–Slm2 complex is required for maintenance of actin cytoskeleton polarity. Additional factors including TORC2 protein kinase
activity further modulate and specify Slm1–Slm2 function in actin
organization.
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PI4,5P2 and TORC2 regulate actin organization
A Audhya et al
1999), became essential for cell viability, suggesting a role
for PI4,5P2 in the response to heat shock. Consistent with
this interpretation, studies have indicated that PI4,5P2 levels
transiently increase following heat shock, potentially required for preventing protein misfolding through activation
of a chaperone complex (Desrivières et al, 1998; Audhya et al,
2000). However, further studies are required to determine
whether PI4,5P2 influences chaperone function during heat
stress.
Additionally, the SGA analysis uncovered a number of
proteins directly involved in actin organization and cell
polarity. Of particular interest was the identification of three
proteins that harbor PH domains, Rom2, Boi2, and Slm1.
Rom2, an exchange factor for the Rho1 GTPase, has been
shown to bind PI4,5P2 and function downstream of Mss4 in
activation of the Rho1–Pkc1 pathway (Audhya and Emr,
2002). Similarly, the PH domain of Boi2, a protein important
for polarized growth and bud formation, has been shown to
interact with phosphoinositides in vitro (Yu et al, 2004).
Additionally, a functionally redundant homolog of Boi2,
Boi1, has been shown to interact with PI4,5P2 via its PH
domain, and this lipid–protein interaction in part mediates its
normal localization and is essential in the absence of Boi2
(Hallett et al, 2002).
Beyond the identification of known PI4,5P2 binding proteins, SGA analysis also identified a new PI4,5P2 effector,
Slm1. Together with its homolog and binding partner Slm2,
these proteins are essential for maintaining proper actin
cytoskeleton organization and cell wall integrity, possibly
through regulation of the Rho1 GTPase (Figure 8). However,
we were unable to identify a direct interaction between Slm1
or Slm2 and known regulators of Rho1, including its exchange factors (Tus1, Rom1, and Rom2) or its activating
proteins (Sac7, Bem2, and Bem3). In an alternative model,
Slm1 and Slm2 may function in parallel to Rho1 in regulation
of actin organization. Genome-wide two-hybrid analysis suggests that Slm2 interacts with both subunits of calcineurin,
a Ca2 þ /calmodulin-dependent protein phosphatase required
during environmental stress, which may function in actin
organization (Uetz et al, 2000; Ito et al, 2001; Cyert, 2003).
Further studies will be necessary to determine the extent to
which calcineurin may function in Slm1/Slm2 signaling.
Coordination of Mss4 and the TORC2 protein kinase
in actin organization
Previous studies in organisms ranging from insects to humans have indicated a relationship between growth factorstimulated phosphoinositide lipid signaling and nutrient-dependent TOR protein kinase signaling in the regulation of cell
growth (Jacinto and Hall, 2003). We have found that this
interaction may also extend to yeast, where PI4,5P2 and Tor2
both function to control the actin cytoskeleton. Yeast express
two forms of TOR: TORC1, which contains either Tor1 or
Tor2, mediates rapamycin-sensitive growth in response to
nutrients, while TORC2, which contains Tor2 but not Tor1,
controls polarization of the actin cytoskeleton. However,
substrates of TORC2 that affect actin organization have
remained elusive. Our findings support a role for Slm1 and
Slm2 in this capacity. Additionally, the physical interaction
between Slm1 and Avo2, the only nonessential component of
either TORC complex, suggests a role for this component of
TORC2 in the recruitment of effector proteins. In the absence
& 2004 European Molecular Biology Organization
of Avo2, PI4,5P2 is likely to be sufficient for recruitment of
Slm1 and Slm2 to sites of TORC2 activity. However, loss of
multiple inputs important for TORC2-dependent phosphorylation cannot be tolerated, as demonstrated by the lethality
exhibited by mss4tsavo2D double-mutant cells. Interestingly,
Rom2, an exchange factor for Rho1, has also been identified
as an effector of PI4,5P2 and potentially Tor2, suggesting that
integration of phosphoinositide-dependent recruitment and
regulation by TOR may not be limited to Slm1 and Slm2.
Our analysis has successfully identified two novel proteins
that function as essential regulators of actin polarity downstream of PI4,5P2. We speculate that under conditions of cell
stress such as heat shock, elevated PI4,5P2 levels result in the
increased recruitment and/or activation of effector proteins
including Slm1, Slm2, and Rom2, all necessary for regulation
of actin cytoskeleton organization. Moreover, to ensure both
rapid and specific responses to cell stress, additional factors
are necessary. In the case of PI4,5P2-dependent Rom2 recruitment/activation to the cell cortex, the integral plasma membrane cell surface sensors, Wsc1 and Mid2, appear to serve
this function (Audhya and Emr, 2002). Correspondingly, we
have found that PI4,5P2-dependent Slm1 and Slm2 recruitment requires the parallel activity of the TORC2 protein
kinase complex, potentially also dependent on conditions of
nutrient availability. Interestingly, following heat shock, the
levels of Slm1 and Slm2 phosphorylation were diminished,
likely due to reduced TORC2 activity and/or elevated protein
phosphatase activity. This decreased level of Slm1 and Slm2
phosphorylation may be important for appropriate depolarization of the actin cytoskeleton and secretion observed
following cell stress. Strikingly, phosphorylation of Slm1
and Slm2 recovered in parallel to actin repolarization, suggesting a link between Slm1 and Slm2 activity and actin
cytoskeleton organization. Collectively, these data indicate
that PI4,5P2 functions together with both positive and negative control elements in a cellular stress response system
necessary for organizing the actin cytoskeleton.
Materials and methods
Strains and media
Enzymes used for recombinant DNA techniques were used as
recommended by the manufacturer. Standard recombinant DNA
techniques and yeast genetics methods were performed, and growth
media used have been described elsewhere (Maniatis et al, 1982;
Gaynor et al, 1994). S. cerevisiae strains used in this study are listed
in Table III. All gene disruptions and temperature-sensitive mutant
strains were generated similarly, as described previously (Audhya
and Emr, 2002). Integrated epitope tagging was performed as
described previously (Longtine et al, 1998).
Synthetic genetic array analysis and yeast plasmids
SGA analysis was performed as described previously (Tong et al,
2001). Strains expressing temperature conditional alleles of slm1
were generated similarly as described using error-prone PCR
(Audhya et al, 2000). All point mutations were created using sitedirected mutagenesis.
Metabolic labeling and immunoprecipitation
Cell labeling and immunoprecipitations were performed as described previously (Gaynor et al, 1994; Audhya et al, 2000, 2002).
For 32P labeling, cells were grown in low phosphate media to early
log phase and labeled with 1 mCi 32P-labeled orthophosphate in the
absence of phosphate. Treatment with shrimp alkaline phosphatase
and immunoprecipitation with antiserum against GFP have been
described previously (Audhya and Emr, 2003).
The EMBO Journal
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PI4,5P2 and TORC2 regulate actin organization
A Audhya et al
Table III S. cerevisiae strains used in this study
Strain
Genotype
Reference or source
SEY6210
SEY6210.1
AAY202
SW80-1D
AAY1602
AAY1608
AAY1610
AAY1622
AAY1623
AAY1624
AAY1625
AAY1626
AAY1627
AAY1628
AAY1629
AAY1630
AAY1633
AAY1635
AAY1651
AAY1652
AAY1661
AAY1663
AAY1671
AAY1674
AAY1681
AAY1683
AAY1692
AAY1701
AAY1709
AAY1804
MATa leu2-3,112 ura3-52 his3-D200 trp1-D901 lys2-801 suc2-D9
MATa leu2-3,112 ura3-52 his3-D200 trp1-D901 lys2-801 suc2-D9
SEY6210; mss4DHHIS3MX6 carrying Ycplacmss4-102 (LEU2 CEN6 mss4-102)
MATa leu2 ura3 rme1 trp1 his3D [KANMX] GAL1pHTOR2 AVO2-TAP:HISMX
SEY6210; slm1DHHIS3
AAY202; slm1DHHIS3
SEY6210; slm2DHHIS3
SEY6120; slm1DHHIS3 slm2DHHIS3 carrying pRS415slm1-1 (LEU2 CEN6 slm1-1)
SEY6120; slm1DHHIS3 slm2DHHIS3 carrying pRS415slm1-2 (LEU2 CEN6 slm1-2)
SEY6120; slm1DHHIS3 slm2DHHIS3 carrying pRS415slm1-3 (LEU2 CEN6 slm1-3)
SEY6120; slm1DHHIS3 slm2DHHIS3 carrying pRS415slm1-4 (LEU2 CEN6 slm1-4)
SEY6120; slm1DHHIS3 slm2DHHIS3 carrying pRS415slm1-5 (LEU2 CEN6 slm1-5)
SEY6210; SLM1-GFP:HIS3MX6
SEY6210; SLM2-GFP:HIS3MX6
AAY1602; SLM2-GFP:HIS3MX6
AAY1610; SLM1-GFP:HIS3MX6
SEY6120; slm1DHHIS3 slm2DHHIS3 carrying pRS415slm1KA (LEU2 CEN6 slm1KA)
AAY202; SLM1-GFP:HIS3MX6
SEY6210; GFP-SNC1:URA3
AAY1622; GFP-SNC1:URA3
AAY1622; sac7DHHIS3
SEY6210; slm1DHHIS3 slm2DHHIS3 sac7DHHIS3
SEY6210; AVO2-13MYC:HIS3MX6
AAY1627; AVO2-13MYC:HISMX6
SEY6210; avo2DHHIS3
AAY202; avo2DHHIS3
AAY1628; slm1DHHIS3
SEY6210; ABP1-RFPHHIS3
SEY6210; ABP140-GFPHHIS3
AAY1622; sec4-8
Robinson et al (1988)
Robinson et al (1988)
Stefan et al (2002)
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Fluorescence microscopy
All fluorescence images were observed using a Zeiss Axiovert
S1002TV fluorescent microscope and subsequently processed using
a Delta Vision deconvolution system. For actin localization, cells
were fixed and stained with rhodamine–phalloidin (Molecular
Probes) as described previously (Audhya et al, 2000). To
depolymerize F-actin, latrunculin A was added from a 10 mM
DMSO stock to a final concentration of 200 mM for 30 min.
Tandem affinity purification and mass spectrometric analysis
TAP of Slm1 was performed as described previously (Cheeseman
et al, 2002). Proteins in the SDS gel were stained by silver and
protein bands were excised and digested by trypsin overnight.
Peptides were extracted and analyzed by microcapillary liquid
chromotography and tandem mass spectrometry. An in-house MS
system consisting of an HPLC and LCQ-ion trap mass spectrometer
(Thermo Finnigan) was used. All identified peptides were further
manually inspected and verified.
TORC2 purification and in vitro kinase assay
SW80-1D cells (5 l) expressing either vector alone, HA-TOR2, or
a kinase-inactive form of HA-TOR denoted HA-TOR2KD (Jiang and
Broach, 1999) pregrown to saturation in synthetic raffinose/glycerol
medium were additionally grown at 301C for 16 h in media
containing glucose. Cells were lysed as described previously
(Loewith et al, 2002). Pooled lysates were cleared, normalized to
B50 ml and B375 mg protein and passed over 125 ml of Sepharose
CL-4B (Sigma) previously equilibrated in lysis buffer. To the flow
through was added 150 ml IgG-Sepharose (Amersham Bioscience)
for 2 h. IgG beads were then incubated with either B5 mg GST,
B2 mg GST-Slm2, B2 mg GST-Slm1, or 2 mg PHAS-I (Stratagene) in
50 ml of lysis buffer with 20% glycerol. In all, 6 ml of 10 buffer
(40 mM MnCl2, 100 mM dithiothreitol, 10 Roche protease
inhibitor cocktail–EDTA, 100 mM NaN3, 100 mM NaF, 100 mM pnitrophenylphosphate, 100 mM b-glycerophosphate) was added
before the reaction was started with the addition of 4 ml of ATP
mix (1.2 mM ATP, 2.5 mCi/ml [g32P]ATP (3000 Ci/mmol) in kinase
assay buffer). Samples were then subjected to SDS–PAGE.
Supplementary data
Supplementary data are available at The EMBO Journal Online.
Acknowledgements
We are grateful to Drs Arshad Desai, Mark Lemmon, and Beth
Weaver for many helpful suggestions on this manuscript. Also, we
thank Stephan Wullschleger for strains and Wolfgang Oppliger and
Iain Cheeseman for technical assistance. AA is currently supported
by a Helen Hay Whitney postdoctoral fellowship at the Ludwig
Institute for Cancer Research. RL was the recipient of an EMBO
long-term fellowship, and ABP holds a Natural Sciences and
Engineering Research Council of Canada graduate student fellowship. This work was partially supported by grant CA58689 from the
National Institutes of Health (to SDE), grants from the Canton of
Basel and the Swiss National Science Foundation (to MNH), and
grants from the Canadian Institute of Health Research and Genome
Ontario (to CB). SDE is an investigator of the Howard Hughes
Medical Institute.
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The EMBO Journal
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