Department of Pharmacology, Cornell University, Ithaca, New York 14853
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The Rho-type GTPase Cdc42p has been implicated in diverse cellular functions including cell shape, cell motility, and cytokinesis, all of which involve the reorganization of the actin cytoskeleton. Targets of Cdc42p that interface the actin cytoskeleton are likely candidates for mediating cellular activities. In this report, we identify and characterize a yeast homologue for the mammalian IQGAP, a cytoskeletal target for Cdc42p. The yeast IQGAP homologue, designated Iqg1p, displays a two-hybrid interaction with activated Cdc42p and coimmunoprecipitates with actin filaments. Deletion of IQG1 results in a temperature-sensitive lethality and causes aberrant morphologies including elongated and round multinucleated cells. This together with its localization at the mother-bud neck, suggest that Iqg1p promotes budding and cytokinesis. At restrictive temperatures, the vacuoles of the mutant cells enlarge and vesicles accumulate in the bud. Interestingly, Iqg1p shows two-hybrid interactions with the ankyrin repeat-containing protein, Akr1p (Kao, L.-R., J. Peterson, J. Ruiru, L. Bender, and A. Bender. 1996. Mol. Cell. Biol. 16:168-178), which inhibits pheromone signaling and appears to promote cytokinesis and/or trafficking. We also show two-hybrid interactions between Iqg1p and Afr1p, a septin-binding protein involved in projection formation (Konopka, J.B., C. DeMattei, and C. Davis. 1995. Mol. Cell. Biol. 15:723-730). We propose that Iqg1p acts as a scaffold to recruit and localize a protein complex involved in actin-based cellular functions and thus mediates the regulatory effects of Cdc42p on the actin cytoskeleton.
Key words: IQGAP; IQG1; Cdc42; morphogenesis; cytoskeleton ![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE Rho-type GTPase Cdc42p is structurally and
functionally conserved from yeast to mammals. It
has been implicated in a variety of fundamental cellular activities ranging from cytoskeletal organization (Ridley et al., 1995) to transcriptional activation (Bagrodia et al., 1995
; Coso et al., 1995
), cell proliferation (Olson et al., 1995
; Qiu et al., 1995
), intracellular trafficking (Singer et al., 1995
; Erickson et al., 1996
), and AIDS etiology
(Sawai et al., 1996
). Thus, Cdc42p may require a number
of regulatory factors as well as target molecules to mediate
its diverse cellular functions.
Like all members of the Ras superfamily, Cdc42p cycles
between an inactive GDP-bound state and an active GTP-bound state at rates defined by specific regulatory proteins. These include members of the Dbl family of oncoproteins, which serve as guanine nucleotide exchange
factors that stimulate GTP-GDP exchange (Cerione and Zheng, 1996), the Cdc42-GTPase-activating proteins
(GAPs)1 and other members of the Rho-GAP family
(Barfod et al., 1993
; Zheng et al., 1993
; Lamarche and
Hall, 1994
), and the Rho-GDP dissociation inhibitors
(Rho-GDI) (Leonard et al., 1992
; Koch et al., 1997
).
The GTP-bound form of Cdc42p interacts with a number of different target/effectors and initiates downstream
signaling cascades that result in biological responses.
Among the best-known targets are members of the family
of p21-activated serine/threonine kinases (Paks) that are
stimulated upon the binding of activated Cdc42p or Rac,
and are thought to initiate signaling pathways that lead to
the nucleus and the activation of two stress-responsive nuclear Map kinases, the c-Jun kinase (JNK1) and p38 (Bagrodia et al., 1995; Coso et al., 1995
; Minden et al., 1995
;
Nobes and Hall, 1995
). More recently, various other putative targets for Cdc42p have been identified and proposed
as possible interfaces between Cdc42p and the actin cytoskeleton including WASP (Symons et al., 1996
) and the
IQGAPs (Brill et al., 1996
; Hart et al., 1996
; McCallum et al.,
1996
; Erickson et al., 1997
).
The IQGAPs are especially interesting because they
contain a number of different motifs, suggesting that these
molecules may function as signaling scaffolds. These include a RasGAP homology domain that appears to contain the binding site for Cdc42p, a calponin homology domain (CHD) that most likely accounts for the binding of
IQGAP to F-actin (Bashour et al., 1997; Erickson et al.,
1997
), and IQ motifs implicated in binding calmodulin.
The binding of calmodulin to IQGAP has been suggested
to weaken its affinity for F-actin (Bashour et al., 1997
) and
Cdc42p (Joyal et al., 1997
).
Another interesting feature of the IQGAPs is that we
have recently found that they are localized to the Golgi, as
well as the plasma membrane and cytosol in mammalian
cells (McCallum et al., 1998). The Golgi localization may
reflect the presence of Cdc42p, which we earlier showed
was predominantly present in the Golgi membranes of
most cells, and whose cellular localization was influenced
by the Arf GTPase (Erickson et al., 1996
). This, taken together with the fact that Cdc42p can act with Arf to synergistically activate the Golgi membrane-associated phospholipase D (Brown et al., 1993
), raises the possibility that
in addition to influencing the cytoskeletal architecture and
events in the nucleus, Cdc42p may participate in some aspect of intracellular trafficking.
Cdc42p may also play a role in cytokinesis (Dutartre et al.,
1996). Transfection of dominant-active Cdc42p produces
giant multinucleated cells defective in cytokinesis after the
reorganization of F-actin. Targets for Cdc42p that interface actin microfilaments may mediate this role in cytokinesis and related processes involving the reorganization of
the actin cytoskeleton. IQGAP is especially well suited for
this role because, aside from its multidomain feature, it
also appears to be a predominant target/effector for Cdc42p in most cells (Erickson et al., 1997
). An understanding of its function and regulation could provide important insights into the biological actions of Cdc42p.
However, thus far it has been difficult to study IQGAP
function in mammalian cells and so we have turned to
yeast, where a number of genetic studies have already
been applied to Cdc42p and its regulators and targets (Adams et al., 1990
; Johnson and Pringle, 1990
; Chant and
Herskowitz, 1991
; Ziman et al., 1993
; Stevenson et al.,
1995
; Evangelista, 1997). In this report, we characterize
the yeast homologue of IQGAP, designated Iqg1p, and
present evidence suggesting that it provides a link between
Cdc42p and pathways involved in yeast cell polarity, morphogenesis, and cytokinesis.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Strains, Media, and Genetic Manipulations
The Escherichia coli strain used in this study for routine cloning was
DH5 (GIBCO BRL, Gaithersburg, MD). Yeast strains used in this study
are listed in Table I; plasmids used are listed in Table II. Yeast growth,
media, and genetic techniques were performed as described in Guthrie
and Fink (1991)
. Yeast transformation was done using a modified lithium
acetate method (Elble, 1992
).
|
|
Cloning and Strain Construction
To construct the IQG1 deletion strain, a method described by Baudin et al.
(1993) was followed. Briefly, two primers were designed (DEL5: 5'-GCTAGCAACAGTTCTGCGACAATTTTGTCAAAAAAAGTAGAAAGTTCC-GCTCTTGGCCTCCTCTAG-3', and DEL3: 5'-GCTTTGTGTTCCATT-TAAACTTCATTCCCTGCAATTCAGAACGTTCTCTTCTCGTTCAGAATGACACG-3'). Each contained sequences for deleting most of the
IQG1 gene by homologous recombination followed by sequences for amplification of the HIS3 gene as a selectable marker. The plasmid YDp was
used to amplify the HIS3 gene and the product (Iqg1
::HIS3) was purified from agarose gel, transformed into the MO1 diploid strain, and then selected on plates lacking histidine. The resultant heterozygous diploid was
sporulated and the tetrads were dissected and analyzed. The tetrads
showed 2:2 segregation of the His+:His
marker at room temperature. To
confirm the IQG1 deletion in the stable His+ colonies, genomic DNA was
prepared from both His+ (including an a [MO2] and an
[MO3] colony) and
His
colonies and analyzed by PCR using primers for the flanking sequences of IQG1 (YQ5: 5'-ATGACAGCATATTCAGGCTCTCCTTCG-3' and
YQ3: 5'-TTACAAAG CGTTCCTTTTATAG-3'). As expected, genomic
DNA from three independent His+ colonies yielded a 1.6-kb fragment
corresponding to the marker gene plus some sequences of the IQG1 ORF.
The His
colonies produced a 4.5-kb fragment corresponding to the IQG1 ORF. The confirmed a and
IQG1-deleted strains were mated and zygotes were selected to produce a homozygous diploid (MO4). The primers, YQ5 and YQ3, were used in combination with different restriction
site sequences to clone the IQG1 gene into the plasmids listed in Table II.
The IQG1 contained within these plasmids fully complements the phenotypes of the His+ colonies. To construct double mutants of IQG1 and each
of AKR1 and AFR1, the iqg1::HIS3-deleting fragment was transformed
into the haploid strains Y975 (akr-
1), 211-5-3 (AFR1 wild-type), and JK26 (afr1::URA3) and double mutants were selected on plates lacking histidine and uracil. To construct afr1
akr1-1 and afr1
akr1
1 double
mutants, the afr1::URA3 fragment was isolated from the pJK38 plasmid
by digesting with BamHI and SalI and transformed into both akr1-1 and
akr1
1 haploid strains to delete the AFR1 gene and double mutants were
selected on plates lacking uracil.
Factor Arrest and Halo Assays
Haploid cells were grown to early log phase, factor was added to 4 µg/ml,
and growth was continued for 3 h. Cells were fixed directly in the growth
medium by adding 37% formaldehyde to 3.7% and incubated for another
3 h; the cells were then briefly sonicated and processed for immunofluorescence or visualized by light microscopy. For halo assays, 15 µl of 40 µg/ml
factor were spotted on sterile filter disks placed on lawns of cells, plates
were incubated for 3 d at room temperature, and then photographed.
Indirect Immunofluorescence
Formaldehyde-fixed diploid (MO1 and MO4) cells were processed by procedures previously described (Kilmartin and Adams, 1984; Ziman et al.,
1993
). For Cdc42p immunofluorescence, an antibody sandwich technique
was used as described in Ziman et al. (1993)
; affinity-purified anti-Cdc42p
(1:50) was followed by AffiniPure goat anti-rabbit (1:1,000), rabbit anti-
goat (1:1,000), and rhodamine-labeled goat anti-rabbit (1:80) antibodies.
AffiniPure secondary antibodies were obtained from Jackson Immuno
Research Laboratories (West Grove, PA). Affinity-purified anti-calmodulin antibody (1:200) was a gift from Dr. T. Davis (Washington University,
St. Louis, MO) and was used according to the procedure published in
Brockerhoff and Davis (1992)
. For Iqg1p localization, pA1 plasmid (HA-IQG1), which fully complements the iqg1
cell phenotypes, was transformed into both wild-type and iqg1
diploid cells. Transformants growing in medium lacking leucine were stained using the above-mentioned sandwiching technique and anti-hemagglutinin (HA) mAb (1:50) 12CA5 (Berkeley Antibody Corp., Richmond, CA). To stain for tubulin, rat anti-
yeast tubulin antibodies (Yol1/34; a gift from Dr. T. Huffacker, Cornell
University, Ithaca, NY) and FITC goat anti-rat IgG antibodies were used.
Coimmunoprecipitation of Iqg1p and Actin
A modification of the procedure described in Erickson et al. (1997) was
used to detect immune complexes containing Iqg1p and F-actin. Cells
transformed with HA-tagged IQG1 (plasmid pA1) were grown on selective media to an OD600 of 0.8, pelleted, washed in cold lysis buffer (50 mM
Tris-HCl, pH 7.5, 1 mM EDTA, 0.1% Triton X-100, 5% glycerol), and
then resuspended in the same buffer containing 20 µl of Fungal Protease
Inhibitor Cocktail (Sigma Chemical Co., St. Louis, MO). Cold acid-washed glass beads were added and the suspension was vortexed (four
times) at 4°C (1 min each with 2-min intervals of cooling). The supernatant was collected by centrifugation and 0.5 ml was added to 1 µg of anti-HA polyclonal antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz,
CA), together with 25 µM phalloidin, and then incubated at 4°C overnight
with rocking. 20 µl of protein A-Sepharose beads equilibrated in the same
buffer were added to the immune complexes and incubated at 4°C for 1 h
with rocking. The beads were collected by centrifugation at 14,000 g for 20 s
and extensively washed (four times) with 1 ml lysis buffer and resuspended in 2× SDS sample buffer, boiled for 5 min, and then loaded onto a
10% SDS-polyacrylamide gel. Western blotting was carried out as described (Ausubel et al., 1992
), and the filters were stained with either anti-
yeast actin antibodies (1:2,000; a gift from Dr. T. Bretscher, Cornell University) or with a polyclonal anti-HA probe (Santa Cruz Biotechnology, Inc.).
Fluorescence Microscopy
Nuclear DNA staining was performed with ~2 × 107 cells, which were
grown at log phase, harvested, fixed in 70% ethanol, and then stained with
DAPI (4',6'-diamidino-2-phenylindole) according to Futcher (1993).
When staining actin with phalloidin, cells were fixed in the growth media
by the addition of 37% formaldehyde solution to 3.7% final concentration
for 3 h. Staining with rhodamine-conjugated phalloidin (Molecular
Probes, Eugene, OR) was carried out as described by Adams and Pringle
(1991)
. To visualize chitin deposition on the cell wall, cells were collected
and stained with Calcofluor (Fluorescent Brightener; Sigma Chemical
Co.) as described by Pringle (1991)
.
Flow Cytometry
Yeast cells were grown to early log phase (OD600 = 0.3) at 23°C and incubated at 23°, 30°, and 37°C for 4 h. Cells were stained with propidium iodide as described by Futcher (1993) and analyzed with the FACS® in the
Cornell Biotechnology Flow Cytometry and Imaging Facility.
Two-Hybrid Analysis
PCR-amplified IQG1 gene was cloned, in frame, into pBTM116 and
pACT2 to produce LexA- and Gal4-Iqg1 fusion proteins respectively for
two-hybrid analysis. These plasmids complement the phenotypes of the
IQG1 deletion strain. Cdc42 fusion proteins were previously described
(Stevenson et al., 1995). Plasmids were cotransformed into the CTY10-5D
strain and transformants were selected on medium lacking tryptophan,
histidine, and uracil. To detect interactions with Afr1p and Akr1p, plasmids pLAF and pPB659, respectively, were used for cotransformation of
L40 strain with pA1 (pACT2-IQG1) and transformants were selected on
medium lacking histidine, tryptophan, and leucine. Colonies thus isolated
were cultured into liquid medium lacking the same amino acids to OD600
of 1.0.
-Galactosidase assays were performed according to the BIO101
protocol.
Electron Microscopy
Exponentially growing cells at 30°C were shifted to 37°C for 2 h and directly fixed in glutaraldehyde. Cells were then prepared for electron microscopy using permanganate fixation as detailed in Kaiser and Schekman
(1990), then processed and sectioned in the Cornell Integrated Microscopy Center (CIMC).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The Existence of a Saccharomyces cerevisiae Homologue, IQG1, for the Mammalian IQGAPs
A search of the yeast Saccharomyces cerevisiae genome
database revealed a gene (these sequence data are available from GenBank/EMBL/DDBJ under accession number Z67751) predicted to encode a protein homologous to
the mammalian IQGAPs. We have cloned this homologue and designated it Iqg1p. Alignment (Fig. 1) of the predicted protein sequence for Iqg1 with the mammalian
IQGAP1 protein revealed significant homology over the
entire coding region, with most of the sequence motifs
from the mammalian IQGAPs being conserved within
Iqg1p (Fig. 1 A). These include the region of homology
with RasGAP (Fig. 1 C), which appears to be responsible
for high affinity binding to the human Cdc42p (Cdc42Hs)
but not Ras, the CHD (Fig. 1 A), a putative actin-binding
site, and IQ motifs that represent potential calmodulin-binding sites. The WW motif, which is present in the mammalian IQGAPs and represents a potential binding site for
proline-rich regions (Sudol et al., 1995), appears to be absent
or less conserved in Iqg1p. The IQG1 sequence was assigned
GenBank/EMBL/DDBJ accession number AF019644.
|
Two-Hybrid Interactions between Iqg1p and the Saccharomyces cerevisiae Cdc42p
Given that Iqg1p contains the RasGAP homology domain,
which appears to be responsible for the binding of mammalian IQGAP to activated Cdc42Hs, we used the two-
hybrid analysis (Fields and Song, 1989; Gyuris et al., 1993
)
to detect interactions between Iqg1p and Cdc42p. The results in Table III show that a significant interaction was
detected between the Iqg1 molecule and the GTPase- defective yeast Cdc42p (Cdc42pG12V,C188S), which was also
mutated at position 188 to prevent membrane localization
and thus facilitate the nuclear localization of the GTP-binding protein. A similar interaction was observed between another dominant-active, GTPase-defective Cdc42p
mutant (Cdc42pQ61L,C188S) and Iqg1p. In contrast, neither
the dominant-negative Cdc42 mutant (Cdc42pD118A, C188S)
nor the Cdc42pC188S showed significant interactions with
Iqg1p compared with the activated forms.
|
Iqg1p Concentrates at Sites of Yeast Cell Growth and at the Septum
As an initial characterization of Iqg1p, we used indirect
immunofluorescence to determine its localization in yeast
cells. The distribution of Iqg1p in an asynchronous cell culture appeared to vary with the cell cycle. In cells that have
not yet undergone a complete budding event, the Iqg1p localized as a patch at the region of the cell from which the
bud was emerging (Fig. 2 A). In cells with smaller buds,
Iqg1p appears more diffuse and is located throughout the
bud. Cells with larger buds, which have presumably completed mitosis, show that Iqg1p is localized at the neck between the mother and daughter cells at cytokinesis (Fig. 2
B). In the majority of the round unbudded cells, the distribution of Iqg1p appeared as punctate and/or diffuse (Fig. 2
C). This pattern was not observed in cells transformed
with vector alone. Overall, the localization of Iqg1p to the
bud site and to the septum is similar to what has been reported for Cdc42p (also, see below) and proteins involved
in cytokinesis (Kim et al., 1991; Brockerhoff and Davis,
1992
; Ziman et al., 1993
; Konopka et al., 1995
).
|
Deletion of IQG1 Affects Yeast Cell Growth and Polarity
To begin to examine the in vivo function of Iqg1p, homologous recombination was used to replace one copy of
IQG1 amino acids 38-1,382 in a wild-type diploid yeast
strain as described in Materials and Methods. Tetrad analysis revealed four viable spores per tetrad at room temperature. The His+:His marker segregated 2:2 indicating that
Iqg1p is not essential at least in this strain background.
Growth of the diploid (MO1) and the homozygous diploid
iqg1
::HIS3 (MO4) were examined at different temperatures ranging from 18° to 37°C. At 18°C, the deletion strain
grew on plates similar to wild-type strains (data not
shown). At 30°C, the deletion strain culture grew more
slowly than the corresponding wild-type strain but was unable to grow at 37°C indicating that the deletion of IQG1
is temperature sensitive.
iqg1 cells grown at 23°, 30°, and 37°C were examined
by light microscopy. At 23° and 30°C, the cells did not exhibit a uniform phenotype in liquid culture. Approximately 5% of the cells appeared normal, such that their
size was comparable to those of the isogenic wild-type
strain. However, the majority of the iqg1
cells were typically larger in size, rounder in shape, or elongated (Fig. 3
A, b-g; also see Figs. 4 E, 5 D, 8 B, and 9 C, below) and defective in budding. In some cases at 30°C, an elongated
bud, tubular in shape, was attached to a large mother cell
(Fig. 3 A, b). A fraction of the cells appeared amorphous.
|
|
|
|
|
After 2 h at 37°C, the majority of the iqg1 cells were
large and round with a large vacuole that occupied nearly
the entire volume of the cell (Fig. 3 C). Some of the iqg1
cells at 37°C still exhibited an elongated cell phenotype
similar to that caused by mutations in proteins involved in
cytokinesis (Haarer and Pringle, 1987
; Ford and Pringle,
1991
; Kim et al., 1991
; Field et al., 1996
; Longtine et al.,
1996
). As the incubation continued over 3 h at 37°C, the
majority of the cells appeared amorphous or lysed.
To further examine the issue of polarity in iqg1 cells,
haploid iqg1
cells (MO2) and their isogenic wild-type
cells were treated with
factor. Under conditions where
wild-type cells were arrested at G1 (Fig. 3 D), a small population of iqg1
cells formed at least two projections,
whereas the majority of the mutant cells were extensively
elongated or misformed (Fig. 3, E-G; also see Figs. 4 B
and 7 B). Halo assays (Fig. 3 H) confirmed that iqg1 cells
are more sensitive to pheromone than their respective wild-type cells. Thus, the machinery for polarized growth
appears to be hyperactive or otherwise deregulated in the
absence of Iqg1p. This phenotype was rescued by the pA1
plasmid encoding an HA-Iqg1 fusion protein.
|
Localization of Cdc42p in iqg1 Cells
Because Iqg1p is a putative cytoskeletal target for Cdc42p,
we examined the cellular localization of Cdc42p in iqg1
cells. In wild-type cells treated with
factor, Cdc42p is localized to the tip of the shmoo (Fig. 4 A), whereas in budding cells, Cdc42p is found in the bud neck, along the sides
of the bud, and in the plasma membrane (Fig. 4 C) as previously reported (Ziman et al., 1993
). In the isogenic iqg1
cells, Cdc42p was located throughout the
factor-treated
cells (Fig. 4 B), and is found primarily in the plasma membrane but not in the necks of budded cells (Fig. 4 D). However, in iqg1
cells that exhibited the elongated bud (Fig. 4
F), Cdc42p localized to ridges along the bud as well as at
the neck between the mother-daughter cells. The Cdc42p
localization in these long buds resembles that of actin and
chitin in iqg1
cells (see below).
Iqg1p Coprecipitates with F-Actin and Is Essential for Proper Actin Filament Localization
Because the predicted protein sequence of Iqg1 harbors a
potential binding site for F-actin in its NH2 terminus similar to that found in -actinin and filamin (Lebart et al.,
1994
; Castresana and Saraste, 1995
), we examined both
the ability of Iqg1p to interact with actin and the organization of actin filaments in iqg1
cells. Under non-permissive conditions, actin mutants arrest as unbudded cells and
enlarge uniformly without directing material to the bud
(Drubin, 1990
). Similarly, cdc42 mutants grow isotropically and delocalize actin filaments (Adams et al., 1990
; Ziman et al., 1991
). We examined the organization of the actin filaments in iqg1
cells using rhodamine-phalloidin
staining. As expected, actin patches in wild-type cells are
concentrated in the small bud and at the tip of the cells
during polarized growth (Fig. 5 A), and at the septum during cytokinesis. By contrast, actin filaments in iqg1
cells
appear to be randomly distributed throughout the mother
cell and the bud (Fig. 5, B and C). However, in iqg1
cells
that exhibit extremely elongated buds, actin patches, while still randomly scattered in the large mother cell, appear to
be concentrated at ridges along the elongated bud (Fig. 5
D). We propose that these ridges represent presumptive
sites for the septa in aborted separation of the mother and
daughter cells. Despite the apparent correct localization of
actin and other cell materials such as chitin (see below)
and Cdc42p (above) at these presumptive septa locations,
the buds grew as tubular projections and showed no obvious constrictions. This phenotype is similar to that observed for mutations in proteins implicated in cytokinesis (Holtzman et al., 1993
; Konopka, 1993
; Bi and Pringle,
1996
; Kao, et al., 1996), raising the possibility that Iqg1p is
involved in some of the early steps of cytokinesis and is required for the polarized distribution of actin filaments during cell growth.
To examine whether Iqg1p interacts with actin in vitro,
an HA-tagged Iqg1p was immunoprecipitated from total
yeast cell lysates and then the resuspended precipitate was
Western blotted to detect F-actin. As shown in Fig. 6,
F-actin coprecipitates with Iqg1p (compare lanes 1 and 2) in
the presence of phalloidin, which induces a net increase in
polymerized actin filaments (Estes et al., 1981). Beads incubated with HA-tagged Iqg1p in total cell lysates and phalloidin did not retain an F-actin band (Fig. 6, lane 3). Similarly, expression of the HA tag without Iqg1p did not precipitate
an F-actin band. Fig. 6, lane 4 shows actin in total cell lysates. Thus, as reported for the mammalian IQGAP (Bashour et
al., 1997
; Erickson et al., 1997
), Iqg1p also appears to interact with F-actin.
|
Calmodulin Is Delocalized in iqg1 Cells
Calmodulin is involved in bud growth, cytokinesis, and
chromosome segregation (Davis, 1992) and localizes to
sites of cell growth similar to the polarity establishment
proteins and overlaps actin (Brockerhoff and Davis, 1992
).
The mammalian IQGAP binds calmodulin (Brill et al.,
1996
; Bashour et al., 1997
; Joyal et al., 1997
), which modulates the interactions of IQGAP with both F-actin and
Cdc42Hs (Joyal et al., 1997
). Because Iqg1p contains at
least four IQ motifs and thus is likely to bind calmodulin, we examined whether the localization of calmodulin was
affected in iqg1
cells using indirect immunofluorescence
in
factor-arrested cells. In wild-type cells, calmodulin
concentrated at the tip of the forming shmoo (Fig. 7 A) as
previously described (Brockerhoff and Davis, 1992
). In
isogenic iqg1
haploid cells, calmodulin was located
throughout the mis-shapen cell (Fig. 7 B). These results suggest that Iqg1p is involved in mediating the correct localization of calmodulin at growth sites.
Chitin Is Delocalized in iqg1 Cells
In S. cerevisiae, chitin is essential for cell growth and is localized at the incipient bud site, bud neck, and bud scars (Bulawa, 1993). During cytokinesis, chitin is localized to
the primary septum between the mother and daughter
cells. Polarity establishment proteins participate in the organization of chitin in the cell wall. Mutations in cdc42
(Adams et al., 1990
) cause actin delocalization as well as
affect chitin deposition. However, how these effects are
mediated by Cdc42p is not well understood. Because
Iqg1p is a putative target for Cdc42p, we examined
whether the absence of Iqg1p affects chitin deposition.
Calcofluor staining of an asynchronous cell culture showed
that in wild-type cells, chitin is correctly deposited at the
opposite poles, and at the incipient bud site, the septum,
and the lateral wall (Fig. 8 A). In some iqg1
cells (~5%),
chitin appeared to be correctly deposited. However, more
typically, chitin was found over the entire surface of the
iqg1
cells (Fig. 8, B and C), yielding a similar phenotype to what has been observed for mutants defective in actin
(Novick and Botstein, 1985
) and in mutants that affect actin function (Liu and Bretscher, 1992
). In large iqg1
cells
bearing elongated buds, chitin was localized throughout
the cell wall, but was concentrated at sites such as the presumptive septa and some bud scars (Fig. 8 C). These results suggest that Iqg1p is required for directed deposition
of chitin in the cell wall.
iqg1 Cells Accumulate Nuclei
Mutations in cdc42 (Adams et al., 1990; Hart et al., 1996
)
and in other proteins that influence the actin cytoskeleton
accumulate nuclei to varying degrees (Holtzman et al.,
1993
; Bi and Pringle, 1996
; Li, 1997
). When stained with
DAPI, as described in Materials and Methods, iqg1
cells
also accumulate nuclei. At 23°C, 20% of the cells appeared
to be bi- or multinucleated. This was especially clear in the
elongated cells (compare Fig. 9, B and D). However, the
majority of the cells appeared to have masses of DNA,
which was difficult to score as bi-nucleate or multinucleate. Many of the rounded cells had a mass of DNA either
at one or both sides of the cell (Fig. 9, E and F). Therefore,
we suspected that 20% (n = 200) may represent an underestimation of the number of multinucleated iqg1
cells. To
more clearly delineate the amount of DNA contained in
these cells, we used flow cytometry to measure the DNA
content of individual iqg1
cells at various temperatures.
As shown in Fig. 10, wild-type cells contained both 1C and
2C DNA peaks, whereas iqg1
cells contained only 2C
and 4C DNA at all temperatures tested. We obtained
identical results comparing haploid or diploid strains. One
possible explanation for these results is that the iqg1
cells have all diploidized. However, because we can detect cells
with multiple nuclei (Fig. 9), we suspect that the results
shown in Fig. 10 may reflect a situation where DNA replication and nuclear division continue in these cells, but
both budding and cytokinesis are blocked in the absence
of Iqg1p, thereby causing the cells to appear polyploidal.
To examine whether tubulin orientation was affected in
iqg1
cells, we performed immunofluorescence studies with anti-tubulin antibodies. The spindles appeared to be
fully extended but curved along or across the cell axis and
were typically observed along one periphery of the cell,
thus demonstrating that they were misoriented in the absence of Iqg1p (compare Fig. 9, G [wild-type] and H [mutant]). This finding resembles that of actin mutations and
points to a possible role for Iqg1p in organizing the actin
cytoskeleton.
|
Genetic and Physical Interactions between Iqg1p and Proteins Involved in Cytokinesis
Because the phenotypes of iqg1 cells, namely the elongated cells and the accumulation of nuclei, appeared to resemble those of many mutants involved in cytokinesis, we
reasoned that Iqg1p may be part of a complex involved in
organizing the actin cytoskeleton during cell cycle progression. One such candidate, Akr1p, contains six ankyrin repeats suggesting that it is a cytoskeletal protein. The deletion of AKR1 is conditionally lethal and at restrictive temperatures produces an elongated cell phenotype (Kao
et al., 1996
) reminiscent of the IQG1 deletion. By contrast,
the deletion of AFR1 has no discernible phenotypic effect
but the overexpression of Afr1p produces a phenotype
(Konopka, 1993
) similar to that caused by iqg1
::HIS3,
akr1
-1, and septin mutations. Thus, Afr1p may also act to
regulate the functions of Iqg1p and Akr1p, similar to its
proposed actions on the septins (Konopka et al., 1995
). In
addition, Afr1p localizes to the neck similar to Iqg1p and interacts with Cdc12p (Konopka et al., 1995
), a septin involved in neck filament formation (Haarer and Pringle,
1987
), thus providing an intimate link to cytokinesis. We
examined the possible interactions between these two proteins and Iqg1p using double mutant and two-hybrid analyses. We transformed the iqg1
::HIS3 deleting fragment
into both JK26 (afr1
) and JK211-5-3 (AFR1) strains. No
transformants were recovered from the afr1
strain, whereas
a high number of transformants was recovered from its parental wild-type strain. Thus, the double deletion of IQG1
and AFR1 appears to result in synthetic lethality at room
temperature, suggesting a physical interaction between
these two proteins that we then confirmed by the two-
hybrid system (Table IV). The double mutation of iqg1
and akr1
-1 resulted in slower growth and an enhanced
cytokinetic defect with extremely elongated and large cells
at room temperature, similar to the akr1
-1 phenotype at
37°C (Kao et al., 1996
; and our unpublished results) suggesting functional synergy between Iqg1p and Akr1p. Indeed, an in vivo interaction was detected in the two-hybrid
system between Iqg1p and Akr1p (Table IV).
|
iqg1 Cells Accumulate Post-Golgi Vesicles
Yeast cells initiate growth at a specific site on the cell surface and undergo polar growth because of the localized fusion of vesicles with the plasma membrane (Sloat et al.,
1981). There is mounting evidence that an aberrant actin
cytoskeleton results in delocalized growth and accumulation of secretory vesicles (Novick and Botstein, 1985
; Liu
and Bretscher, 1992
, Mulholland et al., 1997
). Cdc42p controls cellular polarity and actin cytoskeleton organization
(Adams et al., 1990
; Johnson and Pringle, 1990
; Johnson, 1993
), and its localization to the plasma membrane is essential for its function (Ziman et al., 1991
; Stevenson et al.,
1995
). To examine whether the iqg1
phenotypes are due
to effects at the plasma membrane, we visualized cells with
electron microscopy as described in Materials and Methods. After shifting to the restrictive temperature (37°C),
many of the iqg1
cells displayed a scalloped shape membrane structure (not shown), with large vacuoles as previously revealed by Nomarski optics (Fig. 3 C) and CDCFDA staining (not shown). As shown in Fig. 11, a
population of iqg1
cells (10 out of 21 cells with small
buds), that appeared to be less severely affected or lysed,
accumulated a large number of vesicles in the bud. Presumably, these represented post-Golgi secretory vesicles at the polarized cell surface. By contrast, isogenic wild-type cells did not accumulate these vesicles (0 out of 75 small-budded cells). These results may explain the slow
growth phenotype and cytokinesis defect of iqg1
cells.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The Rho-type GTP-binding protein, Cdc42p, has been implicated in various functions both in yeast and mammalian cells, such as cell polarity, cell motility, and cytokinesis, by reorganizing the actin cytoskeleton, presumably as an outcome of its interactions with cytoplasmic targets. Despite numerous microinjection, genetic, and biochemical studies, little is known about the downstream signals that mediate the roles of Cdc42p in cell cycle-dependent morphological changes. In this study, we provide evidence that Iqg1p, a homologue of the mammalian IQGAPs, mediates Cdc42p function in cytokinesis and other actin-based cellular processes in yeast.
It should be noted that recently, two other studies have
appeared which describe the same homologue (Epp and
Chant, 1997; Lippincott and Li, 1998
). In both of these
studies, the deletion of IQG1 (also called CYK1 by Lippincott and Li, 1998
) was reported to be lethal. At present,
we do not know the underlying genetic basis for the differences in these studies versus our own, which indicate
IQG1 deletion to be conditionally lethal. However, this conditional lethality has allowed us to examine how the
loss of Iqg1p expression impacts on its suspected binding
partners including Cdc42p and actin.
Iqg1p May Mediate Cdc42p Functions by Reorganizing the Actin Cytoskeleton and Localizing Cdc42p
By homology with the mammalian IQGAP protein, we
have identified and cloned the yeast homologue, Iqg1 protein. Our results suggest that Iqg1p may mediate the functions of Cdc42p in reorganizing the actin cytoskeleton, as
well as help localize Cdc42p to sites of cell growth (Table
III; Figs. 4-6). Further support for this idea comes from
earlier biochemical studies with mammalian cells showing that Cdc42Hs, IQGAP, and actin form a ternary complex
(Erickson et al., 1997; Fukata et al., 1997
). Moreover,
Iqg1p and its mammalian homologue, IQGAP, each contain a CHD. These motifs have been previously implicated
in cross-linking actin filaments into bundles (Brown et al.,
1995a
,b; Bashour et al., 1997
) and in binding and recruiting signaling proteins to the interface between actin and
the plasma membrane (Brown et al., 1995a
,b).
In addition, as is the case for the mammalian IQGAPs,
Iqg1p is likely to bind calmodulin (Brill et al., 1996; Joyal
et al., 1997
). Iqg1p contains conserved IQ motifs at positions corresponding to the mammalian IQGAP motifs
(Brill et al., 1996
; Hart et al., 1996
; Joyal et al., 1997
) and
we have shown that calmodulin distribution is diffuse in
cells lacking Iqg1p (Fig. 7), implying that Iqg1p mediates
the proper localization of calmodulin. In yeast, calmodulin colocalizes with actin and its function is affected in actin
mutants (Davis, 1992
). Furthermore, results from mammalian cells have shown that calmodulin modulates the interactions between Cdc42p and actin (Bashour et al., 1997
;
Joyal et al., 1997
).
Iqg1p Promotes Cytokinesis
Cytokinesis is the final stage of the cell cycle that produces
two cells. Before cytokinesis, cells cease polarized growth and assume an isotropic expansion. Numerous proteins
are thought to be involved in this process by virtue of their
localization and the phenotypes of their mutations. However, the signals that regulate this process are thus far unknown. Our results suggest that Iqg1p is required for the
completion of cytokinesis in yeast cells perhaps by transducing a signal from Cdc42p. The elongated cell phenotype (Figs. 3 A, 4 E, 6 D, 8 C, and 9 C), the accumulation of
nuclei in iqg1 cells (Figs. 9 and 10), and the localization of Iqg1p at the mother-bud neck (Fig. 2 B) at cytokinesis
support this view. The localization of Iqg1p to sites of cell
growth and the septum appears to overlap that of actin,
Cdc42p, and calmodulin. These putative Iqg1p-binding
partners are also implicated in morphogenesis and cytokinesis (Drubin, 1990
; Davis, 1992
; Dutartre et al., 1996
). Immunofluorescence experiments performed on iqg1
cells showed elongated buds containing ridges of localized
proteins at positions we proposed to be septa locations.
Namely, Cdc42p (Fig. 4 F), and actin (Fig. 5 D) localized
to ridges across the tubular buds that displayed no constriction formation that precedes cell separation. Whereas
Cdc42p, actin, and chitin all appeared to be correctly localized to the presumptive septum, the absence of Iqg1p
alone apparently accounts for the defect in the completion
of the cell cycle and the separation of the mother and
daughter cells. Whether the mislocalization of other proteins involved in cytokinesis, such as the septins, also contribute to this defect in cytokinesis needs to be further investigated.
Additional support for the involvement of Iqg1p in cytokinesis came from genetic and two-hybrid interactions
with Akr1p and Afr1p (Table IV). The deletion of the
ankyrin repeat-containing Akr1p results in a similar elongated cell phenotype as the Iqg1p deletion, and the double
mutants displayed significant growth and cytokinesis defects compared with each of the single mutant iqg1 and
akr1
cells, thus suggesting synergy of function between these proteins. Afr1p appears to antagonize the functions
of Iqg1p and Akr1p, as suggested by the fact that the ectopic expression of Afr1p produces phenotypes (Konopka
et al., 1995
) similar to the deletions of AKR1 and IQG1. In
addition, Afr1p localizes to the septum and interacts with
a septin, Cdc12p, thus lending a further connection to cytokinesis.
Recently, three IQGAP homologues (Faix and Dittrich,
1996; Adachi et al., 1997
; Lee et al., 1997
) were identified
in Dictyostelium. The three molecules also appear to be involved in cytokinesis at different levels. Both Cdc42Hs
(Dutartre et al., 1996
) and its Dictyostelium relative RacE
(Larochelle et al., 1996
) were previously implicated in cytokinesis. More recently, two reports (Epp and Chant,
1997
; Lippincott and Li, 1998
) have also described cytokinesis as a primary function for the yeast Iqg1p. Together, these findings suggest that Iqg1p function is well conserved among organisms.
Similarly, calmodulin has been implicated in cytokinesis
both in yeast and Dictyostelium (Davis, 1992; Liu et al.,
1992
). Our results suggest that Iqg1p participates in localizing calmodulin to sites of active growth (Fig. 7) supporting the view that Iqg1p recruits and maintains a larger protein complex to execute its functions.
A Possible Role for Iqg1p in the Regulation of Cell Polarity
The localization of Iqg1p at the site of the incipient bud
(Fig. 2) points to a possible involvement in bud morphogenesis. This localization overlaps that of many proteins
involved in bud formation such as actin, Cdc42p, calmodulin, and the septins (Drubin, 1990; Brockerhoff and Davis,
1992
; Ziman et al., 1993
). These proteins may use the same
signal to localize at the site of bud formation. Based on our
findings, it would appear that the role of Iqg1p is to promote isotropic growth of the bud and subsequently cytokinesis. Two pieces of evidence support this suggestion; the localization of Iqg1p throughout the small buds and the
hyperpolarization and pheromone sensitivity of iqg1
cells (Fig. 3, E-H). The hyperpolarization displayed by
iqg1
cells treated with
factor also suggests that Iqg1p
may actually inhibit projection formation, perhaps through an interaction with Akr1p. It appears that Akr1p inhibits
signaling in the pheromone response pathway in cooperation with Ste4p, the G
subunit of the pheromone receptor-coupled G protein (Kao et al., 1996
). Thus, Iqg1p and
Akr1p may work in synergy to inhibit pheromone signaling by Cdc42p. However, the expression of another Iqg1p-binding partner, Afr1p, is induced by mating pheromone
and cells lacking AFR1 are defective in
factor-induced
projections (Konopka, 1993
). The ectopic expression of
Afr1p in vegetative cells, where Afr1p localizes to the
neck, causes abnormal morphologies (Konopka et al., 1995
)
similar to those caused by mutations in Iqg1p and Akr1p.
Taken together, these findings suggest that interactions between Iqg1p, Afr1p and Akr1p (Fig. 12) may result in a
complex regulation of projection formation.
|
Iqg1p May Influence Trafficking
The large vacuole phenotype of iqg1 cells (Fig. 3 C), the
accumulation of vesicles at the growing bud (Fig. 11), and
the interaction of Iqg1p in the two-hybrid system with
Akr1p (Table IV) all suggest a possible involvement of
Iqg1p in secretion or some aspect of protein trafficking.
The accumulation of vesicles is analogous to that caused
by actin mutants (Mulholland et al., 1997
) and mutations
affecting actin functions (Li, 1997
). It seems likely that
these phenotypic defects may result because actin filaments become disorganized in the absence of Iqg1p and in
turn perturb actin-based secretion. However, the two-
hybrid interaction between Iqg1p and Akr1p suggests a
more direct role for Iqg1p in secretion. Akr1p is involved in
the constitutive endocytosis of Ste3p, the a factor receptor
(Givan and Sprague, 1997
). Akr1p also displays two-hybrid
interactions with Gcs1p (Kao et al., 1996
), a GTPase-activating protein for Arf1p. Biochemical studies have shown
that Gcs1p can activate the intrinsic GTPase activity of both yeast and mammalian Arfs (Poon et al., 1996
). Further, the mammalian IQGAP binds to Golgi membrane-
associated Cdc42p (McCallum et al., 1996
, 1998
), and we
have previously shown that the Golgi localization of Cdc42p
was influenced by the Arf GTPase (Erickson et al., 1996
).
This, together with the fact that mammalian Cdc42p acts
with Arf to synergistically activate the Golgi membrane- associated phospholipase D (Brown et al., 1993
), supports
the possibility that Cdc42p may participate in some aspect
of intracellular trafficking through Iqg1p.
Interestingly, the large vacuole (Fig. 3 C) resembles the
phenotype caused by csd4-3::LEU2, a mutant allele of
CHS4, which encodes an activator of chitin synthase III
and interacts with the septin Cdc10p (DeMarini et al.,
1997), and thus this phenotypic similarity could be significant in terms of secretion and cell wall deposition. The fact
that chitin is mislocalized in iqg1
cells (Fig. 8), septin mutants and in cells ectopically expressing Afr1p (Konopka
et al., 1995
) imply that these proteins may all participate in
some aspects of cell wall deposition mediated by actin-based cellular trafficking.
Mechanism of Action of Iqg1p
We propose that the Iqg1p is involved in recruiting and maintaining the organization of a number of cytoskeletal proteins at sites of cell growth and therefore, may act as a scaffold. The kinetics of an Iqg1p-mediated recruitment of different
proteins would likely be important. Our immunofluorescence studies show that the elongated buds contain ridges
of localized proteins that we propose to represent septa locations. The fact that the various proteins that we have examined were not correctly localized at the outset (compare
Figs. 4, D and F, and 5, C and D) suggest that there might
be a delay in their localization. This delayed localization
may explain the slow growth phenotype of iqg1 cells. The
lethality of iqg1
cells at 37°C may reflect the instability of
a protein complex involved in growth and cytokinesis.
The multidomain structure of Iqg1p suggests that it can
interact with a variety of proteins to negatively regulate
cell polarization and promote isotropic growth and subsequently cytokinesis. This is especially apparent by the hyperpolarized phenotypes exhibited by iqg1 cells and suggested by the observed two-hybrid interactions between
Iqg1p, Akr1p, and Afr1p. Given that Akr1p appears to inhibit the pheromone response pathway (Kao et al., 1996
)
and promote cytokinesis, we suspect that it may serve to
mediate the effects of Cdc42p and Iqg1p on these events.
The two-hybrid interactions detected between Iqg1p and
both Akr1p and Afr1p point to a complicated scheme by
which Cdc42p and Iqg1p may regulate a number of fundamental processes in yeast (Fig. 12). Future studies will be
directed at exploring the interplay between Iqg1p, Akr1p,
and Afr1p in more detail and in particular examining the
possibilities that Akr1p and Afr1p represent positive and
negative effectors, respectively, for the actions of Cdc42p
and Iqg1p.
![]() |
Footnotes |
---|
Received for publication 4 December 1997 and in revised form 28 May 1998.
Address all correspondence to Richard A. Cerione, Department of Molecular Medicine, Veterinary Medical Center, Cornell University, Ithaca, NY 14853-6401. Tel.: (607) 253-3888. Fax: (607) 253-3659. E-mail: rac1{at}cornell.eduWe thank Dr. T. Huffacker for strains, tubulin antibodies, and the microscope. We also thank Drs. A. Bender, J. Konopka, S. Elledge, C. Boone, and J. Sprague for strains and plasmids. We thank Dr. T. Davis for calmodulin antibodies. We thank Drs. E. Hong for critically reading the manuscript, D. Manor for his help, J. Erickson for helpful suggestions, N. Nassar for pointing out conserved boxes 1-3 on the sequence alignment, R. Collins for discussion, and C. Westmiller for expert technical assistance.
This work was supported by National Institutes of Health grant GM47458.
![]() |
Abbreviations used in this paper |
---|
CHD, calponin homology domain; GAP, Cdc42-GTPase-activating protein; HA, hemagglutinin.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Adachi, H.,
Y. Takahashi,
T. Hasebe,
M. Shirouzu,
S. Yokoyama, and
K. Sutoh.
1997.
Dictyostelium IQGAP-related protein specifically involved in the completion of cytokinesis.
J. Cell Biol.
137:
891-898
|
2. | Adams, A.E.M., and J.R. Pringle. 1991. Staining of actin with fluorochrome-conjugated phalloidin. Methods in Enzymology. 194:729-731. |
3. | Adams, A.E.M., D.I. Johnson, R.M. Longnecker, B.F. Sloat, and J.R. Pringle. 1990. CDC42 and CDC43, two additional genes involved in budding and the establishment of cell polarity in the yeast Sacharomyces cerevisiae. J. Cell Biol. 111: 131-142 [Abstract]. |
4. | Ausubel, F.M., R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl. 1992. Current Protocols in Molecular Biology. John Wiley and Sons Ltd., New York. |
5. |
Bagrodia, S.,
B. Derijard,
R.J. Davis, and
R.A. Cerione.
1995.
Cdc42 and PAK-mediated signaling leads to jun kinase and p38 mitogen-activated protein kinase activation.
J. Biol. Chem.
270:
27995-27998
|
6. |
Barfod, E.T.,
Y. Zheng,
W.-J. Kuang,
M.J. Hart,
T. Evans,
R.A. Cerione, and
A. Ashkenazi.
1993.
Cloning and expression of a human CDC42 GTPase-activating protein reveals a functional SH3-binding domain.
J. Biol. Chem.
268:
26059-26062
|
7. |
Bashour, A.-M.,
A.T. Fullerton,
M.J. Hart, and
G.S. Bloom.
1997.
IQGAP1, a
Rac- and Cdc42-binding protein, directly binds and cross-links microfilaments.
J. Cell Biol.
137:
1555-1566
|
8. | Baudin, A., O. Ozier-Kalogeropoulos, A. Denouel, F. Lacroute, and C. Cullin. 1993. A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res. 21: 3329-3330 |
9. | Berben, G., J. Dumont, V. Gilliquet, P. Bolle, and F. Hilger. 1991. The YDp plasmids: a uniform set of vectors bearing versatile gene disruption cassettes for Saccharomyces cerevisiae. Yeast. 7: 475-477 |
10. | Bi, E., and J.R. Pringle. 1996. ZDS1 and ZDS2, genes whose products may regulate Cdc42 in Saccharomyces cerevisiae. Mol. Cell. Biol. 16: 5264-5275 [Abstract]. |
11. | Brill, S., S. Li, C.W. Lyman, D.M. Church, J.J. Wasmuth, L. Weissbach, A. Bernards, and A.J. Snijders. 1996. The Ras GTPase-activating-protein-related human protein IQGAP2 harbors a potential actin binding domain and interacts with calmodulin and Rho family GTPases. Mol. Cell. Biol. 9: 4869-4878 . |
12. | Brockerhoff, E.S., and T.N. Davis. 1992. Calmodulin concentrates at regions of cell growth in Saccharomyces cerevisiae. J. Cell Biol. 118: 619-629 [Abstract]. |
13. | Brown, A., G. Berneir, H. Mathieu, J. Rossant, and R. Kothary. 1995a. The mouse dystonia musculorum gene is a neural isoform of bullous pemphigoid antigen 1. Nat. Genet. 10: 301-306 |
14. | Brown, A., G. Dalpe, M. Mathieu, and R. Kothary. 1995b. Cloning and characterization of the neural isoforms of human dystonin. Genomics. 29: 777-780 |
15. | Brown, H.A., S. Gutowski, C.R. Moomaw, C. Slaughter, and P.C. Sternweis. 1993. ADP-ribosylation factor, a small GTP-dependent regulatory protein, stimulates phospholipase D activity. Cell. 75: 1137-1144 |
16. | Bulawa, C.E.. 1993. Genetics and molecular biology of chitin synthesis in fungi. Annu. Rev. Microbiol. 47: 505-534 |
17. | Castresana, J., and M. Saraste. 1995. Does Vav bind to F-actin through a CH domain? FEBS (Fed. Eur. Biochem. Soc.) Lett. 374: 149-151 . |
18. | Cerione, R.A., and Y. Zheng. 1996. The Dbl family of oncogenes. Curr. Opin. Cell Biol. 8: 216-222 |
19. | Chant, J., and I. Herskowitz. 1991. Genetic control of bud site selection in yeast by a set of gene products that constitute a morphogenetic pathway. Cell. 65: 1203-1212 |
20. | Coso, O.A., M. Chiariello, J. Yu, H. Teramoto, P. Crespo, N. Xu, T. Miki, and J.S. Gutkind. 1995. The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway. Cell 81: 1137-1146 |
21. | Davis, T.N.. 1992. A temperature-sensitive calmodulin mutant loses viability during mitosis. J. Cell Biol. 118: 607-617 [Abstract]. |
22. |
DeMarini, D.J.,
A.E.M. Adams,
H. Fares,
C. De Virgilio,
G. Valle,
J.S. Chuang, and
J.R. Pringle.
1997.
A septin-based hierarchy of proteins required for localization deposition of chitin in the Saccharomyces cerevisiae cell wall.
J.
Cell Biol.
139:
75-93
|
23. | Drubin, D.G.. 1990. Actin and actin-binding proteins in yeast. Cell Motil. Cytoskeleton. 15: 7-11 |
24. |
Dutartre, H.,
J. Davoust,
J.-P. Gorvel, and
P. Chavrier.
1996.
Cytokinesis arrest
and redistribution of actin-cytoskeleton regulatiory components in cells expressing the Rho GTPase CDC42Hs.
J. Cell Sci.
109:
367-377
|
25. | Elble, R.. 1992. A simple and efficient procedure for transformation of yeasts. Biotechniques. 13: 18-20 |
26. | Epp, A., and J. Chant. 1997. An IQGAP-related proteincontrols actin ring formation and cytokinesis in yeast. Curr. Biol. 7: 921-929 |
27. |
Erickson, J.W.,
C-J. Zhang,
R.A. Kahn,
T. Evans, and
R.A. Cerione.
1996.
Mammalian Cdc42 is a brefeldin A-sensitive component of Golgi apparatus.
J. Biol. Chem.
271:
26850-26854
|
28. |
Erickson, J.W.,
R.A. Cerione, and
M.J. Hart.
1997.
Identification of an actin cytoskeletal complex that includes IQGAP and the Cdc42 GTPase.
J. Biol.
Chem.
272:
24443-24447
|
29. | Estes, J.E., L.A. Selden, and L.C. Gershman. 1981. Mechanism of action of phalloidin on the polymerization of muscle actin. Biochemistry. 20: 708-712 |
30. |
Evangelista, M.,
K. Blundel,
M.S. Longtine,
C.J. Chow,
N. Adames,
J.R. Pringle,
M. Peter, and
C. Boone.
1997.
Bni1, a yeast formin linking Cdc42p and
the actin cytoskeleton during polarized morphogenesis.
Science.
276:
118-122
|
31. | Faix, J., and W. Dittrich. 1996. DGAP1, a homologue of rasGTPase activating proteins that controls growth, cytokinesis, and development in Dictyostelium discoideum. FEBS (Fed. Eur. Biochem. Soc.) Lett. 394: 251-257 . |
32. | Field, C.M., O. Al-Awar, J. Rosenblatt, M.L. Wong, B. Alberts, and T.J. Mitchison. 1996. A purified Drosophila septin complex forms filaments and exhibits GTPase activity. J. Cell Biol. 133: 605-616 [Abstract]. |
33. | Fields, S., and O-K. Song. 1989. A novel genetic system to detect protein-protein interactions. Nature. 340: 245-246 |
34. | Ford, S.K., and J.R. Pringle. 1991. Cellular morphogenesis in the Saccharomyces cerevisiae cell cycle: localization of the CDC11 gene product and the timing of events at the budding site. Dev. Genet. 12: 281-292 |
35. |
Fukata, M.,
S. Kuroda,
K. Fujii,
T. Nakamura,
I. Shoji,
Y. Matsuura,
K. Okaya,
A. Iwamatsu,
K. Kikuchi, and
K. Kaibuchi.
1997.
Regulation of cross-linking
of actin filament by IQGAP1, a target for Cdc42.
J. Biol. Chem.
272:
29579-29583
|
36. | Futcher, B. 1993. Analysis of the cell cycle in Saccharomyces cerevisiae. In The Cell Cycle. P. Fantes, and R. Brooks, editors. IRL Press Oxford University Press, Oxford, England. 69-92. |
37. | Givan, S.A., and G.F. Sprague Jr.. 1997. The ankyrin repeat-containing protein Akr1p is required for the endocytosis of yeast pheromone receptors. Mol. Biol. 8: 1317-1327 . |
38. | Guthrie, C., and G.R. Fink. 1991. Guide to yeast genetics and molecular biology. Methods Enzymol. 194: 1-933 |
39. | Gyuris, J., E. Golemis, H. Chertkov, and R. Brent. 1993. Cdil, a human G1 and S phase protein phosphatase that associates with Cdk2. Cell. 75: 791-803 |
40. | Haarer, B.K., and J.R. Pringle. 1987. Immunofluorescence localization of the Saccharomyces cerevisiae CDC12 gene product to the vicinity of the 10-nm filaments in the mother-bud neck. Mol. Cell. Biol. 7: 3678-3687 |
41. | Hart, M.J., M.G. Callow, B. Souza, and P. Polakis. 1996. IQGAP1, a calmodulin-binding protein with a rasGAP-related domain, is a potential effector for Cdc42Hs. EMBO (Eur. Mol. Biol. Organ.) J. 15: 2997-3005 [Abstract]. |
42. | Holtzman, D.A., S. Yang, and D.G. Drubin. 1993. Synthetic-lethal interactions identify two novel genes, SL1 and SLA2, that control membrane cytoskeleton assembly in Saccharomyces cerevisiae. J. Cell Biol. 122: 635-644 [Abstract]. |
43. |
James, P.,
J. Halladay, and
E.A. Craig.
1996.
Genomic libraries and a host
strain designed for highly efficient two-hybrid selection in yeast.
Genetics.
144:
1425-1436
|
44. | Johnson, D.I., and J.R. Pringle. 1990. Molecular characterization of CDC42, a Saccharomyces cerevisiae gene involved in the development of cell polarity. J. Cell Biol. 111: 143-152 [Abstract]. |
45. | Johnson, D.I. 1993. CDC42: a member of the ras superfamily involved in the control of cellular polarity during the Saccharomyces cerevisiae cell cycle. In The ras Superfamily of GTPases. J.C. Lacal, and F. McCormick, editors. CRC Press, Boca Raton, FL. 297-312. |
46. |
Joyal, J.L.,
R.S. Annan,
Y.-D. Ho,
M.E. Huddleston,
S.A. Carr,
M.J. Hart, and
D.B. Sacks.
1997.
Calmodulin modulates the interaction between IQGAP1
and Cdc42.
J. Biol. Chem.
272:
15419-15425
|
47. | Kaiser, C.A., and R. Schekman. 1990. Distinct sets of SEC genes govern transport vesicle formation and fusion early in the secretory pathway. Cell. 61: 723-733 |
48. | Kao, L.-R., J. Peterson, J. Ruiru, L. Bender, and A. Bender. 1996. Interactions between the ankyrin repeat-containing protein Akr1p and the pheromone response pathway in Saccharomyces cerevisiae. Mol. Cell. Biol. 16: 168-178 [Abstract]. |
49. | Kilmartin, J.V., and A.E.M. Adams. 1984. Structural rearrangements of tubulin and actin during the cell cycle of the yeast Saccharomyces. J. Cell Biol. 98: 922-933 [Abstract]. |
50. | Kim, H.B., B.K. Haarer, and J.R. Pringle. 1991. Cellular morphogenesis in the Saccharomyces cerevisiae cell cycle: localization of the CDC3 gene product and the timing of events at the budding site. J. Cell Biol. 112: 535-544 [Abstract]. |
51. | Koch, G., K. Tanaka, T. Masuda, W. Yamochi, H. Nonaka, and Y. Takai. 1997. Association of the Rho family small GTP-binding proteins with Rho GDP dissociation inhibitor (Rho GDI) in Saccharomyces cerevisiae. Oncogene. 15: 417-422 |
52. |
Konopka, J.B..
1993.
AFR1 acts in conjunction with the ![]() |
53. | Konopka, J.B., C. DeMattei, and C. Davis. 1995. AFR1 promotes polarized apicalmorphogenesis in Saccharomyces cerevisiae. Mol. Cell. Biol. 15: 723-730 [Abstract]. |
54. | Lamarche, N., and A. Hall. 1994. GAPs for rho-related GTPases. Trends Genet. 10: 436-440 |
55. | Larochelle, D.A., K.K. Vithalani, and A. De Lozanne. 1996. A novel member of the rho family of small GTP-binding proteins is specifically required for cytokinesis. J. Cell Biol. 133: 1321-1329 [Abstract]. |
56. |
Lebart, M.-C.,
C. Méjean,
D. Casanova,
E. Audemard,
J. Derancourt,
C. Roustan, and
Y. Banyamin.
1994.
Characterization of the actin binding site
on smooth muscle filamin.
J. Biol. Chem.
269:
4279-4284
|
57. |
Lee, S.,
R. Escalante, and
R.A. Firtel.
1997.
A RasGAP is essential for cytokinesis and spatial patterning in Dictyostelium.
Development (Camb.).
124:
983-996
|
58. |
Leonard, D.,
M.J. Hart,
J.V. Platko,
A. Eva,
W. Henzel,
T. Evans, and
R.A. Cerione.
1992.
The identification and characterization of a GDP-dissociation
inhibitor (GDI) for the CDC42Hs Protein.
J. Biol. Chem.
267:
22860-22868
|
59. |
Li, R..
1997.
Bee1, a yeast protein with homology to Wiscott-Aldrich syndrome
protein, is critical for the assembly of cortical actin cytoskeleton.
J. Cell Biol.
136:
649-658
|
60. |
Lippincott, J., and
R. Li.
1998.
Sequential assembly of myosin II, an IQGAP-like protein, and filamentous actin to a ring structure involved in budding
yeast cytokinesis.
J. Cell Biol.
140:
355-366
|
61. | Liu, H., and A. Bretscher. 1992. Characterization of TPM1 disrupted yeast cells indicates an involvement of tropomyocin in directed vesicular transport. J. Cell Biol 118: 285-299 [Abstract]. |
62. | Liu, T., J.G. Williams, and M. Clarke. 1992. Inducible expression of calmodulin antisense RNA in Dictyostelium cells inhibits the competition of cytokinesis. Mol. Biol. Cell. 3: 1403-1413 [Abstract]. |
63. | Longtine, M.S., D.J. DeMarini, M.L. Valencik, O.S. Al-Awar, H. Fares, C. De Virgilio, and J.R. Pringle. 1996. The septins: roles in cytokinesis and other processes. Curr. Opin. Cell Biol. 8: 106-119 |
64. |
McCallum, S.J.,
W.J. Wu, and
R.A. Cerione.
1996.
Identification of a putative
effector for Cdc42Hs with high sequence similarity to the RasGAP-related
protein IQGAP1 and a Cdc42Hs binding partner with similarity to
IQGAP2.
J. Biol. Chem.
271:
21732-21737
|
65. | McCallum, S.J., J.W. Erickson, and R.A. Cerione. 1998. Characterization of the association of the actin-binding protein, IQGAP, and activated Cdc42 with Golgi membranes. J. Biol. Chem. In press. |
66. | Minden, A., A. Lin, F.-X. Claret, A. Abo, and M. Karin. 1995. Selective activation of the JNK signaling cascade and c-jun transcriptional activity by the small GTPases Rac and Cdc42Hs. Cell. 81: 1147-1157 |
67. | Mulholland, J., A. Wesp, H. Riezman, and D. Botstein. 1997. Yeast actin cytoskeleton mutants accumulate a new class of Golgi-derived secretory vesicle. Am. Soc. Cell Biol. 8: 1481-1499 . |
68. | Nobes, C.D., and A. Hall. 1995. Rho, Rac, and Cdc42 GTPases regulate the assembly of multicellular focal complexes associated with actin stress fibers, lamillipodia, and filipodia. Cell. 81: 53-62 |
69. | Novick, P., and D. Botstein. 1985. Phenotypic analysis of temperature sensitive yeast actin mutants. Cell. 40: 405-416 |
70. | Olson, M.F., A. Ashworth, and A. Hall. 1995. An essential role for Rho, Rac and Cdc42 GTPases in cell cycle progression through G1. Science. 269: 1270-1272 |
71. |
Poon, P.P.,
X. Wang,
M. Rotman,
I. Huber,
E. Cukierman,
D. Cassel,
R.A. Singer, and
G.C. Johnston.
1996.
Saccharomyces cerevisiae Gcs1 is an ADP-ribosylation factor GTPase-activating protein.
Proc. Natl. Acad. Sci. USA.
93:
10074-10077
|
72. | Pringle, J.R.. 1991. Staining of bud scars and mother cell wall chitin with Calcofluor. Methods Enzymol. 194: 732-735 |
73. | Qiu, R.-G., J. Chen, D. Kirn, F. McCormick, and M. Symons. 1995. An essential role for Rac in Ras transformation. Nature. 374: 457-459 |
74. | Ridley, A.J., P.M. Comoglio, and A. Hall. 1995. Regulation of scatter factor/ hepatocyte growth factor responses by Ras, Rac and Rho in MDCK cells. Mol. Cell. Biol. 15: 1110-1122 [Abstract]. |
75. | Sawai, E.T., I.H. Khan, P.M. Montbriand, B.M. Peterlin, C. Cheng-Mayer, and P.A. Luciw. 1996. Activation of PAK by HIV and SIV Nef: importance for AIDS in rhesus macaques. Curr. Biol. 6: 1519-1527 |
76. |
Singer, W.D.,
H.A. Brown,
G.M. Bokoch, and
P.C. Sternweis.
1995.
Resolved
phospholipase D activity is modulated by cytosolic factors other than Arf.
J.
Biol. Chem.
270:
14944-14950
|
77. | Sloat, B.F., A. Adams, and J.R. Pringle. 1981. Roles of the CDC24 gene product in cellular morphogenesis during the Saccharomyces cerevisiae cell cycle. J. Cell. Biol. 89: 395-405 [Abstract]. |
78. | Stevenson, B.J., B. Ferguson, C. De Virgilio, E. Bi, J.R. Pringle, G. Ammerer, and G.F. Sprague Jr.. 1995. Mutation of RGA1, which encodes a putative GTPase-activating protein for the polarity establishment protein Cdc42p, activates the pheromone-response pathway in the yeast Saccharomyces cerevisiae. Genes. Dev. 9: 2949-2963 [Abstract]. |
79. | Sudol, M., W.I. Chen, C. Bougeret, A. Einbond, and R. Bork. 1995. Characterization of a novel protein binding module: the WW domain. FEBS (Fed. Eur. Biochem. Soc.) Lett. 369: 67-71 . |
80. | Symons, M., J.M. Derry, B. Karlak, S. Jiang, V. Lemahieu, F. McCormick, U. Francke, and A. Abo. 1996. Wiskott-Aldrich syndrome protein, a novel effector for the GTPaseCdc42Hs, is implicated in actin polymerization. Cell. 84: 723-734 |
81. |
Weissbach, L.,
J. Settleman,
M. Kaladay,
A. Snjiders,
A. Murthy,
Y. Yan, and
A. Bernards.
1994.
Identification of a human RasGAP-related protein containing calmodulin-binding motifs.
J. Biol. Chem.
269:
20517-20521
|
82. |
Zheng, Y.,
M.J. Hart,
K. Shinjo,
T. Evans,
A. Bender, and
R.A. Cerione.
1993.
Biochemical comparisons of the Saccharomyces cerevisiae Bem2 and Bem3
proteins.
J. Biol. Chem.
268:
24629-24634
|
83. | Ziman, M., J.M. O'Brien, L.A. Ouellette, W.R. Church, and D.I. Johnson. 1991. Mutational analysis of Cdc42Sc, a Saccharomyces cerevisiae gene that encodes a putative GTP-binding protein involved in the control of cell polarity. Mol. Cell. Biol. 11: 3537-3544 |
84. | Ziman, M., D. Preuss, J. Mulholland, J.M. O'Brien, D. Botstein, and D.I. Johnson. 1993. Subcellular localization of Cdc42, a Saccharomyces cerevisiae GTP-binding protein involved in the control of cell polarity. Mol. Biol. Cell. 4: 1-10 |