Department of Cell Biology, Harvard Medical School, Boston, MA 02115
Yeast protein, Bee1, exhibits sequence homology to Wiskott-Aldrich syndrome protein (WASP),
a human protein that may link signaling pathways to
the actin cytoskeleton. Mutations in WASP are the primary cause of Wiskott-Aldrich syndrome, characterized by immuno-deficiencies and defects in blood cell
morphogenesis. This report describes the characterization of Bee1 protein function in budding yeast. Disruption of BEE1 causes a striking change in the organization of actin filaments, resulting in defects in budding
and cytokinesis. Rather than assemble into cortically associated patches, actin filaments in the buds of bee1
cells form aberrant bundles that do not contain most of
the cortical cytoskeletal components. It is significant
that
bee1 is the only mutation reported so far that
abolishes cortical actin patches in the bud. Bee1 protein
is localized to actin patches and interacts with Sla1p, a
Src homology 3 domain-containing protein previously implicated in actin assembly and function. Thus, Bee1
protein may be a crucial component of a cytoskeletal
complex that controls the assembly and organization of
actin filaments at the cell cortex.
Proteins that are conserved between yeast and mammals are likely to carry out fundamental cellular
functions. The complete yeast genome database
provides a facile route for the identification of these proteins, which can then be studied directly in yeast through
combined genetic and biochemical approaches. In recent years, yeast has been increasingly attractive for studying
actin cytoskeleton function in cell morphogenesis. During
mitotic growth, yeast cells undergo highly reproducible
changes in cell shape that are accompanied by actin cytoskeleton rearrangements (Adams and Pringle, 1984 In addition to the conserved actin-binding proteins,
building blocks of protein interactions common to mammalian cytoskeletal and signaling molecules are also found
in yeast. Sla1p, a protein implicated in actin assembly in
yeast (Holtzman et al., 1993 A search of the yeast genome database revealed an open
reading frame that encodes a protein with sequence homology to Wiskott-Aldrich syndrome protein (WASP) (Symons et al., 1996 WASP homologues share several functional domains.
WASP homology domain 1 (WH1), an NH2-terminal domain that exhibits sequence similarity to pleckstrin homology domains, can interact with phospholipids in vitro
(Miki et al., 1996 The yeast WASP-like protein contains all of the functional regions listed above except the GBD that interacts
with Cdc42p (Symons et al., 1996 Strains and Media and Genetic Manipulations
Yeast strains used in this work are listed in Table I. Yeast cell culture and
genetic techniques were carried out by methods described by Sherman
et al., 1974 Table I.
Yeast Strains
; Kilmartin and Adams, 1984
). Actin assembly and organization in yeast are modulated by a set of actin-binding proteins similar to those that operate in mammalian cells
(for review see Welch et al., 1994
). Examples of these actin-binding proteins include Sac6p, a fimbrin homologue
that bundles actin filaments (Adams et al., 1989
); Cap1p
and Cap2p, subunits of a protein complex that caps the
barbed (high-affinity) ends of actin filaments (Amatruda
et al., 1990
); and Cof1p, a cofilin homologue that severs
actin filaments (Moon et al., 1993
). These actin-binding proteins localize to cortical actin patches, membrane-associated structures that are concentrated at sites of cell surface
growth (Adams and Pringle, 1984
; Kilmartin and Adams,
1984
). It has been speculated that the cortical patches may
be involved in membrane activities, such as exocytosis and
endocytosis (for review see Bretscher et al., 1994
).
; Li et al., 1995
), contains three
Src homology 3 (SH3)1 domains and a COOH-terminal repeat structure similar to a region in bindin, a sea urchin
sperm adhesion protein. Disruption of SLA1 results in aberrant morphology of actin patches and temperature-sensitive cell growth. Despite the fact that a significant number of yeast actin cytoskeletal components have been
identified, little is known about how they interact with
each other in vivo to control actin filament assembly and
organization.
). Mutations in WASP result in WiskottAldrich syndrome (WAS) (Derry et al., 1994
, 1995), a
severe immuno-deficiency and platelet deficiency disease
(for review see Kirchhausen and Rosen, 1996
; RemoldO'Donnell et al., 1996). Lymphocytes from WAS patients
are morphologically abnormal with decreased size and
density of microvilli (Kenney et al., 1986
; Molina et al.,
1992
), suggesting that WASP may be required for blood
cell morphogenesis. Recently, a more ubiquitously expressed homologue of WASP, N-WASP, was identified
(Miki et al., 1996
). Overexpression of either WASP in tissue
culture cells induces the formation of actin-rich structures
to which the overexpressed WASP localizes, suggesting
that WASP may interact directly with actin cytoskeletal components (Miki et al., 1996
; Symons et al., 1996
).
). Both WASP and N-WASP contain a
GTPase-binding domain (GBD) that interacts with the activated form of Cdc42 (Kolluri et al., 1996
; Symons et al.,
1996
), a small GTP-binding protein involved in regulating
actin cytoskeleton rearrangements (for review see Nobes
and Hall, 1995
). Downstream from the GBD, there is a
proline-rich region that interacts with the SH3 domains of several signaling proteins (Rivero-Lezcano et al., 1995
; Banin et al., 1996
; Bunnell et al., 1996
; Miki et al., 1996
). The
presence of these functional regions in WASP suggests
that WASP may act as a molecular scaffold that links various signaling pathways to the actin cytoskeleton.
). I have named this protein Bee1 because of its similarity to WASP. Reported
here are the results of studies on the cellular function of
Bee1 protein (Bee1p) in yeast. Bee1p is a component of
the cortical actin cytoskeleton and plays an essential role
in the organization of actin filaments at the cell cortex.
BEE1-disrupted cells are defective in budding and cytokinesis, most likely as a result of the inability to assemble
cortical actin patches. Thus, Bee1 protein may share a
structural function with WASP.
Materials and Methods
.
Cloning, Plasmid, and Strain Construction
Yeast genomic DNA was prepared from strain RLY1 as described (Hoffman and Winston, 1987). A 2.4-kb DNA fragment containing the BEE1
coding region (NCBI accession No. 1101757) and 282-bp 5
and 180-bp 3
flanking sequences was amplified from yeast genomic DNA by PCR using
a 5
primer, 5
-BamHI-TACTTGAAATTGTGTCTCTG-3
(Primer 1), and
a 3
primer, 5
-XbaI-ATCATTGTAGCCCGACTATT-3
(Primer 2). This
fragment was cut with BamHI and XbaI and cloned into pRS316 (Sikorski
and Hieter, 1989
) and Bluescript SK to yield pRL101 and pRL88, respectively. To construct the BEE1 knock-out plasmid, pRL88 was cut with
BsmI, removing 84% of the BEE1 coding region (amino acids 102-633
and 11 bp of 3
UTR), and blunted and ligated with a DNA fragment containing the LEU2 gene (Berben et al., 1991
), yielding pRL90. pRL102, a
yeast vector for COOH-terminal 6-myc tagging was constructed by ligating the BamHI-SnaBI fragment that contains six copies of myc epitope
from CS+MT (Roth et al., 1991
) into vector pRS306 (Sikorski and Hieter,
1989
) between BamHI and XbaI (blunt). To construct a plasmid that expresses COOH-terminal myc-tagged Bee1p, a BEE1 fragment was PCRamplified from yeast genomic DNA using Primer 1 and a 3
primer of sequence BamHI-CCAATCATCACCATTGTCC (Primer 3). The latter
corresponds to the COOH terminus of Bee1p. This fragment was cut with
BamHI and ligated into the BamHI site of pRL102. The orientation of the
insert was determined, and the resulting plasmid in which the myc tag was
at the 3
end of BEE1 was named pRL108. pRL108 was cut with PvuII
and the fragment that contains BEE1-myc was ligated into pRS423
(Sikorski and Hieter, 1989
) between the PvuII sites, yielding pRL111.
pRL111 complements the
bee1 mutation.
To construct bee1 strain, PRL90 was cut with XbaI and BamHI and
transformed into a Leu2
diploid yeast strain. The diploid was sporulated
and the tetrads were dissected and analyzed. The tetrads showed 2:2 segregation for the Leu+ phenotype. To confirm BEE1 deletion in the Leu+
colonies, genomic DNA was prepared from a tetrad and analyzed by PCR
using Primers 1 and 3. Genomic DNA from the Leu
colonies gave a 2.2kb fragment, corresponding to the segment flanked by the two primers.
Genomic DNA from the Leu+ colonies did not yield any PCR product, as
expected for BEE1 deletion since the sequence corresponding to Primer 3 was within the deleted part of BEE1 gene in PRL90. In addition, the phenotypes of the Leu+ colonies can be fully complemented by PRL101, a
centromere-containing plasmid carrying full-length BEE1.
Immunofluorescence
Cells were fixed directly in growth media by the addition of 37% formaldehyde to 5% final concentration. Immunofluorescence staining was carried
out essentially as described (Drubin et al., 1988). Rhodamine-conjugated
donkey anti-goat and FITC-conjugated donkey anti-rabbit and donkey
anti-mouse secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA).
Phalloidin Staining of Yeast Cells
Cells were fixed directly in growth media by the addition of 37% formaldehyde to 5% final concentration. Staining with rhodamine-labeled phalloidin (Molecular Probes, Eugene, OR) was carried out as described (Lillie and Brown, 1994).
Permeabilization of Yeast Cells and In Vitro Actin Assembly
Cell growth and permeabilization, rhodamine-actin preparation, and in
vitro rhodamine-actin assembly assay were carried out as described (Li et al.,
1995).
Fluorescence Microscopy
Fluorescence imaging was performed on a microscope (model Axiophot; Carl Zeiss, Inc., Thornwood, NY) with a HB100 W/Z high-pressure mercury lamp and a 100× Plan Neofluar oil immersion objective (Carl Zeiss, Inc.). Image acquisition was performed using Northern Exposure (Phase 3 Imaging Systems, Milford, MA).
Video Microscopy
5 µl of an exponentially growing yeast culture was mixed with 5 µl of 1.6% low melting agarose in YPD (kept at 37°C) in a well of a multitest slide (ICN Biomedicals, Inc., Costa Mesa, CA). The slide was covered with a glass coverslip and sealed with nail polish. The cells were observed with a 63×/1.4NA plan-Apochromat objective on a microscope (model Axiovert 135; Carl Zeiss, Inc.). Images were collected with a cooled CCD (Photometics Ltd., Tucson, AZ). Image acquisition and data analysis were carried out using Metamorph 2.0 (Universal Imaging Corp., West Chester, PA).
Electron Microscopy
10 ml of exponentially growing cells were harvested and the cell pellet was resuspended directly in 1 ml of 1% glutaraldehyde in 200 mM cacodylate and 100 mM KPO4 buffer, pH 6.5. Cells were fixed for 30 min at room temperature and were washed three times with 100 mM KPO4, pH 7.5. Cells were treated with 0.2 mg/ml zymolyase 20T (Seikagaku Corp., Tokyo, Japan) for 30 min at 37°C. Cells were washed twice in 200 mM cacodylate buffer, pH 7.4, and incubated at room temperature for 1 h in 1.5% potassium ferrocyanide/1% osmium tetroxide (in water). The cells were washed with water (3 × 15 min), incubated for 1 h in 2% uranyl acetate (in water, at room temperature), washed again in water (3 × 15 min), and dehydrated in ethanol 70%/90%/100% (15 min each). The samples were then infiltrated in Epon/Araldite mixed 1:1 with propyleneoxide for 2 h, transferred to pure Epon/Araldite in an embedding mould, and polymerized overnight at 60°C. Ultra-thin sections were cut on a Reichert Ultracut S microtome (Leica Inc., Deer Lake, IL), stained with uranyl acetate and lead citrate, and examined in a transmission electron microscope (model 1200 EX; JEOL U.S.A., Inc., Peabody, MA) at 80 kV.
Yeast Extract Preparation and Immunoprecipitation
Yeast cells were lysed by the liquid nitrogen-grinding method (Sorger and
Pelham, 1987) in UBT (50 mM KHepes, pH 7.5, 100 mM KCl, 3 mM
MgCl2, 1 mM EGTA, 0.5% Triton X-100) supplemented with protease
inhibitors as described (Li et al., 1995
). The cell lysate was centrifuged at
300,000 g for 60 min. The resulting high-speed supernatant was usually
at a concentration of ~10 mg/ml. 40 µl high-speed extract was incubated,
for 1 h at 4°C, with 20 µl protein A-Sepharose beads (Pharmacia LKB
Biotechnology, Piscataway, NJ) bound with mouse anti-myc monoclonal
antibody (ascites; Evan et al., 1985
) or, as a control, mouse anti-hemagglutinin monoclonal antibody (ascites; BABCO, Berkeley, CA), or affinitypurified rabbit anti-Sla1 antibody (a gift from D. Drubin, University of
California, Berkeley, CA) or, as a control, affinity-purified rabbit anti- glutathione S-transferase antibody. The beads were washed four times
with UBT and once with UBT + 0.5 M KCl and then incubated with 50 µl
UBT + 1 M KCl for 10 min. The eluted proteins were precipitated with
10% trichloroacetic acid and resuspended in 20 µl protein gel sample
buffer. 10 µl of each sample and 5 µl of the extract were loaded onto a
12.5% polyacrylamide gel. Immunoblot analysis was carried out using the
enhanced chemiluminescence detection kit (Amersham Corp., Arlington
Heights, IL).
BEE1 Is Required for Budding and Cytokinesis
A 2.4-kb yeast genomic DNA fragment was cloned that
contains the BEE1 coding region and its flanking sequences. A knock-out construct was made in which 84%
of the BEE1 coding region was replaced by the LEU2
gene. The knock-out construct was transformed into a
Leu2 diploid yeast strain. The diploid was sporulated and
the tetrads were dissected and analyzed. When the spores
were grown at room temperature, all of the tetrads had
two colonies that grew at a wild-type rate and two colonies
that grew much more slowly (Fig. 1 A). The fast growing
colonies all had a Leu
phenotype, whereas the slow growing ones were all Leu+ (data not shown), indicating that
the slow growth results from the integration of the knockout construct. Deletion of BEE1 gene in the Leu+ strains
was further confirmed by PCR analysis and complementation of the slow growth by a centromere plasmid carrying
BEE1 gene (see Materials and Methods). At elevated
temperatures (34°C and above),
bee1 cells are unable to
grow (Fig. 1 B). The temperature-sensitive growth phenotype is common to many mutations affecting actin cytoskeleton function (e.g., Adams et al., 1991
; Holtzman et
al., 1993
).
bee1 cells grown at 23°C were examined by light microscopy. The cells were heterogeneous in size and morphology, and many were larger than wild-type cells (Fig.
2 A). About 37% of
bee1 cells had more than one nucleus. A significant fraction of the cells (~24%, compared
to only 3% in wild-type population) were large budded
with divided nuclei. There were also clumps of cells that
could not be separated with a microdissection needle. These morphological characteristics suggest that the
bee1 mutation may affect budding and cytokinesis. In addition to the
above morphologies, a small fraction (~5%) of the cells
appeared amorphous and lysed. Cell death does not appear to be the main cause of the slow colony growth at
23°C, however, because >85% of
bee1 cells can form colonies (data not shown).
To better understand the bee1 deficiency, the rates of
bud emergence, bud growth, and cytokinesis were compared between
bee1 and wild-type cells. Because many
bee1 cells are in clumps, it was easier to observe the
above events on solid media. 40 unbudded
bee1 or wildtype cells of average sizes were aligned on agar with a dissection needle, and bud emergence was scored every 30 min. Surprisingly,
bee1 cells did not show any apparent
defect in bud emergence (Fig. 2 B). The slightly faster bud
emergence rate of
bee1 cells can be explained by the
larger sizes of
bee1 G1 cells, which presumably result from
a cytokinesis delay in the previous cell cycle (see below).
To estimate the rate of bud growth, individual budding
cells (starting from a bud size of ~1 µm) were followed by
video microscopy. Bud length was measured and plotted
as a function of time. Fig. 2 C shows bud growth of two
bee1 and wild-type cells. Bud length increased linearly
before reaching about 2.5 µm. The estimated rates of bud
length increase within the linear period were 19 ± 6 nm/
min for
bee1 cells (sample size: 17 cells) and 53 ± 16 nm/
min for wild-type cells (sample size: 16 cells). Since the buds in both strains are roughly spherical, the rate at
which the surface area increases in
bee1 cells was about
1/7 of that in wild-type cells.
The increase in the fraction of large-budded cells with
divided nuclei in bee1 liquid culture suggests that there
might be a delay in cytokinesis. But it was also possible
that the large-budded cells were actually two contacting
G1 cells. To directly test whether
bee1 causes a delay in
cell division, 40 large-budded (bud size >1/2 of mother
size)
bee1 or wild-type cells were aligned on agar, and
cell division was determined by attempting to separate the
bud from the mother with a microdissection needle. The
rate at which cell separation completes in
bee1 cells, estimated from the data shown in Fig. 2 D, was only 40% of
that in wild-type cells. This delay in cell division was not
likely to result from an S phase or a mitotic block, since in
bee1 liquid cell culture, there was not an increase in the
fraction of large-budded cells with undivided nuclei, characteristics of S or M phase-arrested cells.
Bee1p Is Essential for the Assembly of Cortical Actin Patches
Because actin is involved in both budding and cytokinesis,
the organization of actin filaments in bee1 cells was examined by rhodamine-phalloidin staining. In wild-type
cells, actin patches are concentrated in the bud during polarized growth (Fig. 3 A, a) and at the septum during cytokinesis (Fig. 3 B, a). In
bee1 cells, actin filaments appear to be assembled at the right regions, i.e., the bud or
the septum. However, actin is organized into structures
strikingly different from those in wild-type cells. Rather
than form cortical patches, actin filaments (F-actin) in
bee1 cells assemble into thick cables that do not seem to
be restricted to the cell cortex (Fig. 3 A, b and B, b). A few
scattered actin patches are seen in some cells, but these
patches are in the mother rather than the bud. Bars of actin are also found in the mother of some
bee1 cells (not
shown). The amount of F-actin in
bee1 buds, judged by
the intensity of rhodamine fluorescence, appears to be
higher than that in wild-type buds. This result suggests that
Bee1p plays an important role in the organization of actin
filaments but is not required for the polarized distribution of F-actin.
Actin assembly in the cell is regulated by many actinbinding proteins. The altered F-actin organization and
level in bee1 cells may correlate with changes in the localization of cellular actin-binding proteins. In wild-type
cells, fimbrin (Sac6p), cofilin (Cof1p), and capping protein
(Cap2p) are all localized in cortical actin patches (Drubin
et al., 1988
; Amatruda and Cooper, 1992
; Moon et al.,
1993
). In
bee1 cells, Sac6p is associated with the actin
bundles in the buds, whereas Cof1p and Cap2p do not appear to be enriched in the buds (Fig. 4). This suggests that
the actin bundles in the buds of
bee1 cells have a different biochemical composition from that of cortical actin
patches.
bee1 Cells Accumulate Post-Golgi Vesicles in the Bud
To further understand the bee1 phenotype, the cells were
examined by electron microscopy. As shown in Fig. 5 B,
bee1 mutant cells accumulate a large number of vesicles
mostly in the bud. Wild-type cells, by contrast, do not accumulate any vesicles (Fig. 5 A). In some
bee1 buds, the
vesicles appear to form linear arrays (Fig. 5 C), perhaps
along the aberrant actin bundles. The vesicles accumulated in
bee1 cells are similar in size to post-Golgi vesicles (80-100 nm) (Novick et al., 1980
). The accumulation
of these vesicles may reflect an exocytosis defect, which
would explain the slow rates of bud growth and cytokinesis in
bee1 cells.
Bee1p Localizes to Cortical Actin Patches
To ask whether Bee1p interacts directly with the cortical
actin cytoskeleton, Bee1p cellular localization was determined by using a multicopy plasmid that expresses myctagged Bee1p. This plasmid complements the slow growth
and temperature-sensitive phenotypes of the null mutant
(not shown), suggesting that myc-tagged Bee1p is functional. Immunofluorescence staining with an anti-myc antibody showed that Bee1p exhibits patchlike staining. This
staining pattern is not seen in cells that do not express
myc-tagged Bee1p (Fig. 6 A) or in cells that express an unrelated myc-tagged protein (data not shown), suggesting
that the staining is specific for myc-Bee1p. Double staining
using anti-actin and anti-myc antibodies showed that the
majority of Bee1p patches colocalize with actin patches
(Fig. 6 B), indicating that Bee1p is in fact a component of
the cortical actin patches. Bee1p does not appear to be enriched on actin cables in the mother.
Bee1p Interacts with Sla1p, an SH3 Domain-containing Cortical Cytoskeletal Component
The domain structure of WASP suggests that the WASP
family of proteins may provide a molecular scaffold that
brings together various structural and signaling proteins. I
tested whether Bee1p interacts with any known components of cortical actin patches. Myc-tagged Bee1p was immunoprecipitated from detergent-solubilized high-speed
supernatant of myc-Bee1p-expressing cells using protein A beads bound with anti-myc antibody. Proteins that coprecipitated with myc-Bee1p and were eluted from the
beads by 1 M KCl were analyzed by immunoblotting using
antibodies against yeast proteins required for actin cytoskeletal function, including Actin, Sac6p, Cap2p, Cof1p,
Sla1p, and Bem1 (Pringle et al., 1995). Only Sla1p and actin were detected in the high-salt eluate of anti-myc beads but not detected in the eluate of the control anti-hemagglutinin epitope beads (Fig. 7 a).
Anti-Sla1p antibody-bound protein A beads also precipitated myc-Bee1p (Fig. 7 b). Actin, however, was not detected in the Sla1p immunoprecipitate (not shown). Sla1p
is an SH3 domain-containing protein involved in cortical
actin assembly and organization (Holtzman et al., 1993; Li
et al., 1995
). The interaction between Bee1p and Sla1p
may be mediated through the SH3 domains of Sla1p and the proline-rich domain of Bee1p. Bem1p, a SH3 domain-
containing protein involved in budding (Bender and Pringle, 1991
), did not coimmunoprecipitate with Bee1p (Fig. 7
a), indicating that not all SH3-containing proteins interact
with Bee1p.
Bee1p Is Required for In Vitro Actin Assembly in Permeabilized Yeast Cells
We previously established a permeabilized cell assay for
cortical actin assembly in vitro (Li et al., 1995). Permeabilized
sla1 cells fail to assemble actin into the bud, suggesting that Sla1 may be involved in stimulating actin polymerization at cortical patches.
bee1 cells should also exhibit
an in vitro actin assembly defect since Sla1 is not localized
to the bud in these cells (Li, R., unpublished result). Permeabilized
bee1 and wild-type cells were prepared and
the rhodamine-actin assembly assay was carried out as described (Li et al., 1995
). While >60% of permeabilized wild-type cells were able to incorporate rhodamine-actin
into patches in the bud, permeabilized
bee1 were completely deficient in actin assembly in vitro (Fig. 8).
The Function of Bee1 Protein in Cortical Actin Patch Assembly
The results presented in this report showed that Bee1p is
a component of cortical actin patches and is essential for
the organization of actin filaments. Although null mutations in many cytoskeletal proteins result in a less polarized cortical actin distribution, accumulation of aberrant
actin structures, and disappearance of actin cables, bee1
is the only mutation reported so far that abolishes cortical
actin patches in the bud. Electron microscopy studies on
wild-type cells showed that at least some cortical actin
patches are membrane invaginations associated with highly organized actin filaments (Mulholland et al., 1994
). The
actin structures that replace cortical patches in the buds of
bee1 cells are bundles of actin filaments that, under light
microscope, do not appear to be restricted to the cell periphery. These aberrant actin structures lack many cortical
patch components including cofilin, capping protein, and
Sla1 and therefore are fundamentally different from actin
patches.
What might be the role of Bee1p in cortical actin patch
assembly? WH1, shared between WASP and Bee1p, can
potentially interact with phospholipids in the cell (Miki et al.,
1996). One simple model is that Bee1p localizes to the cell
cortex via WH1 and brings together a set of cytoskeletal
proteins that locally organize actin filaments into defined
membrane-associated structures. Cofilin is an actin-depolymerizing factor usually associated with dynamic actin in
the cell (Bamburg and Bray, 1987
; Yonezawa et al., 1987
), whereas capping protein can prevent elongation from the
barbed ends of actin filaments (Caldwell et al., 1989
; Amatruda and Cooper, 1992
). The lack of these actin-binding
proteins may contribute to the alteration of actin structures in the buds of
bee1 cells. There is no evidence that
Bee1p can directly recruit cofilin or capping protein. The
interactions of cofilin and capping protein with actin are
inhibited, at least in vitro, by phosphatidylinositol 4,5-bisphosphate (PIP2) (Yonezawa et al., 1990
; Amatruda and
Cooper, 1992
). Cofilin activity is also regulated by phosphorylation (for review see Moon and Drubin, 1995
). It is
possible that Bee1p can locally modulate the level of PIP2
or the phosphorylation state of cofilin by interacting with
the cofilin kinase or phosphatase.
Understanding how Bee1p functions will depend on the
identification of its interacting proteins. So far, there is
good evidence that one cytoskeletal component, Sla1,
physically interacts with Bee1p. It is not known whether
this interaction is direct, but an interaction between the
three SH3 domains of Sla1 with the proline-rich domain of
Bee1p is an appealing possibility. The proline-rich domain
of Bee1p may also interact with profilin, a possibility that
is currently under examination. The action of Bee1p, however, can not be mediated entirely through its interaction with Sla1, since the phenotype of an SLA1 null mutant is
less severe than that of bee1.
sla1 cells grow at a near
wild-type rate at 23°C and contain cortical actin patches,
although often in large clusters.
Bee1p, like N-WASP (Miki et al., 1996), also coimmunoprecipitates with actin. I have not been able to detect
any interaction between Bee1p and F-actin. It is likely that
Bee1p and N-WASP are actin monomer-binding proteins,
which may explain the in vitro F-actin-depolymerizing activity of N-WASP (Miki et al., 1996
). The result that actin
does not coimmunoprecipitate with Sla1p suggests that actin and Sla1p may be in different Bee1p-containing complexes.
In vitro analysis in permeabilized yeast cells revealed an
activity that promotes actin assembly in the bud. Although
the exact biochemical nature of this activity is not completely understood, the activity is distinct from the ends of
actin filaments and is dependent on Sla1p (Li et al., 1995).
The result that permeabilized
bee1 cells are also defective in actin assembly in vitro suggests that this activity
may depend on the interaction between Sla1p and Bee1p.
If Sla1p and Bee1p are important for the polarized actin
assembly, why is the F-actin content in
bee1 buds higher than that in the mothers? One explanation might be that
actin cables in
bee1 cells remain oriented toward the bud.
The cables may provide a transport system (for review see
Bretscher et al., 1994
) that delivers cytoskeletal proteins
and/or actin monomers to the bud, maintaining the polarity of actin distribution. In the absence of Bee1p, actin assembly in the bud may be stimulated by an aberrant mechanism that was not detected in the permeabilized cells.
The Role of Cortical Actin in Budding and Cytokinesis
The accumulation of cortical actin patches at growth sites
and at the plane of cytokinesis led to the view that these
actin structures are somehow required for cell surface generation (Adams and Pringle, 1984; Kilmartin and Adams,
1984
). The lack of cortical patches but not actin cables in
bee1 cells provides an opportunity to better define the
role of cortical patches in polarized cell growth and division. The results reported here suggest that disruption of
cortical patches does not prevent cell polarity establishment but slows down polarized cell surface growth and cytokinesis.
The phenotypes of act1 mutants first implicated actin in
exocytosis (Novick and Botstein, 1985), but the mechanism of this involvement is unclear. Vesicle accumulation
was also observed in other cytoskeletal mutants, including
tpm1 (tropomyosin gene disruption) (Liu and Bretscher,
1989
) and myo2-66 (a mutation in MYO2 which encodes a
class V myosin) (Johnston et al., 1991
).
tpm1 and myo266 cells accumulate vesicles predominantly in the mother,
probably reflecting a defect in vesicle transport into the
bud due to the lack of actin cables in
tpm1 cells or the loss of Myo2 motor function in myo2-66 cells.
bee1 cells,
by contrast, accumulate post-Golgi vesicles in the bud like
late sec mutants (Novick et al., 1980
). This suggests that
the lack of cortical actin patches in
bee1 cells does not affect the transport of vesicles to the bud but may affect the
membrane fusion step of exocytosis. It is also possible,
however, that the vesicles are sequestered by the aberrant
actin bundles in
bee1 buds and are unable to fuse with
the plasma membrane. Experiments are currently being
carried out to test the above possibilities and to identify the cargo of
bee1 vesicles.
The Relation between Bee1p and WASP
Is Bee1p a true homologue of WASP? The phenotype of
bee1 null mutants resembles, at a superficial level, some of
the cellular defects of WAS. For example, T cells from
WAS patients are characterized by size heterogeneity, reduced number of the actin-rich microvilii, and the appearance of abnormal surface structures. Overexpression of
N-WASP in Cos7 cells reduces the number of actin stress
fibers but results in F-actin accumulation in cortical areas
(Miki et al., 1996), consistent with a role for N-WASP in
cortical actin assembly. Expression of WASP in yeast,
however, does not rescue the
bee1 defect (Li, R., and T. Kirchhausen, unpublished result), suggesting that the functions of Bee1p and WASP, like their sequences, are not
completely homologous. The most significant difference between WASP and Bee1p is the lack of a recognizable Cdc42binding motif in the sequence of the latter. This is not surprising considering that yeast cells lacking Cdc42 function
are unable to polarize (Adams et al., 1990
), whereas
bee1
cells can, suggesting that Cdc42p and Bee1p may not be involved in the same pathway. Recent studies in tissue culture cells showed that the interaction between Cdc42p and
WASP is probably not required for the effects of Cdc42p on actin organization (Lamarche et al., 1996
). Therefore,
the significance of Cdc42-WASP interaction awaits further investigation.
Received for publication 2 October 1996 and in revised form 4 December 1996.
I am very grateful to Tom Kirchhausen for communicating the sequence similarity between Bee1p and WASP before its publication and for his encouragement throughout these studies. I am indebted to David Drubin, John Cooper, and John Pringle for generously providing antibodies. I am grateful to Susanna Rankin and Le Ma for their help in light microscopy, and Maria Ericsson for her expert assistance in electron microscopy. I am grateful to the members of my laboratory, and Dan Sun and Tika Li for support and encouragement. I thank Christine Field, Dan Finley, Ryn Miake-Lye, Tim Mitchison, David Pellman, Tom Rapaport, and Dirk Winter for their comments on the manuscript.This work was supported by Harcourt General Charitable Foundation.
Bee1p, Bee1 protein; GBD, GTPasebinding domain; SH3, Src homology 3; WAS, Wiskott-Aldrich syndrome; WASP, Wiskott-Aldrich syndrome protein; WH1, WASP homology domain 1.