Molecular Biology of the Cell
Vol. 10, 4121–4133, December 1999
The Rho GTPase Rho3 Has a Direct Role in Exocytosis
That Is Distinct from Its Role in Actin Polarity
Joan E. Adamo,*†Guendalina Rossi,* and Patrick Brennwald*†‡
*Department of Cell Biology and†Graduate Program in Molecular Biology, Cell Biology, and
Genetics, Weill Medical College of Cornell University, New York, New York 10021
Submitted June 16, 1999; Accepted October 7, 1999
Monitoring Editor: Juan Bonifacino
Budding yeast grow asymmetrically by the polarized delivery of proteins and lipids to specific
sites on the plasma membrane. This requires the coordinated polarization of the actin cytoskeleton
and the secretory apparatus. We identified Rho3 on the basis of its genetic interactions with
several late-acting secretory genes. Mutational analysis of the Rho3 effector domain reveals three
distinct functions in cell polarity: regulation of actin polarity, transport of exocytic vesicles from
the mother cell to the bud, and docking and fusion of vesicles with the plasma membrane. We
provide evidence that the vesicle delivery function of Rho3 is mediated by the unconventional
myosin Myo2 and that the docking and fusion function is mediated by the exocyst component
Exo70. These data suggest that Rho3 acts as a key regulator of cell polarity and exocytosis,
coordinating several distinct events for delivery of proteins to specific sites on the cell surface.
Delivery of newly synthesized proteins and lipids to specific
sites on the cell surface is critical for the polarized growth
observed in many eukaryotic cells. Budding yeast show a
high degree of polarity and polarized growth during several
stages of their cell cycle (Welch et al., 1994; Lew and Reed,
1995). They are polarized to the presumptive bud site in
unbudded cells. Polarized growth occurs at the bud tip in
small budded cells, then expands in larger buds, and finally
is directed to the mother–daughter neck in cells about to
undergo cytokinesis (Lew and Reed, 1995). Protein secretion
is also highly polarized and corresponds to the pattern of
bud growth (Tkacz and Lampen, 1973; Field and Schekman,
1980). This polarization of a yeast cell involves distinct re-
arrangements of the actin cytoskeleton and the transport of
vesicles to the site of polarized growth (Novick and Botstein,
Genetic analysis in yeast has identified a number of gene
products required for the terminal stages of exocytic traf-
ficking to the cell surface in yeast. These include the uncon-
ventional type V myosin Myo2 (Johnston et al., 1991), the rab
GTPase Sec4 (Salminen and Novick, 1987), a number of gene
products that form a multisubunit complex known as the
exocyst (TerBush et al., 1996; Finger et al., 1998; Guo et al.,
1999), and the SNAP receptor (SNARE) proteins Snc1/2,
Sso1/2, and Sec9 (Aalto et al., 1993; Protopopov et al., 1993;
Brennwald et al., 1994). Myo2 is thought to be involved in
the polarized delivery of vesicles from the mother cell to the
bud (Govindan et al., 1995; Pruyne et al., 1998), whereas the
rab, exocyst, and SNARE components all appear to function
at the docking and fusion step (Novick and Brennwald,
The rho family of GTPases is thought to have a central role
in the polarized growth process (Ridley, 1995; Drubin and
Nelson, 1996). Yeast cells contain five rho family members:
RHO1, RHO2, RHO3, RHO4, and CDC42. These genes, sim-
ilar to their mammalian counterparts, play a variety of roles
in cell polarity and polarized cell growth (Johnson, 1999;
Kroschewski et al., 1999). RHO1 is essential and is thought to
be involved in regulating the synthesis and integrity of the
cell wall (Drgonova et al., 1996). RHO2 is not essential and
has overlapping functions with RHO1 (Madaule et al., 1987).
RHO3 and RHO4 may have some partially overlapping
functions (Matsui and Toh-e, 1992a,b; Kagami et al., 1997)
and are thought to play a role in bud formation (Imai et al.,
1996). CDC42 and its guanine nucleotide exchange factor
CDC24 are thought to be the most upstream components in
the pathway for polarized bud formation (Pringle et al., 1995;
Richman et al., 1999).
A previous report (Imai et al., 1996) suggested a possible
connection between Rho3 and the exocytic apparatus by
identifying SEC4 as a multicopy suppressor of a rho3?
strain. However in the analysis of a temperature-sensitive
allele of RHO3 reported in this same study, they were unable
to detect any significant defect in the secretion of the exo-
cytic marker invertase (Imai et al., 1996). Here we show a
large number of additional genetic interactions between
Rho3 and components of the exocytic apparatus, and we
demonstrate that Rho3 has a direct role in exocytosis that is
independent of its role in regulating actin polarity. Further-
‡Corresponding author. E-mail address: firstname.lastname@example.org.
© 1999 by The American Society for Cell Biology4121
more, analysis of a conditional mutant indicates that the role
of Rho3 in exocytosis likely has two components: 1) a myo-
sin (Myo2)-mediated function in the transport of post-Golgi
vesicles from the mother cell to the bud and 2) an exocyst
(Exo70)-mediated function in the docking and fusion of
vesicles with the plasma membrane.
MATERIALS AND METHODS
Yeast Strains, Reagents, and Genetic Techniques
Cells were grown in YPD media containing 1% bacto-yeast extract,
2% bacto-peptone, and 2% glucose, all from Difco (Sparks, MD). For
all assays performed, 25°C was the permissive temperature,
whereas 14°C was used as the restrictive temperature. Room tem-
perature (RT) was ?22°C.
Sorbitol, sodium azide (NaN3), N-ethylmaleimide (NEM), ?-mer-
captoethanol, o-dianasidine, glucose oxidase, peroxidase, Cal-
cofluor, tetramethylrhodamine isothiocyanate (TRITC)-conjugated
phalloidin, Triton X-100, and 3-amino-1,2,4-triazole were obtained
from Sigma Chemical (St. Louis, MO). Zymolyase (100T) was from
Seikagaku (Tokyo, Japan). BSA and yeast nitrogen base were from
US Biologicals (Swampscott, MA). Formaldehyde (37%), gluteralde-
hyde, and Spurr’s resin were from Electron Microscopy Sciences (Ft.
Transformations for suppression analysis were performed using
the lithium acetate method described by Ito et al. (1983). Strain
crossing, tetrad dissection, diploid sporulation, and mating-type
determination were performed as described by Guthrie and Fink
Generation of Mutations in RHO3
Oligo-directed mutagenesis was performed to create the GTP- and
GDP-bound forms of RHO3, as well as the pool of effector domain
mutations (Kunkel et al., 1987). Fourteen synthetic oligonucleotides
were used to change each of the 14 amino acids in the effector
domain to other random amino acids. Each oligonucleotide had a
mixture of bases at the position where different amino acids were
desired and was designed to contain at least 10 bp of homology on
both sides of the mixed residue. Resulting mutants were then se-
quenced to determine the amino acid sequence.
To determine the phenotype of each of the rho3 mutants as the
only source of Rho3, we transformed the constructs into a diploid
yeast strain heterozygous for a chromosomal deletion of RHO3.
BY506 (a/?; GAL?/GAL?; ura3-52/ura3-52; leu2-3112/leu2-3112; his3-
?200/his3-?200; rho3?::LEU2/RHO3) has one copy of RHO3 dis-
rupted by the insertion of the LEU2 marker. The plasmids contain-
ing the rho3 mutant were cleaved within the URA3 gene to target
chromosomal integration at the URA3 locus. The transformants
were colony purified and sporulated, and tetrads were dissected
with a micromanipulator on YPD plates. The plates were grown at
25°C. The haploid progeny were then analyzed by replica plating
for the presence of the integrated mutants (scored as ura?), for the
presence of a disrupted copy of rho3 (scored as leu?), and for
conditional growth at 37 or 14°C.
Cells were grown in YPD to midlog phase, diluted back, and pre-
shifted to the restrictive temperature for 1 h (or kept at the permis-
sive temperature). From these cultures, two aliquots of 0.8 OD599U
were pelleted. One-half was resuspended in 10 mM NaN3and kept
on ice, while the other one-half was resuspended in low-glucose
(0.1%) YPD and shifted to 14°C for 5 or 10 h or to 25°C for 1.5 h.
After the shift, the fractions were resuspended in NaN3, and ali-
quots from both fractions were spheroplasted for 30 min at 37°C in
a 1.4 M sorbitol and Tris-buffered solution containing ?-mercapto-
ethanol and 0.1 mg/ml 100T zymolyase. Spheroplasted internal
fractions were then resuspended in 0.5% Triton X-100.
All internal and external samples were assayed at 37°C in a
sodium acetate and NEM buffer by adding 25 ?l of 0.5 M sucrose
and then stopping the reaction after 15 min with 150 ?l of 0.2 M
K2HPO4. The addition of an assay mix containing NEM, o-dianasi-
dine, glucose oxidase, and peroxidase in a 0.1 M KPO4buffer causes
a color change in the presence of glucose. This end-point assay was
fully stopped and developed by the addition of 6 N HCl. Spectro-
photometric readings were taken at A540, and units of activity were
calculated and reported as micromoles of glucose released per
minute per OD599of cells.
Invertase, Bgl2, and Carboxypeptidase Y (CPY) blots
RHO3, rho3-V51, and sec4-P48 cells were grown overnight to midlog
phase at 25°C in YPD. Strains were preshifted to the restrictive
temperature of 14°C for 1 h before being transferred to YP media
containing low (0.1%) glucose for 5 h. In addition, the late-secretory
temperature-sensitive mutant sec18-1 was preshifted to 37°C for 1 h
before a 3-h shift into low glucose. Whole-cell glass bead lysates
were prepared and boiled with 2? sample buffer. Samples were
subjected to SDS-PAGE, transferred to nitrocellulose, and probed
with affinity-purified ?-invertase antibody at 1:200 or affinity-puri-
fied ?-CPY antibody at 1:1000. For Bgl2 blots, cells were sphero-
plasted, and the internal and external fractions were separated.
Internal fractions were boiled in 2? sample buffer, and the external
fractions were boiled in 6? sample buffer. Samples were subjected
to SDS-PAGE, transferred to nitrocellulose, and probed with affin-
ity-purified ?-Bgl2 antibody at 1:100.
Wide-Field Fluorescence and Imaging
For actin staining, cells were grown overnight in YPD media to ?0.5
OD599. They were then either shifted to the restrictive temperature
of 14°C for 5 h or kept at the permissive temperature before fixative
(formaldehyde to 3.7%) was added directly to the media. A second
round of fixation was performed in KPO4buffer (pH 6.5), and the
cells were transferred to a sorbitol buffer for overnight storage at
The next day cells were permeabilized for 10 min in 0.1% Triton
X-100 and washed twice with PBS. Cells were resuspended in 100 ?l
of PBS and stained in the dark for 25 min with 35 ?l of 3.3 ?M
TRITC-phalloidin dissolved in methanol. Cells were washed six
times with PBS and then resuspended in the appropriate volume of
mounting media (90% glycerol with 4?,6-diamidino-2-phenylindol
to visualize DNA and o-phenylenediamine to retard photobleach-
ing). To study the deposition of chitin in the bud scars, cells from the
same round of fixation were not permeabilized, and 3 ?l of 1 mg/ml
Calcofluor was added instead of labeled phalloidin. Stained cells
was viewed on a Nikon Eclipse E600 microscope (Garden City, NY);
images were captured with a Princeton Instruments charge-coupled
device camera (Trenton, NJ) and Metamorph Imaging software
(Universal Imaging, West Chester, PA).
Thin-Section Electron Microscopy
Cells were grown overnight in YPD media to midlog phase and
diluted back the next day, and then one-half of the culture was shifted
(Rochester, NY) filter, washed with 0.1 M cacodylate, and resuspended
in a 0.1 M cacodylate and 3% gluteraldehyde fixative solution. Cells
were then allowed to fix for 1 h at RT and then overnight at 4°C. Cells
were spheroplasted for 40 min at 37°C in a KPO4-buffered solution of
0.3 mg/ml zymolyase and then washed with cold cacodylate. Then 1.5
OD U was pelleted in a 1.7-ml microfuge tube and incubated on ice for
1 h at RT with a solution of 2% osmium tetroxide and 0.1 M cacodylate.
J.E. Adamo et al.
Molecular Biology of the Cell 4122
aqueous solution of uranyl acetate for 1 h at RT in the dark. Each pellet
was rinsed twice with dH2O and taken through the following 10-min
ethanol. The pellet was rinsed once with 100% acetone and then covered
with a 50% acetone and 50% Spurr’s resin mixture. This remained at
RT for 5 h, the acetone and Spurr’s resin mixture was replaced with
100% Spurr’s resin, and these remained at RT overnight. The 100%
Spurr’s resin was changed the next day, and the pellets were baked in
Spurr’s resin for 24 h at 80°C. Thin sections were cut and layered onto
an uncoated copper grid and poststained with lead citrate and uranyl
acetate before viewing. Cells were viewed on a JEOL 100CXII Electron
microscope (Tokyo, Japan) and photographed at 60 kV.
The constructs were prepared by a recombinational cloning method
(Hudson et al., 1997) in which the indicated Rho3 fragments were
PCR amplified with 70 bp extensions on each end. These extensions
then directed the in vivo incorporation of the fragment into a
NcoI/PvuII-digested pOBD.CYH vector. Rho3 and activated Rho3-
V25 were amplified by PCR, whereas the Rho3-V25,V51 double
mutant was generated by fusion PCR, and all were confirmed by
DNA sequence analysis. The C-terminal CAAX box was deleted by
placing a termination codon in place of the cysteine residue. The
GAL4AD constructs were made in a similar way and inserted into
the activating domain vector pOAD by recombinational cloning. All
constructs were tested to verify that they did not interact when
paired with the opposing empty vector. Transformants were shown
to express the expected size of the GAL4BD-RHO3 fusion by West-
ern blotting and then transformed with the GAL4AD-MYO2 or
GAL4AD-EXO70 constructs. Constructs were transformed into the
strain PJ694? that has the HIS3 gene as a reporter (James et al.,
1996). To reconfirm that similar amounts of Rho3 fusion proteins
were present, immunoblotting of the two-hybrid transformants
was performed on whole-cell lysates as described above. Blots
were probed with affinity-purified Rho3 antibodies and detected
Identification of RHO3 as a Suppressor of the
We identified previously a number of genes, including the
plasma membrane SNARE SEC9, on the basis of their ability
to act as suppressors of the cold-sensitive Rab GTPase mu-
tant sec4-P48 (Brennwald et al., 1994). Although SEC9 was
the most potent multicopy suppressor, we describe here the
analysis of a second suppressor, HSS43, whose strength of
suppression was nearly as strong as that of SEC9. Analysis
of subclones of HSS43 (Figure 1A) identified the 2.2-kb
SalI-XhoI fragment that contained the entire open reading
frame of RHO3 as necessary and sufficient to achieve the
suppression attributed to HSS43 (Figure 1B). To determine
the specificity of Rho3 in this pathway, we looked at the
suppression capabilities of all five of the yeast RHO family
members. The results shown in Figure 2A demonstrate
clearly that this property is highly specific to Rho3 because
none of the other RHO genes, RHO1, RHO2, RHO4, or
CDC42, were able to interact genetically with Sec4 in the
same way that Rho3 did.
Rho3 is a member of the Ras superfamily of small GTPases
that function as molecular switches (Bourne et al., 1991). By
analogy to the ras oncogene, mutations were made in Rho3
that predict a constitutively active (GTP-bound) or a consti-
tutively inactive (GDP-bound) form. We made two muta-
tions, RHO3-V25, equivalent to the activating ras-V12 mu-
RHO3. (A) A restriction map of the
10-kb HSS43-suppressing fragment
and the subclones derived from it
are shown. (a) HSS43 (10 kb). (b)
PvuII fragment. (c) SalI fragment.
(d) SalI-SmaI fragment. (e) SalI-XhoI
2.2-kb fragment. (B) The SalI-XhoI
RHO3 open reading frame (ORF) is
exclusively responsible for the sup-
pression seen by HSS43.
Rho3 Function in Exocytosis
Vol. 10, December 19994123
tant, and RHO3-N30, analogous to the dominant-inhibitory
ras-N17 mutant. We would predict that if the GTP-bound
form is the active form in this pathway, there would be
suppression at much lower doses of RHO3-V25 compared
with wild type and that the GDP-bound form would fail
to suppress. We therefore examined the effect of each of
these on single copy (CEN) plasmids and tested their
ability to suppress the sec4-P48 mutant (Figure 2B). Inter-
estingly, a single extra copy of wild-type RHO3 results in
only partial suppression of the cold sensitivity. However
activated RHO3-V25 on a single copy shows suppression
equivalent to that observed with wild-type RHO3 on high
copy. In contrast, the RHO3-N30 allele, predicted to be in
the GDP-bound form, not only fails to suppress but is
RHO3 Shows Multiple Genetic Interactions with
Components of the Exocytic Apparatus
In addition to testing the ability of RHO3 to suppress sec4-
P48, we examined the ability of the original clone HSS43 and
the RHO3 subclone to suppress all of the 10 late-acting sec
genes. We found that HSS43 and RHO3 show a significant
level of suppression of both sec15-1 and sec8-9 (Brennwald et
al., 1994) (our unpublished results). Both of these genes are
components of the exocyst, a peripherally associated plasma
membrane complex thought to aid docking and fusion of
vesicles (TerBush and Novick, 1995).
In addition, a number of late-acting secretory genes have
been found to suppress rho3 mutants. Both SEC9 and SEC4
act as high-copy suppressors of rho3 (Imai et al., 1996; Leh-
man et al., 1999). SRO7 and SRO77 were first identified as
high-copy suppressors of a rho3 mutant (Kagami et al., 1998).
We have shown recently that Sro7 and Sro77 are Sec9-
binding proteins and that loss of these gene products results
in a block in Golgi-to-cell-surface transport (Lehman et al.,
To look for additional connections between rho3 and the
exocytic apparatus, we used a galactose turn-off assay to
identify genes that could rescue the extremely slow growth
phenotype of rho3? cells. Cells with the sole copy of RHO3
under the control of a Gal promoter were transformed with
a genomic library prepared in a 2? vector. Transformants
were selected on glucose-containing media to repress ex-
pression of Rho3. Plasmids were isolated from colonies
showing growth on glucose, retested for suppression, and
sequenced. This resulted in the identification of four distinct
suppressing loci (in addition to RHO3). Two of these con-
tained genes shown previously to suppress rho3 mutants,
SEC4 and BEM1, a gene shown to be involved in bud for-
contained overlapping segments of the t-SNARE SSO2;
subcloning revealed this gene to be necessary and suffi-
cient for the suppressing activity. The fourth gene is still
being characterized and will be described elsewhere. In
summary, four distinct components of the exocytic ma-
chinery, Sec4, Sec9, Sro7, and Sso2, can act as suppressors
of rho3? mutants. This demonstrates that loss of Rho3
function is most readily assisted by increasing the func-
tion of the exocytic machinery. These numerous genetic
interactions strongly suggest that Rho3 plays a critical
role in exocytosis.
Three independent clones
Mutational Analysis of the RHO3 Effector Domain
We hypothesized that the above genetic evidence suggests
an additional critical role for Rho3 in exocytosis, indepen-
dent of its role in actin polarity maintenance. This is consis-
tent with the rapidly growing body of evidence that Rho
GTPases often have a large number of effectors that can
cover a diverse range of functions (Tapon and Hall, 1997;
Aspenstro ¨m, 1999). In an attempt to reveal a direct role of
Rho3 in exocytosis and to distinguish this role from the role
of RHO3 in actin polarity, we initiated an extensive mu-
tagenesis of the effector domain. This highly conserved re-
gion has been shown to be critical for interactions between
ras GTPase superfamily members and their downstream
effectors (Bourne et al., 1991).
The region of Rho3 known as the effector domain can be
aligned with the same conserved domains from other
proteins such as H-ras and Cdc42 (Figure 3A). The results
of the oligo-directed mutagenesis are listed in Figure 3B.
Mutant alleles were introduced into yeast and tested for
their ability to function as the only copy of RHO3. Of the
46 mutants examined, all were recessive, and 30 were
indistinguishable from wild type under the conditions
tested. Eleven mutants failed to rescue the severe growth
bound form is the active form. (A) RHO3 is the only one of the five
RHO genes, RHO1, RHO2, RHO3, RHO4, or CDC42, that can sup-
press the cold sensitivity of sec4-P48. (B) Activated alleles of RHO3
suppress at a single copy. RHO3-V25 has amino acid 25 changed
from a glycine to a valine that is analogous to the ras-V12-activating
mutation. RHO3-N30 has the threonine at amino acid 30 changed to
an asparagine, analogous to the ras-N17 dominant-inhibitory muta-
tion. Each of these was introduced into sec4-P48 at a single copy, and
transformants were tested for suppression of the cold sensitivity.
RHO3 is a specific suppressor of sec4-P48, and the GTP-
J.E. Adamo et al.
Molecular Biology of the Cell4124
defect associated with the rho3? deletion, and 1 mutant
rescued slightly but gave constitutively slow growth un-
der all conditions tested. Four mutants showed relatively
normal growth at 25°C but showed significantly reduced
growth at 14°C (Figure 3, these last four are shown in bold
type). Figure 3C shows the growth of four cold-sensitive
mutants at 14°C as compared with wild type and com-
pared with their growth at the permissive temperature of
25°C. None of the mutants showed sensitivity to growth
at high temperature (37°C) in this study. Bright-field anal-
ysis of all four of the mutants revealed an increase in cell
size, a phenotype often associated with defects in cell
polarity (Novick and Botstein, 1985). But this enlargement
was not dependent on the temperature shift in any of the
cells, and the most dramatic size increase was seen in the
mutant rho3-C40 (our unpublished results).
Analysis of the Actin Cytoskeleton in rho3 Mutants
Loss of Rho3 function has been shown previously to cause
an altered distribution in the normally highly polarized actin
cytoskeleton (Imai et al., 1996). We therefore examined the
status of the actin cytoskeleton in the four cold-sensitive rho3
mutants by rhodamine phalloidin staining. Both actin fila-
ments and cortical actin patches show a very high degree of
polarity in yeast (Welch et al., 1994; Lew and Reed, 1995;
Karpova et al., 1998). In wild-type cells, cortical actin is
highly polarized and found almost exclusively in the bud
(Welch et al., 1994; Lew and Reed, 1995). Only as the cell
nears cytokinesis do the cortical patches become signifi-
cantly apparent in the mother cell. Actin cables are normally
oriented along the mother–bud axis.
Cells were examined after growth at 25°C or after a 5-h shift
to 14°C. As expected, several of the effector mutants demon-
domain. (A) Alignment of the 14 amino acids
of the effector domains of Rho3, H-ras, and
Cdc42. (B) Forty-six single-point mutations
generated by oligo-directed
Growth at each temperature was recorded as
follows: (?) ? wild-type growth, (?/?) ?
borderline growth, (?/?) ? slight growth
above background, and (?) ? no growth. (C)
Growth of the rho3 mutants at the permissive
and restrictive temperature. Plates at 25°C
were incubated for 4 d, and plates at 14°C
plates were incubated for 7 d.
Mutagenesis of the Rho3 effector
Rho3 Function in Exocytosis
Vol. 10, December 1999 4125
the mutants show a loss of the normal actin patterns. In par-
ticular the most severe defects were seen in rho3-C40 and
rho3-G46. They showed almost complete loss of cables and
random distribution of cortical patches. rho3-G49 showed a
partial loss of cables and a disruption of cortical patch polar-
ization. In all three of these mutants, the actin polarity defects
appeared to be similar at both temperatures.
In contrast, the rho3-V51 mutant had a normal and polar-
ized actin cytoskeleton that was indistinguishable from the
polarized actin in wild-type cells at both 25 and 14°C. As
with wild-type cells, the cortical actin patches were well
polarized to the bud. The cables were clearly present, well
organized, and of normal length relative to cell size (i.e.,
because rho3-V51 cells are larger, the cables are proportion-
ally longer). We also examined diploids homozygous for the
rho3-V51 mutation and found they also showed staining
patterns identical to that of wild-type diploids at both the
permissive (our unpublished results) and restrictive temper-
atures (Figure 4).
The proper formation of chitin scars at bud sites is depen-
dent on the polarization of actin (Chant and Pringle, 1991).
We therefore looked for bud site selection defects in a
subset of these rho3? mutants by staining chitin with the
fluorescent dye Calcofluor. The results demonstrate that
rho3-V51 exhibited normal budding patterns in both hap-
loids and homozygous diploids, and in contrast, the rho3-
C40 mutant and, to a somewhat lesser extent, the other
mutants showed aberrant bud site selection and delocal-
ized chitin deposition (our unpublished results). This pro-
vides additional evidence that out of the four cold-sensi-
tive effector mutants, only rho3-V51 is fully functional in
regulation of the actin cytoskeleton.
The rho3-V51 Mutant Has a Post-Golgi Secretory
To determine whether the actin-independent growth defect
associated with the rho3-V51 mutant was caused by a defect
in the exocytic function of these cells as predicted by the
cells were subsequently permeabilized and stained as described in MATERIALS AND METHODS. Bar, 5 ?m.
Actin localization in wild-type and rho3 mutant cells. Cells were grown at 25°C or shifted to 14°C for 5 h before fixation. Fixed
J.E. Adamo et al.
Molecular Biology of the Cell4126
abundance of genetic data described above, we examined
the ability of these strains to export two periplasmic pro-
teins, invertase and Bgl2. Both of these proteins are depen-
dent on the classical secretory pathway for their delivery to
the cell surface (Kaiser et al., 1996). RHO3, rho3-V51, and
sec4-P48 strains were shifted to low glucose to derepress the
show a defect in the secretion for rho3-V51 at both the permissive and restrictive temperature. Secretion assays are presented as a percentage
of invertase that is found internally. This is a positive measure of the secretory defect associated with a particular strain. This percentage of
internal invertase is calculated by internal/(internal ? external), where these values are measured as micromoles per minute per OD599. The
data represent the average ? SD for five assays. (B) Immunoblot analysis of Bgl2 protein distribution shows a defect in secretion for rho3-V51
at both the permissive and restrictive temperature. The distribution of the secreted protein Bgl2 was determined by immunoblot analysis
using affinity-purified antibodies raised against the C terminus of Bgl2. The band corresponding to Bgl2 is indicated. Top, quantitation of the
bands was done using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). The results are presented as a percentage of the total Bgl2
that is found internally. The data represent the average ? SD for three experiments. Bottom, a representative Bgl2 blot is shown. (C)
Immunoblot analysis of invertase reveals the glycosylation state of invertase in these cells. (D) Transport of the vacuolar protein CPY is not
affected in rho3-V51, because it is found exclusively in the mature form (mCPY, 61 kDa) at both temperatures. A sec18-1 mutant was included
as a size marker and exhibits accumulation of the core glycosylated form (p1, 67 kDa). glycos., glycosylated.
The rho3-V51 mutant is defective in the secretion of the periplasmic enzymes invertase and Bgl2. (A) Invertase enzyme assays
Rho3 Function in Exocytosis
Vol. 10, December 1999 4127
production of invertase at either 25 or 14°C. The amount of
internal and external enzyme activity was determined in five
separate experiments, and the amount of invertase in the
internal fraction is shown in Figure 5A. As expected, the
sec4-P48 mutant shows a defect at 14°C but secretes well at
25°C. In agreement with the prediction that Rho3 has a
direct role in exocytosis, the rho3-V51 cells display a pro-
nounced defect in invertase secretion similar to that of sec4-
P48. Although wild-type cells were able to secrete 92% of
invertase and retained only 8% in the internal fraction at
14°C, the sec4-P48 mutant retained 33%, and rho3-V51 re-
tained 27% in the internal fraction at the restrictive temper-
ature. Surprisingly, when tested at 25°C, the rho3-V51 mu-
tant showed a significant defect in exocytosis at the
permissive temperature (21% internal invertase for rho3-V51
compared with only 5% for wild-type cells).
We also examined the ability of the other mutants to
secrete invertase after a 5-h shift to 14°C. They were found to
the mother to the bud. (A) Three panels of rho3-V51 at both 25 and 14°C as well as single panels of RHO3 at 25 and 14°C are shown.
Preparation of the cells is described in MATERIALS AND METHODS. (B) Cells were scored for the location of accumulated vesicles, and bar
graphs show the percentage of cells with vesicles in the bud/neck region, in the mother cell, and in the entire cell. Approximately 30 cells
were counted for rho3-V51 at each temperature, and 20 cells were counted for the RHO3 cells (wt). Only small budded cells were counted.
Bars, 1 ?m.
Electron microscopy of rho3-V51 cells reveals accumulation of post-Golgi vesicles and a conditional defect in vesicle delivery from
J.E. Adamo et al.
Molecular Biology of the Cell 4128
be significantly less defective in secretion: 17 ? 9.8% (rho3-
C40), 10 ? 6.5% (rho3-G46), and 16 ? 9.6% (rho3-G49). How-
ever, because defects in actin have been shown previously to
cause defects in secretion (Novick and Botstein, 1985), it is
unclear whether these partial defects are associated with a
direct effect on the secretory capacity versus an indirect
effect resulting from a depolarized actin cytoskeleton.
showed that virtually all of the invertase in the cells
migrated as the fully glycosylated form, demonstrating
that the defect is likely to be at the post-Golgi stage of
secretion (Figure 5C). Consistent with a post-Golgi defect,
maturation and transport of CPY appear to be unaffected.
Immunoblots of these cells show that only the mature
form is present in rho3-V51 as in wild-type and sec4-P48
(Figure 5D). In addition, pulse-chase analysis of CPY mat-
uration demonstrated that there was no defect in the
kinetics of transport of CPY to the vacuole (our unpub-
Two classes of vesicles have been demonstrated to be
involved in post-Golgi delivery to the plasma membrane
(Harsay and Bretscher, 1995). Invertase is specific to the
lighter class of vesicles, and Bgl2, a cell wall endoglu-
canase, has been shown to be specific to the denser class of
vesicles. To determine whether both classes of vesicles
were affected, we examined the secretion of Bgl2 in the
rho3-V51 and sec4-P48 cells. Immunoblots were used to
probe the internal and external fractions at both the per-
missive and restrictive temperatures. Figure 5B shows the
results of Bgl2 secretion assays that are expressed as the
percentage of Bgl2 found internally. In agreement with
the results seen for the secretion of invertase above, rho3-
V51 shows a pronounced defect in the secretion of Bgl2 at
both 25 and 14°C. Although wild-type RHO3 cells show
small internal pools of only 13 and 19% at 25 and 14°C, the
rho3-V51 cells show much larger internal pools of 38 and
40%, respectively. The quantitation is the mean ? SD of
three assays, and a representative blot is shown (Figure
5B, bottom). The identity of the bottom band seen in all
the external fractions is unknown and was not included in
the quantitation. This defect in Bgl2 secretion indicates
that the rho3-V51 mutant is affecting both classes of vesi-
cles. Interestingly, the sec4-P48 mutant appears to be less
temperature dependent in its secretion of Bgl2 than inver-
tase because it shows defects at both temperatures.
rho3-V51 Shows a Cold-Sensitive Defect in the
Delivery of Vesicles from Mother to Bud
To examine the exocytic defect in greater detail, we exam-
ined rho3-V51 mutant cells by electron microscopy. All of the
post-Golgi–blocked secretory mutants show an accumula-
tion of 80- to 100-nm vesicles. These vesicles are only rarely
seen in wild-type cells because their rate of fusion with the
bud tip plasma membrane is very rapid. Because the rho3-
V51 mutants had secretory defects at both ambient and low
temperatures, we examined the morphology of cells grown
with and without a 5-h shift to 14°C. Consistent with the
secretory defect seen at 25°C, the rho3-V51 mutant shows a
large accumulation of 80- to 100-nm vesicles. The vesicles
appear to be present primarily in the bud and neck region of
the cells (Figure 6A, left). In contrast, although rho3-V51 cells
grown at 14°C had a slight increase in the total number of
vesicles per cell, a dramatic shift was observed in the loca-
tion of the vesicles within the cell. Although mother cells
with large numbers of vesicles were rare at 25°C, an abun-
dant accumulation of vesicles in the mother cells was ob-
served after a 5-h shift to 14°C (Figure 6A, middle). Wild-
type RHO3 cells are also shown in Figure 6A, right. The
quantitation of the vesicle accumulation phenotype is shown
as a bar graph (Figure 6B). At 14°C, there is a dramatic shift
in the distribution of vesicles within the cell; 60% of the
mother cells contained ?60 vesicles, whereas ?20% of the
cells at the permissive temperature fall into this category.
This demonstrates that the rho3-V51 mutant has a cold-
sensitive defect in the transport of vesicles from the mother
cell into the bud.
The V51 Mutation in Rho3 Results in a Loss of the
Interaction with Myo2 and Exo70
A recent study identified fragments of Exo70- and Myo2-
coding sequences in a two-hybrid screen for potential
effectors of Rho3 (Robinson et al., 1999). To determine
whether the exocytic defects associated with rho3-V51
might be a result of a defect in interaction with these
Figure 6 (cont.)
Rho3 Function in Exocytosis
Vol. 10, December 19994129
candidate effectors, we examined the effect of the rho3-V51
mutation on this interaction. We made use of the same
regions of MYO2 and EXO70 identified by Robinson et al.
(1999), fused to the activation domain of GAL4, whereas
the RHO3 alleles were fused to the DNA-binding domain
of GAL4. In this assay, we made use of the HIS3 gene as a
reporter of activation by the two-hybrid constructs. Acti-
vation of this reporter results in growth in the absence of
histidine. The strength of interaction can be monitored in
part by the sensitivity of growth to the presence of 3-ami-
notriazole, an inhibitor of the histidine biosynthetic path-
way. As can be seen in Figure 7A, a portion of the MYO2
neck and tail region (amino acids 871-1204) interacts spe-
cifically with the activated allele of RHO3, RHO3-V25.
However, the introduction of the V51 effector mutation
into RHO3-V25 completely abolishes the interaction with
MYO2. Like MYO2, the C-terminal portion of EXO70
shows both a strong interaction with the activated RHO3-
V25 as well as a weaker interaction with the wild-type
form of RHO3. The strength of the interaction between
activated RHO3-V25 and EXO70 is apparent because
growth occurs when 5 mM 3-aminotriazole is present and
persisted even in the presence of 40 mM 3-aminotriazole
(Figure 7A; our unpublished results). As was the case
with the MYO2 interaction the presence of the V51 muta-
tion in the activated form of RHO3 leads to a complete
loss of growth even on ?His media lacking 3-aminotria-
zole (Figure 7A). Therefore, the rho3-V51 mutation leads
to a loss of the ability of Rho3 to interact with both Myo2
and Exo70. This is consistent with the vesicle delivery
defects we observe at 14°C being attributable to a loss of
interaction with Myo2 and with the docking and fusion
of Rho3, Rho3-V25, and Rho3-V25,V51 mutants on the interaction with Myo2 and Exo70. Constructs containing GAL4BD-RHO3 and the
GAL4AD-MYO2 (encoding residues 871-1024) or EXO70-GAL4AD (encoding residues 238–623) were transformed into PJ694?. Four inde-
pendent transformants were replicated onto media selecting for both plasmids (?Trp, ?Leu) or media that require activation of the HIS3
reporter (?His), and interactions were assayed by the ability of the two GAL4 fusions to activate the reporter gene HIS3. Growth on media
lacking histidine or on media lacking histidine with 5 mM 3-aminotriazole (3-AT), which functions as an inhibitor of the histidine biosynthetic
pathway and requires a higher level of HIS3 activation to support growth, is shown. (B) Western blot analysis of Rho3 protein levels in the
two-hybrid transformants using affinity-purified ?-Rho3 antibodies. Cotransformants were examined for the expression of the Gal4-binding
domain (BD) fusion proteins. The endogenous Rho3 protein migrates at ?29 kDa, and a larger fusion protein of the expected size is visible
migrating at ?48 kDa.
Two-hybrid analysis of the effect of the V51 mutation on the interaction of Rho3 with Myo2 and Exo70. (A) Two-hybrid analysis
J.E. Adamo et al.
Molecular Biology of the Cell4130
defects observed at 25°C being attributable to defects in
interaction with the Exo70 component of the exocyst com-
plex (TerBush et al., 1996).
Analysis of an extensive collection of mutants in the effector
domain of Rho3 allowed us to separate the exocytic function
of Rho3 from its role in regulating actin localization. Our
results demonstrate that Rho3 acts as a key regulator of cell
polarity by coordinating two important cellular processes: 1)
maintenance and localization of the actin cytoskeleton and
2) the polarized delivery, docking, and fusion of post-Golgi
secretory vesicles with the plasma membrane. Genetic evi-
dence gave us the first clues concerning a possible role for
Rho3 in exocytosis, but it was the mutagenesis of the effector
domain in particular that allowed us to discriminate clearly
its role in exocytosis from its role in actin polarity. Of the
four loss-of-function mutations that we studied, three
showed clear actin polarity defects, whereas the fourth
showed no defects in actin polarity but had an exocytic
defect. It was especially important to distinguish between
these two possibilities because actin mutants are known to
cause defects in post-Golgi trafficking (Novick and Botstein,
Our results suggest that Rho3 regulates cell polarity by
simultaneously directing the rearrangements of the actin
cytoskeleton and the polarized delivery and fusion of the
vesicles to specific sites on the cell surface. This is clearly
important because these are both dynamic processes in yeast
cells. Although the bud tip is the major destination of po-
larized growth early in the cell cycle, growth becomes iso-
tropic later in the cell cycle. The site of actin polarity and
delivery reorients itself to the mother–bud neck, just before
cytokinesis. Rho3 could be acting to maintain the proper
synchronized actions of actin and vesicle transport; both in
specifying a new site and in maintaining delivery to the
In this view, it is especially interesting to find evidence
that Rho3 functions in two separate steps of exocytosis: the
delivery of vesicles and the docking and fusion of the vesi-
cles. Rho3 shows a number of strong genetic interactions
with gene products involved in this second step of the
docking and fusion of secretory vesicles at the plasma mem-
brane. In particular, rho3? was suppressed by the two
t-SNAREs Sec9 and Sso2, as well as by the Sec9-binding
protein Sro7 (Kagami et al., 1998; Lehman et al., 1999). In
addition, there are strong interactions with two subunits of
the exocyst complex and the Rab GTPase Sec4. Recent data
have suggested that the Rab GTPase and exocyst complex
function as tethering factors and work just upstream of
SNAREs in driving the vesicles to initiate the fusion reaction
(Pfeffer, 1999). Presumably the genetic interactions between
these components reflect the role of Rho3 in the docking and
With respect to the vesicle delivery step, a conditional
defect was apparent when electron microscopy revealed that
a shift to 14°C resulted in a very clear accumulation of
vesicles in the mother cell. Because vesicles are thought to be
primarily produced in the mother cell, they must be deliv-
ered to their site of fusion in the bud, so an accumulation of
vesicles in the mother cell is a result of a defect in this
vectorial delivery process. This cold-sensitive delivery phe-
notype of Rho3 is strikingly similar to the phenotype of an
unconventional type V myosin Myo2 mutant (myo2-66) that
shows a conditional accumulation of vesicles in the mother
cell at the nonpermissive temperature (Govindan et al.,
Recently, Robinson et al. (1999) isolated two candidate
effectors of Rho3 using a two-hybrid screen. This resulted in
the identification of Myo2 and Exo70 as proteins that spe-
cifically interact with the GTP-bound form of Rho3. Our
analysis of the rho3-V51 mutant gives strong evidence that
Myo2 and Exo70 are likely to be bona fide effectors of Rho3
function in exocytosis. In particular the loss of binding in-
teraction, as measured by two-hybrid analysis, between
rho3-V51 and both MYO2 and EXO70 strongly supports the
idea that it is a defect in interaction with these two factors
that is responsible for the exocytic defects we observe in this
mutant. In addition the constitutive exocytic defect of the
rho3 mutants may be caused by a defect in the activation of
the exocyst complex via Exo70. The electron microscopy of
the mutant under permissive conditions supports this idea.
The accumulation of vesicles in the bud and not in the
mother in rho3-V51 cells at 25°C suggests a defect at the
docking and fusion stage. This is similar to the phenotype
observed in temperature-sensitive mutations in components
exocytosis. GTP-bound Rho3 appears to have three distinct effector
pathways. Previous work (Matsui and Toh-e, 1992b; Imai et al.,
1996) and results presented here show a role for Rho3 in regulating
the polarized distribution of the actin cytoskeleton. Here we dem-
onstrate a role for Rho3 in exocytosis that is independent of its effect
on the actin cytoskeleton. In particular, genetic and electron micros-
copy data suggest that Rho3 acts both in the delivery of Golgi-
derived vesicles from the mother to the bud (Step 1) and in the
docking and fusion of these vesicles with the plasma membrane
(Step 2). The data presented here provide strong support for the
model suggested by the two-hybrid interactions identified by Rob-
inson et al. (1999). Therefore our analysis of the rho3-V51 effector
domain mutant supports the model that the vesicle delivery func-
tion of Rho3 (Step 1) is mediated by Myo2 and that the vesicle
docking and fusion function of Rho3 (Step 2) is mediated by Exo70.
A model for the involvement of Rho3 in two steps of
Rho3 Function in Exocytosis
Vol. 10, December 19994131
of the exocyst complex after a brief shift to 37°C (Govindan
et al., 1995).
Taken together, these data suggest a model, shown in
Figure 8, in which Rho3 acts as a key regulator of polarized
trafficking by coordinating three distinct effector pathways.
One pathway involves the regulation of the actin cytoskel-
eton, whereas the other two pathways involve exocytic
transport to the cell surface. This includes the regulation of
a Myo2-dependent delivery of vesicles from the mother cell
to the bud (Figure 8, Step 1) and regulation of an Exo70-
dependent docking and fusion of vesicles at specific sites on
the plasma membrane (Step 2). These data give us an outline
of the function of Rho3 in exocytosis and cell polarity and
place it as a key step in the coordinate regulation of these
two essential processes. Future work will no doubt unravel
the precise mechanisms by which Rho3 effects these func-
The authors are especially grateful to Dr. Geri Kreitzer and Dr.
Enrique Rodriguez-Boulan for allowing us use of their microscope
and their expertise and assistance with the fluorescence microscopy
and image analysis. We also thank Henry Hamilton for generation
of the RHO3-V25 and RHO3-N30 mutants, Linda Burg-Freedman
and Lee Cohen-Gould for assistance with electron microscopy, Dr.
Doug Johnson and Dr. Mike Snyder for plasmids, and Dr. Ruth
Collins, Dr. Tim McGraw, Luba Katz, Mayya Maksimova, and Greg
St. John for critical reading of the manuscript. This work was
supported by grants from the Mathers Charitable Foundation and
the Pew Scholars in Biomedical Sciences Program and by the Na-
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