Verprolin function in endocytosis and actin organization
Roles of the Las17p (yeast WASP)-binding domain and a novel
C-terminal actin-binding domain
Thirumaran Thanabalu
1,2
, Rajamuthiah Rajmohan
2
, Lei Meng
2
, Gang Ren
4,5
, Parimala R. Vajjhala
4
and Alan L. Munn
1,3,4,6
*
1 Institute of Molecular and Cell Biology, A*STAR Biomedical Science Institutes, Singapore
2 School of Biological Sciences, Nanyang Technological University, Singapore
3 Department of Biochemistry, Yong Loo Lin School of Medicine, The National University of Singapore, Singapore
4 Institute for Molecular Bioscience and ARC Special Research Centre for Functional and Applied Genomics, The University of Queensland,
St Lucia, Australia
5 UMR7156, CNRS, Universite Louis Pasteur, Strasbourg, France
6 School of Biomedical Sciences, The University of Queensland, St Lucia, Australia
The actin cytoskeleton is a complex and highly dynamic
intracellular protein network with essential roles in
cell polarity and morphogenesis. Much of our under-
standing of the actin cytoskeleton has come from
genetic studies using the unicellular eukaryote
Saccharomyces cerevisiae (budding yeast). Actin cyto-
skeleton components and regulators first discovered in
S. cerevisiae have often subsequently been found to

WASP). The LBD localizes C-Vrp1p to membranes and the cortical actin
cytoskeleton. Intriguingly, the LBD is sufficient to restore endocytosis and
growth at elevated temperature to Vrp1p-deficient cells. The CABS also
restores these functions, but only if modified by a lipid anchor to provide
membrane association. Our findings highlight the role of Las17p binding
for Vrp1p membrane association, suggest general membrane association
may be more important than specific targeting to the cortical actin cytoske-
leton for Vrp1p function in endocytosis and cell growth, and suggest that
Vrp1p binding to individual effectors may alter their physiological activity.
Abbreviations
CABS, C-terminal actin-binding submodule; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; GST, glutathione S-transferase;
LBD, Las17p-binding domain; LY, Lucifer yellow; PVDF, poly(vinylidene difluoride); VH2-C, verprolin homology 2 C-terminal domain; VH2-N,
verprolin homology 2 N-terminal domain; WASP, Wiskott–Aldrich syndrome protein; WIP, WASP-interacting protein.
FEBS Journal 274 (2007) 4103–4125 ª 2007 The Authors Journal compilation ª 2007 FEBS 4103
organism for functional analysis of actin cytoskeleton
components.
The basic elements of the yeast actin cytoskeleton
are cortical actin patches and cytoplasmic actin
cables. Actin patches are spots whose subcellular dis-
tribution is polarized towards sites of surface growth
during the cell cycle, i.e. nascent bud sites, the tips of
small buds, isotropically in large buds, and on either
side of the bud neck during cytokinesis. Actin cables
are thick filaments that align along the mother–bud
axis with their tips focused at sites of actin-patch
polarization [1–5]. Actin patches undergo rapid move-
ment at the cortex [6–9]. Some of these movements
correlate with endocytic cargo internalization, consis-
tent with a role for cortical actin patches in endocyto-
sis [10–15].

proteins (the sole yeast member is Las17p ⁄ Bee1p)
[20,25–29]. Interactions with actin monomers and
WASP-family proteins are key features shared with
human WIP [30–33]. Both N- and C-terminal Vrp1p
modules also bind type I myosins [22,28,34,35]. Eluci-
dating the physiological role of these interactions is
essential to understand the molecular basis of Vrp1p
function.
Like Vrp1p, Las17p and type I myosins localize to
cortical patches with a polarized distribution and parti-
ally colocalize with cortical actin patches [11,25,27,34].
Las17p and type I myosins are also essential for
both fluid-phase and receptor-mediated endocytosis
[20,27,36]. Like Vrp1p, localization of Las17p to corti-
cal patches is not perturbed by depolymerization of
actin filaments, however, polarization of Las17p pat-
ches requires F-actin [27,29]. Similarly, Las17p local-
ization to cortical patches is not dependent on Vrp1p
but Vrp1p is required for polarization of Las17p pat-
ches [29] (our unpublished data). The localization of
type I myosins to cortical patches is also not depend-
ent on Vrp1p, however, polarization of type I myosin
patches is dependent on Vrp1p [34]. This is consistent
with a role of F-actin and ⁄ or actin polymerization in
the generation or maintenance of a polarized distribu-
tion of cortical patches.
Las17p and type I myosins promote the assembly of
actin monomers into short actin filaments by binding
and stimulating the Arp2 ⁄ 3 complex [28,29,35,37,38].
The Arp2 ⁄ 3 complex is an actin filament nucleation

cortical actin-patch polarization? What is the rela-
tionship among cortical actin-patch polarization,
endocytosis, and growth at elevated temperatures?
Here we address these questions and show that both
a novel C-terminal actin-binding submodule (CABS)
containing a novel actin monomer binding verprolin
homology 2 C-terminal (VH2-C) domain and a sec-
ond submodule comprising the previously character-
ized LBD are essential for cortical actin-patch
polarization. Intriguingly, however, we find that each
of these submodules has the potential to at least par-
tially support endocytosis and growth at elevated
temperatures. We revise the model for Vrp1p func-
tion in the actin cytoskeleton based on these new
findings.
Function of Vrp1p C-terminal module T. Thanabalu et al.
4104 FEBS Journal 274 (2007) 4103–4125 ª 2007 The Authors Journal compilation ª 2007 FEBS
Results
C-Vrp1p residues K485 and R486 are essential for
cortical actin-patch polarization, but not for
localization to patches, endocytosis, or growth
at elevated temperature
To delineate the domains of C-Vrp1p
364)817
(Fig. 1)
responsible for restoration of endocytosis, growth at
elevated temperatures, and full cortical actin-patch
polarization we performed charged-to-alanine scanning
mutagenesis. A hydrophilicity profile of C-Vrp1p
364)817

patch polarization (Fig. 2C, Table 1). None of the
other seven substitutions had any apparent effect on
growth at elevated temperatures or cortical actin-patch
polarization (data not shown). This result highlights
the importance of residues K485R486, especially for
cortical actin-patch polarization.
To determine whether K485R486 are required for
endocytosis in the context of C-Vrp1p
364)817
, we meas-
ured uptake of the membrane-impermeant fluid-phase
endocytic dye Lucifer yellow (LY). vrp1D cells expres-
sing C-Vrp1p
364)817
or C-Vrp1p
364–817K485AR486A
took
up LY at 24 °C (Fig. 2D) and 37 °C (data not shown).
Hence, these charged residues are not essential for
endocytosis. As LY uptake is only a qualitative indica-
tor of endocytosis and not quantitative, it is possible
that the charged residues nevertheless increase the effi-
ciency of endocytosis.
To examine the expression level of each mutant
protein, the genes encoding C-Vrp1p
364)817
and
C-Vrp1p
364)817K485AR486A
were both fused inframe to

614-817
614
364 760
C-Vrp1p
364-760
1 817760
C-Vrp1p
760-817
364 760
C-Vrp1p
364-760
-CAAX
X
1 760
C-Vrp1p
760-817
-GST
1817364
364 760
C-Vrp1p
364-760
-GST
C-Vrp1p
364-817 K485A, R486A
1817364
C-Vrp1p
364-817
-GST
1817364
C-Vrp1p

364)817
proteins, but not GFP only,
were functional in restoring growth at elevated temper-
atures when introduced into vrp1D cells, indicating that
addition of the GFP did not perturb C-Vrp1p
364)817
function (Fig. S1). Total-cell extracts were prepared
from vrp1D cells expressing C-Vrp1p
364)817
–GFP or
C-Vrp1p
364)817K485AR486A
–GFP, the proteins were
resolved by SDS ⁄ PAGE, and immunoblotted with a
polyclonal anti–GFP serum (Fig. 2E). This ana-
lysis revealed that both C-Vrp1p
364)817
–GFP and
C-Vrp1p
364)817K485AR486A
–GFP are expressed at equiv-
alent levels. We were unable to raise a Vrp1p-specific
polyclonal antiserum and therefore could not assess
the expression level of the untagged C-Vrp1p
364)817
and C-Vrp1p
364)817K485AR486A
proteins. However, we
have tested all C-Vrp1p–GFP fusion proteins used in
this study for rescue of vrp1D temperature-sensitive

C-Vrp1p
364)817K485AR486A
(C-Vrp1p
364)817
AA). Each strain was
streaked for single colonies on YPUAD solid medium, incubated
at either 24 or 37 °C, and photographed after 3 days. (B) The
C-Vrp1p
364)817
charged-cluster residues K485R486 are not essential
for growth in liquid medium at elevated temperatures. Growth rate
of vrp1D (AMY88) cells carrying YCplac111 vector (vect), pAM236
expressing C-Vrp1p
364)817
(C-Vrp1p
364)817
), and pAM873 expres-
sing C-Vrp1p
364)817K485AR486A
(C-Vrp1p
364)817
AA). A YPUAD culture
of each strain was grown at 24 °C, diluted to D
600
¼ 0.05 in fresh
YPUAD medium, and incubated at 37 °C. D
600
was monitored
at 1 h intervals. (C) The C-Vrp1p
364)817

AA) were grown in YPUAD to exponential phase at
24 °C and 1 · 10
7
cells were incubated with LY dye for 1 h at
24 °C. Cells were washed and fluorescence was visualized using
fluorescence microscopy. (Upper) Fluorescence optics. (Lower) DIC
optics. Bar ¼ 5 lm. (E) C-Vrp1p
364)817
with charged-cluster resi-
dues K485R486 substituted with alanine is stably expressed. Total
extracts from vrp1D (AMY88) cells carrying pAM241 expres-
sing C-Vrp1p
364)817
fused at its C-terminus to green fluorescent
protein (GFP) (C-Vrp1p
364)817
–GFP) or pAM913 expressing
C-Vrp1p
364)817K485AR486A
–GFP (C-Vrp1p
364)817
AA–GFP) resolved by
SDS ⁄ PAGE, transferred to a PVDF membrane, and immunoblotted
with a polyclonal anti-GFP serum (a-GFP) and with anti-hexokinase
serum as a loading control (a-Hex).
Function of Vrp1p C-terminal module T. Thanabalu et al.
4106 FEBS Journal 274 (2007) 4103–4125 ª 2007 The Authors Journal compilation ª 2007 FEBS
present in full-length Vrp1p, but not C-Vrp1p
364)817
,

of these mutations on localization of C-Vrp1p
364)817
to
cortical patches, but may be due to inefficient polariza-
tion of C-Vrp1p
364)817
cortical patches.
C-Vrp1p residues 465–492 are essential for
cortical actin-patch polarization, but nonessential
for endocytosis and growth at elevated
temperatures
As an independent approach to identify domains
within C-Vrp1p
364)817
important for function we
constructed deletions initiating at the N-terminus of
C-Vrp1p
364)817
(Fig. 1). Five deletion constructs were
introduced into vrp1D (AMY88) cells and its ability
to functionally substitute for full-length Vrp1p was
assessed (Fig. 3A–C). Cells were stained with fluoro-
phore-conjugated phalloidin to visualize their actin
cytoskeleton. Deletion of residues 364–464 of
C-Vrp1p
364)817
had no obvious effect on cortical actin-
patch polarization (Fig. 3C) or on growth at elevated
temperatures (Fig. 3A,B), thus demonstrating that
this region is not essential for either of these

(Fig. 2E). Thus, loss of cortical actin-patch polariza-
tion is likely to be a direct effect of the loss of residues
465–492 rather than an indirect consequence of
reduced C-Vrp1p
493)817
expression levels. We were not
able to assay the expression level of the untagged pro-
teins, but we expect that the relative expression level of
the tagged proteins is indicative of that of the equival-
ent untagged proteins.
To assess the function of these proteins in endo-
cytosis we carried out LY uptake assays on
vrp1D (AMY88) cells expressing C-Vrp1p
465)817
,
C-Vrp1p
493)817
, C-Vrp1p
533)817
, C-Vrp1p
614)817
or
C-Vrp1p
716)817
. All five proteins rescued the endocyto-
sis defect at both 24 °C (Fig. 3E) and 37 °C (data not
shown). This suggests that residues 465–492 are not
essential for endocytosis. This is consistent with our
finding that K485 and R486 are not essential for endo-
cytosis (Fig. 2D). Residues 465–492 may nevertheless

CAAX 5 95 3 97
T. Thanabalu et al. Function of Vrp1p C-terminal module
FEBS Journal 274 (2007) 4103–4125 ª 2007 The Authors Journal compilation ª 2007 FEBS 4107
N-Las17p
1)241
(Fig. S3A). Furthermore, none of the
five deletions (including deletion of residues 364–492)
abolished localization of C-Vrp1p
364)817
to cortical
patches, although all except deletion of residues 364–
464 affected polarization of the cortical patches
(Fig. S3B).
A C-Vrp1p fragment comprising residues 465–533
including the charged cluster KK485R486DDR
interacts with actin
Inspection of the amino acid sequence in the region
bordered by residues 465 and 492 revealed the exist-
C
B
A
E
D
Function of Vrp1p C-terminal module T. Thanabalu et al.
4108 FEBS Journal 274 (2007) 4103–4125 ª 2007 The Authors Journal compilation ª 2007 FEBS
ence of a charged cluster surrounding K485 and R486:
KK485R486DDR (see Vrp1p-VH2-C sequence in
Fig. 4A). This charged cluster has some features in
common with the charged cluster in the N-terminal
WH2 domain of Vrp1p, which is known to bind actin

To further test if binding is direct, we incubated the
beads bearing GST only or the wild-type and
K485R486 mutant C-Vrp1p
465)533
–GST fusion pro-
teins with purified Saccharomyces cerevisiae actin in
G-actin buffer. Bound proteins were analysed as
above. The wild-type C-Vrp1p
465–533
fragment bound
to purified yeast G-actin, however, the K485R486
mutant protein as well as GST alone did not (Fig. 4D,
left). The wild-type Vrp1p fragment also bound
purified G-actin from rabbit skeletal muscle (data not
shown). The wild-type GST–Vrp1p
465)533
fragment did
not cosediment with F-actin from rabbit skeletal
muscle in an F-actin-pelleting assay (data not shown).
Thus the biochemical data are consistent with our
yeast two-hybrid data and suggests that the charged
cluster interacts with G-actin, but not F-actin.
An alignment of the various known and putative
actin-binding sequences in Vrp1p is shown in Fig. 4A.
Vrp1p-WH2-1 is the WH2 domain at the N-terminus
of Vrp1p that has previously been shown to mediate
interaction with G-actin [19]. Vrp1p-WH2-2 is a puta-
tive second WH2 domain identified by sequence align-
ment with other WH2 domains [43]. Note that the
Fig. 3. C-Vrp1p residues 465–492 containing the K485R486 charged cluster are essential for cortical actin-patch polarization, but not endocy-

ium at elevated temperatures. Growth rate of vrp1D (AMY88) cells carrying YCplac111 vector (vect), pAM236 expressing C-Vrp1p
364)817
(C-Vrp1p
364)817
), pAM880 expressing C-Vrp1p
465)817
(C-Vrp1p
465)817
), pAM881 expressing C-Vrp1p
493)817
(C-Vrp1p
493)817
), pAM882 expres-
sing C-Vrp1p
533)817
(C-Vrp1p
533)817
), pAM883 expressing C-Vrp1p
614)817
(C-Vrp1p
614)817
), or pAM884 expressing C-Vrp1p
716)817
(C-Vrp1p
716)817
). A YPUAD culture of each strain was grown at 24 °C, diluted to D
600
¼ 0.05 in fresh YPUAD medium and shifted to 37 °C.
D
600

Cells were grown in YPUAD to exponential phase at 24 °C. Cells were fixed with formaldehyde, permeabilized, and F-actin stained with
Alexa-488-conjugated phalloidin. Stained cells were viewed using fluorescence microscopy. Fields containing small-budded cells were specif-
ically chosen to compare the polarization of cortical actin patches at this stage of the cell cycle. Bar ¼ 5 lm. (D) C-Vrp1p
364)817
residues
465–492 containing the K485R486 charged cluster are not essential for fluid-phase endocytosis. vrp1D (AMY88) cells carrying pAM236
expressing C-Vrp1p
364)817
(C-Vrp1p
364)817
), pAM880 expressing C-Vrp1p
465)817
(C-Vrp1p
465)817
), pAM881 expressing C-Vrp1p
493)817
(C-Vrp1p
493)817
), pAM882 expressing C-Vrp1p
533)817
(C-Vrp1p
533)817
), pAM883 expressing C-Vrp1p
614)817
(C-Vrp1p
614)817
), or pAM884
expressing C-Vrp1p
716)817
(C-Vrp1p

614)817
–GFP), or pAM889 expressing C-Vrp1p
716)817
–GFP (C-Vrp1p
716)817
–GFP), resolved by SDS ⁄ PAGE, transferred to a PVDF
membrane, and immunoblotted with a polyclonal anti-GFP serum (a-GFP) and with a-hexokinase as a loading control (a-Hex).
T. Thanabalu et al. Function of Vrp1p C-terminal module
FEBS Journal 274 (2007) 4103–4125 ª 2007 The Authors Journal compilation ª 2007 FEBS 4109
names D1 and D2 are used by Paunola et al. [43] to
refer to WH2-1 and WH2-2, respectively. Vrp1p-WH2-
2 has not yet been shown to bind actin experimentally
and a fragment comprising Vrp1p residues 70–270 that
includes Vrp1p-WH2-2 does not exhibit two-hybrid
interaction with actin [23]. Vrp1p-VH2-C is the actin-
binding domain identified here containing K485R486.
We have aligned Vrp1p-VH2-C with a sequence within
the fragment comprising residues 270–364 of Vrp1p
(Vrp1p-verprolin homology 2 N-terminal or VH2-N)
which we previously showed does contain an actin-
binding domain (this actin-binding domain has not yet
been mapped) [23]. We name the actin-binding domain
that we have identified VH2-C and VH2-N because it
is not yet clear how closely these domains resemble the
WH2 (also known as VH) domain.
Function of C-Vrp1p in cortical actin-patch
polarization, endocytosis, and growth at elevated
temperatures requires the LBD
Residues 760–817 ⁄ end comprise the LBD of Vrp1p
[20,27–29]. We therefore tested if the LBD is important

actin-patch polarization. The truncated form of
C-Vrp1p
364)817
lacking the LBD also did not comple-
ment the LY uptake defect of vrp1D cells (Fig. 5D).
Hence, the LBD is essential for both cortical actin-
patch polarization and endocytosis.
To test if the LBD is essential for C-Vrp1p
364)817
expression or stability the C-terminus of a trun-
cated form of C-Vrp1p
364)817
lacking the LBD
(C-Vrp1p
364)760
) was tagged with GFP to create
Fig. 4. C-Vrp1p residues 465–533 containing the K485R486 charged cluster directly binds G-actin and residues K485R486 are critical.
(A) Amino acid sequence alignment of actin-binding sequences in Vrp1p. Vrp1p-WH2-1 (D1 in Paunola et al. [43]) is the original WH2 domain
shown to bind actin monomers by [19]. Vrp1p-WH2-2 (D2 in Paunola et al. [43]) is a sequence identified by Paunola et al. [43] as homolog-
ous to a WH2 domain (but whether it binds actin is not yet known). Vrp1p-VH2-C is the actin-binding domain within the longer CABS frag-
ment identified in this study that contains the K485R486 charged cluster. Vrp1p-VH2-N is a sequence within residues 270–364 of Vrp1p,
which we have previously shown contains an actin-binding domain. Note that the domain within residues 270–364 that binds actin has not
been mapped and may be distinct from the sequence VH2-N [23]. We use the nomenclature VH2 rather than WH2 because the sequence
of VH2-C and VH2-N is different from a WH2 domain and it is not yet clear they adopt a structure similar to a WH2 domain. (B) K485R486
are essential for yeast two–hybrid interaction between C-Vrp1p
465)533
and actin. pAM252 expressing Gal4-BD-Act1p (BD-Act1p ) and pAS2-1
BD vector only expressing Gal4-BD (BD-vect) were tested for two–hybrid interaction with pAM253 expressing Gal4-AD-N-Vrp1p
1)70
(AD-N-Vrp1p

crude yeast cell lysate. The beads were washed extensively and the bound proteins were eluted. The eluted proteins were resolved by
SDS ⁄ PAGE and the proteins were transferred to a PVDF membrane and immunoblotted with an anti-actin mAb. Equivalent amounts of
crude yeast cell lysate were used in each binding assay. The lower panel shows GST only and the GST fusion proteins used to coat the
beads used for binding assays subjected to SDS ⁄ PAGE and stained with Coomassie Brilliant Blue. The full-length GST and GST fusion pro-
teins are indicated (arrows). * indicates a protein that copurified with the fusion proteins and is likely to be a degradation product. (D) The
C-Vrp1p
364)817
charged cluster directly binds yeast G-actin in vitro and residues K485R486 are essential. Beads bearing GST (GST),
GST–C-Vrp1p
465)533
(GST–C-Vrp1p
465)533
), and GST–C-Vrp1p
465)533K485AR486A
(GST-C-Vrp1p
465)533
AA), prepared as in (C), were incubated
with purified yeast actin in G buffer. The beads were washed extensively and the bound proteins were eluted. The eluted proteins were
resolved by SDS ⁄ PAGE and the proteins were transferred to a PVDF membrane and immunoblotted with an anti-actin mAb (left). Equivalent
amounts of purified yeast actin were used in each binding assay and an amount representing 10% of the load used in each binding assay is
shown (right). The lower panel shows GST only and the GST fusion proteins used to coat the beads used for binding assays subjected to
SDS ⁄ PAGE and stained with Coomassie Brilliant Blue. The full-length GST and GST fusion proteins are indicated (arrows). * indicates a
protein that copurified with the fusion proteins and is likely to be a degradation product.
Function of Vrp1p C-terminal module T. Thanabalu et al.
4110 FEBS Journal 274 (2007) 4103–4125 ª 2007 The Authors Journal compilation ª 2007 FEBS
C-Vrp1p
364)760
–GFP. This protein was expressed in
vrp1D (AMY88) cells and its steady-state expression
level examined by SDS ⁄ PAGE and immunoblot

AD-vect
B
D-
Ac
t
1
p
B
D-
v
e
c
t
B
B
D
-
Ac
t
1
p
B
D-
v
e
c
t
+His -His
C
-actin

6
5
-
5
3
3
A
A
G
S
T

D
G
S
T
-
C
-
V
r
p
1
p
4
6
5
-
5
3

-
V
r
p
1
p
4
6
5
-
5
3
3
A
A
G
S
T
-
C
-
V
r
p
1
p
4
6
5
-

T
-
C
-
V
r
p
1
p
4
6
5
-
5
3
3
G
S
T
Purified
GST fusion
GST
GST fusion
GST
*
*
T. Thanabalu et al. Function of Vrp1p C-terminal module
FEBS Journal 274 (2007) 4103–4125 ª 2007 The Authors Journal compilation ª 2007 FEBS 4111
(Fig. 6A). C-Vrp1p
364)760

Time (h)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1234567
8
D
600
vect
C-Vrp1p
364-817
C-Vrp1p
364-760
37°C24°C
C-Vrp1p
364-760
C-Vrp1p
364-817
vect
24°C
C-Vrp1p
364-760
C-Vrp1p
364-817
vect

), however, in
contrast, the truncated form C-Vrp1p
364)760
was unable
to interact with N-Las17p
1)241
(Fig. 6B). Hence, in
yeast the Vrp1p C-terminal 56 residues are essential for
interaction with Las17p.
Is the LBD (residues 760–817) sufficient for localiza-
tion of C-Vrp1p
364)817
to cortical patches? To address
this question we tagged the isolated LBD of
Vrp1p with GFP to create C-Vrp1p
760)817
–GFP.
C-Vrp1p
760)817
–GFP was expressed in vrp1D (AMY88)
cells and its subcellular distribution examined by live
cell fluorescence imaging (Fig. 6C, left). Strikingly,
C-Vrp1p
760)817
–GFP has the ability to localize to cor-
tical patches. In contrast, GFP alone exhibited only a
diffuse cytoplasmic localization. We also examined
whether the LBD can mediate cortical patch localiza-
tion in the presence of full-length Vrp1p by examining
the localization of a C-Vrp1p

of that of the equivalent untagged proteins.
Lipid anchoring of C-Vrp1p bypasses the
requirement for the LBD for endocytosis and
growth at elevated temperatures, but not for
cortical actin-patch polarization
Addition of a CAAX box to the C-terminus of pro-
teins confers covalent lipid attachment and efficient
membrane anchoring to proteins that do not normally
associate with membranes [44]. We have previously
shown that the Ras1p CAAX box confers efficient
membrane anchoring on the otherwise cytoplasmic
N-Vrp1p
1)364
fragment [23]. Addition of the CAAX
box also enhances the function of N-Vrp1p
1)364
in
growth at elevated temperature such that it rescues the
temperature-sensitive growth defect of vrp1D with an
efficiency approaching that of C-Vrp1p
364)817
or full-
length Vrp1p [23]. We therefore asked whether using
the same technique to anchor C-Vrp1p
364)760
(CABS)
to membranes would restore function in the absence of
the LBD. C-Vrp1p
364)760
was tagged at the C-terminus

600
was monitored at 1 h intervals. (C) The 57-residue LBD of C-Vrp1p
364)817
is essential for polarization of cortical actin
patches. Cortical actin-patch polarization in vrp1D (AMY88) cells carrying YCplac111 vector (vect), pAM236 expressing C-Vrp1p
364)817
(C-Vrp1p
364)817
), pAM896 expressing C-Vrp1p
364)760
(C-Vrp1p
364)760
). Cells were grown in YPUAD to exponential phase at 24 °C, fixed with
formaldehyde, permeabilized, and F-actin stained with Alexa-488-conjugated phalloidin. Stained cells were viewed by fluorescence microsco-
py. Fields containing small-budded cells were specifically chosen to compare the polarization of cortical actin patches at this stage of the cell
cycle. Bar ¼ 5 lm. (D) The C-Vrp1p
364)817
LBD is essential for endocytosis. vrp1D (AMY88) cells carrying YCplac111 vector only (vect),
pAM236 expressing C-Vrp1p
364)817
(C-Vrp1p
364)817
), pAM896 expressing C-Vrp1p
364)760
(C-Vrp1p
364)760
) or pAM899 expressing
C-Vrp1p
364)760
fused at its C-terminus to the CAAX box of Ras1p (C-Vrp1p

CAAX box allowed the truncated C-Vrp1p
364)760
-
CAAX that lacks a LBD to restore growth at elevated
temperature to vrp1D cells (Fig. 7A,B) and endocytosis
(Fig. 5D). This suggests that one of the functions of
the LBD is to target Vrp1p to cortical patches and
A B
+His
-His
BD-vect
BD-vect
BD-N-Las17p
1-241
AD-C-Vrp1p
364-81
7
AD-C-Vrp1p
364-760
AD-C-Vrp1p
465-81
7
AD-C-Vrp1p
465-760
AD-vect
BD-N-Las17p
1-241
C-Vrp1p
364-760
-GFP

364)817
is essen-
tial for localization to cortical patches. vrp1D (AMY88) cells carrying pAM237 expressing GFP, pAM241 expressing C-Vrp1p
364)817
–GFP
(C-Vrp1p
364)817
–GFP), pAM891 expressing C-Vrp1p
364)760
–GFP (C-Vrp1p
364)760
–GFP) or pAM1003 expressing C-Vrp1p
364)760
–GFP fused at
its C-terminus to the CAAX box of Ras1p (C-Vrp1p
364)760
–GFP-CAAX) were grown in YPUAD to exponential phase at 24 °C and GFP fluores-
cence was visualized in living cells by fluorescence microscopy. (Upper) FITC-fluorescence optics. (Lower) DIC optics. Bars ¼ 5 lm. (B) The
57-residue LBD is essential for C-Vrp1p
364)817
interaction with Las17p. pAM912 expressing Gal4-BD-N-Las17p
1)241
(BD-N-Las17p1–241)
and pAS2-1 BD vector only expressing Gal4-BD (BD-vect) were tested for two–hybrid interaction with pAM902 expressing Gal4-
AD-C-Vrp1p
364)817
(AD-C-Vrp1p
364)817
), pAM909 expressing Gal4-AD-C-Vrp1p
364)760

716)817
–GFP), pAM237 expressing GFP (GFP) or pAM890 expressing C-Vrp1p
760)817
– GFP (C-Vrp1p
760)817
–GFP)
were resolved by SDS ⁄ PAGE, transferred to a PVDF membrane, and immunoblotted with a polyclonal anti-GFP serum (a-GFP) and with
a-hexokinase serum as a loading control (a-Hex).
Function of Vrp1p C-terminal module T. Thanabalu et al.
4114 FEBS Journal 274 (2007) 4103–4125 ª 2007 The Authors Journal compilation ª 2007 FEBS
vect
C-Vrp1p
364-817
C-Vrp1p
364-760
C-Vrp1p
364-760
-CAAX
24°C 37°C
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
12345678
Time (h)
D

364)760
(C-Vrp1p
364)760
), or pAM899 expressing C-Vrp1p
364)760
fused at its C-terminus to the CAAX box of Ras1p (C-Vrp1p
364)760
–CAAX). Each strain was streaked for single colonies on YPUAD solid med-
ium, incubated at either 24 or 37 °C, and photographed after 3 days. (B) Addition of a CAAX box to confer lipid anchoring bypasses the
requirement for the LBD of C-Vrp1p
364)817
for growth in liquid medium at elevated temperature. Growth rate of vrp1D (AMY88) cells carry-
ing YCplac111 vector (vect), pAM236 expressing C-Vrp1p
364)817
(C-Vrp1p
364)817
), pAM896 expressing C-Vrp1p
364)760
(C-Vrp1p
364)760
), or
pAM899 expressing C-Vrp1p
364)760
–CAAX (C-Vrp1p
364)760
–CAAX). A YPUAD culture of each strain was grown at 24 °C, diluted to D
600
¼
0.05 in fresh YPUAD medium and shifted to 37 °C. D
600

to vrp1D cells. vrp1D (AMY88) cells expressing
C-Vrp1p
364)760
–CAAX were stained with fluoro-
chrome-conjugated phalloidin and their cortical actin-
patch polarization was examined (Fig. 7C and
Table 1). Perhaps not surprisingly (given the absence
of the LBD), C-Vrp1p
364)760
–CAAX was not able to
restore a polarized cortical actin-patch distribution to
vrp1D cells.
We next asked whether addition of a CAAX box to
C-Vrp1p
364)760
restores localization to cortical patches.
The DNA sequence encoding C-Vrp1p
364)760
was fused
inframe to sequences encoding GFP–CAAX and
expressed in vrp1D cells (AMY88) (Fig. 6A). The
CAAX sequence targeted C-Vrp1p
364)760
to membranes
including the plasma membrane consistent with
our earlier findings with N-Vrp1p
1)364
[23]. Signifi-
cantly, however, the CAAX sequence failed to
target C-Vrp1p

ding GST (C-Vrp1p
760)817
–GST). Fusion to GST has
been shown to stabilize some proteins to proteolysis
[46]. GST is also known to dimerize [47]. This could
potentially enhance the activity of some proteins it
is fused to. We then expressed this fusion protein in
vrp1D (AMY88) cells. Interestingly, expression of
C-Vrp1p
760)817
–GST, but not GST alone or
C-Vrp1p
364)760
–GST, restored growth at elevated tem-
perature to vrp1D cells moderately well (Fig. 8A,B).
This result demonstrates that C-Vrp1p
760)817
does pos-
sess sufficient information to mediate the growth func-
tion of Vrp1p, but that to perform this function it
must be either stabilized or dimerized by fusion to
GST. C-Vrp1p
760)817
–GST also restored endocytosis
to vrp1D cells (Fig. 8C). However, in contrast,
C-Vrp1p
760)817
–GST lacked the ability to restore corti-
cal actin-patch polarization (Fig. 8D).
Discussion

is sufficient for growth in liquid medium at elevated temperature. Growth
rate of vrp1D (AMY88) cells carrying pAM915 expressing GST only (GST), pAM236 expressing C-Vrp1p
364)817
(C-Vrp1p
364)817
), or pAM895
expressing C-Vrp1p
760)817
–GST (C-Vrp1p
760)817
–GST). A YPUAD culture of each strain was grown at 24 °C, diluted to D
600
¼ 0.05 in fresh
YPUAD medium and shifted to 37 °C. D
600
was monitored at 1 h intervals. (C) The 57-residue LBD of C-Vrp1p
364)817
is sufficient for fluid-
phase endocytosis. vrp1D (AMY88) cells carrying pAM915 expressing GST only (GST), pAM236 expressing C-Vrp1p
364)817
(C-Vrp1p
364)817
),
or pAM895 expressing C-Vrp1p
760)817
–GST (C-Vrp1p
760)817
–GST) were grown in YPUAD to exponential phase at 24 °C and 1 · 10
7
cells

cortical patches. A role for the LBD in cortical-patch
localization is consistent with our previous results
showing that Las17p is required for localization of
A
B
C
C-Vrp1p
364-760
-GST
C-Vrp1p
364-817
-GST
C-Vrp1p
760-817
-GST
GST
24°C 37°C
Time (h)
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
123456789
D
600
C-Vrp1p

C-Vrp1p
465)533
binds endogenous actin in yeast lysates
and directly binds purified yeast and rabbit skeletal
muscle G-actin in vitro (Fig. 4B–D and data not
shown). Mutations of K485 and R486 abolish both
two-hybrid interaction and binding to G-actin in vitro,
showing that these charged residues are critical for
actin binding. Our results suggest that the phenotypes
that arise when the CABS charged cluster or the LBD
are mutated are not simply due to decreased protein
expression or stability. Neither mutation of the CABS
VH2-C domain charged cluster nor deletion of the
LBD significantly affected the steady-state expression
level of GFP-tagged C-Vrp1p
364)817
(Figs 2E and 5E).
Moreover, mutation of the VH2-C domain charged
cluster did not appear to have global effects on
C-Vrp1p
364)817
folding because two-hybrid interaction
with Las17p was not abolished (Fig. S2A). Moreover,
mutation of the VH2-C domain charged cluster did
not affect the ability of C-Vrp1p
364)817
to localize to
cortical patches or to support endocytosis and growth
at elevated temperature (Fig. 2A,B,D and Fig. S2B).
Similarly, deletion of the LBD did not have global

Arp2 ⁄ 3 complex in vitro, resulting in enhanced actin-
filament assembly [11,15,35,37,48]. The Vrp1p VH2-C
domain, by supplying actin monomers for polymeriza-
tion may play a critical role in actin filament assembly
in the actin patch. Binding of the LBD to Las17p may
also stimulate Las17p-dependent activation of the
Arp2 ⁄ 3 complex. De novo actin filament assembly is
critical for actin-patch formation at polarized cortical
sites [29]. Because cortical actin patches are short-lived
structures, continual actin-patch formation at polarized
cortical sites is essential for the maintenance of cortical
actin-patch polarization.
The requirement for the LBD for C-Vrp1p
364)817
localization to cortical patches demonstrates that the
VH2-C domain alone is not sufficient to confer cortical
patch localization on C-Vrp1p
364)817
despite its ability
to interact with actin. Moreover, C-Vrp1p
760)817
comprising the LBD alone is unable to interact with
actin but is still able to localize to cortical patches
(Figs 4B and 6C left, Fig. S4). Thus, interaction with
actin is neither sufficient nor necessary to target
C-Vrp1p
364)817
to cortical patches. This is consistent
with previous reports that Vrp1p persists in cortical
patches even after disassembly of all F-actin using

C-Vrp1p
364)817
–GFP in this respect and localizes to
cortical patches even in Las17p-deficient cells. This is
consistent with previous findings [29] (although in con-
trast to deletion of the WH1 domain only the complete
deletion of Las17p causes these Vrp1p–GFP cortical
patches to become depolarized) [23]. In contrast to
both of these studies, a more recent study found that
full-length Vrp1p–GFP localization to cortical patches
is Las17p dependent [15]. Perhaps these differences
reflect the use of different yeast strain backgrounds or
Function of Vrp1p C-terminal module T. Thanabalu et al.
4118 FEBS Journal 274 (2007) 4103–4125 ª 2007 The Authors Journal compilation ª 2007 FEBS
GFP fusions. In agreement with Sun et al. [15], we find
that Las17p–GFP localizes to cortical patches in
Vrp1p-deficient cells (our unpublished data).
Intriguingly, under certain conditions, the Vrp1p
CABS is able to at least partially restore endocytosis
and growth at elevated temperature to cells lacking
full-length Vrp1p. To supply this function the CABS
must be linked to membranes using a lipid anchor
(Figs 5D, 6A and 7A,B). The Vrp1p LBD alone can
also at least partially substitute for full-length Vrp1p
in endocytosis and growth at elevated temperature.
The longer fragment C-Vrp1p
716)817
retains some ability
to function without any additional sequences, whereas
the shorter fragment C-Vrp1p

study, we showed that at elevated temperature Vrp1p
is essential for localization of the cytokinesis regulator
Hof1p to a ring at the bud neck and for passage
through cytokinesis [22]. Hof1p has been shown to
play an important role in cytokinesis and growth at
elevated temperature [51]. Perhaps endocytosis plays
an important role in Vrp1p-dependent localization of
Hof1p to the bud neck at elevated temperature and
this in turn is important for cytokinesis and cell
growth under these conditions.
Interestingly, mutation of K485 and R486 in the
VH2-C domain charged cluster appears to abolish
interaction with actin but is without severe effects on
growth at elevated temperature (Figs 2A,B and
4B–D). By contrast, deletion of residues 465–492,
which includes the charged cluster, had more severe
effects (Fig. 3A,B). Perhaps other residues within
465–492 are more important than K485 and R486 for
growth at elevated temperature. Alternatively, actin
binding in vivo may be severely reduced but not abol-
ished by substitution of K485 and R486 with alanine.
In this case, residual actin binding may be sufficient
to at least partially restore endocytosis and growth at
elevated temperature, but not to the extent required
for efficient cortical actin-patch polarization. In the
case of the LBD, it is not yet clear if interaction with
Las17p itself or other proteins that may also interact
with the LBD is required for endocytosis and growth
at elevated temperature. More defined mutations
within the LBD that specifically affect interaction

still poorly understood. WIP–WASP-family protein
interaction has been proposed to play a role in recruit-
ment of WASP to sites of actin-filament assembly. For
example, during intracellular motility of vaccinia virus,
recruitment of WIP to the virus is essential for recruit-
ment of N-WASP and Arp2 ⁄ 3-dependent virus move-
T. Thanabalu et al. Function of Vrp1p C-terminal module
FEBS Journal 274 (2007) 4103–4125 ª 2007 The Authors Journal compilation ª 2007 FEBS 4119
ment. In the case of intracellular motility of the bac-
terium Shigella, WASP is important for recruitment of
WIP to the bacterial surface and Arp2 ⁄
3-dependent movement [52]. Our findings suggest an
analogous role for Las17p in recruitment of Vrp1p to
sites of actin-filament assembly in yeast cells. It has
also recently been shown that human WASP–WIP
interaction is essential for human WASP to function-
ally substitute for yeast WASP (Las17p) in yeast [55].
Binding to WIP serves to protect WASP from
degradation [55–57]. In another study, WIP binding
inhibited the ability of the WASP-family protein
(N-WASP) to activate the Arp2 ⁄ 3 complex in vitro,
suggesting that WIP–N-WASP interaction may be
inhibitory [31]. The Schizosaccharomyces pombe ortho-
log of Vrp1p has been found to have neither stimula-
tory nor inhibitory effects on Las17p-dependent
activation of the Arp2 ⁄ 3 complex in vitro [48]. How-
ever, this study used only the Arp2 ⁄ 3-binding domain
of Las17p and not full-length Las17p. Our results sug-
gest that Vrp1p binding may stimulate and ⁄ or stabilize
Las17p in yeast. However, further work is necessary

actin, Hof1p, Rvs167p, and Las17p) are proteins that
when overexpressed have deleterious effects on cell
growth and ⁄ or on cortical actin cytoskeleton organiza-
tion (especially at elevated temperature) and may be
aggregation prone [20,60–63]. Indeed, we proposed
that Vrp1p may act as a chaperone for the Hof1p SH3
domain [24]. Human WIP has been described as a
chaperone for WASP [56]. In the future it will be inter-
esting to test whether Vrp1p acts specifically to regu-
late components of the actin filament assembly
machinery or whether it has a more general role in
stabilizing or altering protein conformation and that
regulation of the actin-filament assembly machinery is
only one example of its cellular roles.
Experimental procedures
Strains, plasmids, media, and reagents
The yeast strains used in this study were: RH1657 (MATa-
lys2 his4 leu2 ura3 bar1) (Riezman lab wild-type strain),
AMY88 (MATa lys2 his4 leu2 ura3 vrp1D::KanMx bar1)
[23], IDY166 (MATa his3 leu2 ura3 trp1 las17D::URA3 )
[20], and PJ69-4A (MATa his3 leu2 ura3 trp1 gal4D gal80D
met2::GAL7-lacZ GAL2-ADE2 LYS2::GAL1–HIS3) [64].
Yeast strain PJ69-4A was a gift from P. James (University
of Wisconsin, USA). YPUAD is 1% yeast extract (Gib-
co ⁄ BRL, Paisley, UK), 2% peptone (Gibco ⁄ BRL), 2%
glucose supplemented with 40 lgÆmL
)1
adenine and
20 lgÆmL
)1

previously [66]. PCR was carried out using Pfu polymerase
(Roche, Mannheim, Germany) and a Thermocycler (Per-
kin–Elmer Biosystems, Foster City, CA).
DNA sequences encoding fragments of Vrp1p were
expressed from a YCplac111-based low-copy-number
Table 2. Plasmids used in this study.
Plasmid Description Ref.
pGEX-KG Vector for expression of GST in E. coli GE Healthcare
YCplac111 LEU2 CEN ARS plasmid [67]
pAS2-1 TRP1 two-hybrid vector with Gal4p DNA-binding domain (bait) Clontech
pACT2 LEU2 two-hybrid vector with Gal4p activation domain (prey) Clontech
pAM236 YCplac111 expressing C-Vrp1p
364)817
under VRP1 promoter [23]
pAM237 YCplac111 expressing GFP under VRP1 promoter [23]
pAM241 YCplac111 expressing C-Vrp1p
364)817
–GFP under VRP1 promoter [23]
pAM252 YCplac22 with GAL4BD-ACT1 [23]
pAM253 pACT2 expressing N-Vrp1p
1)70
[23]
pAM872 YCplac111 expressing C-Vrp1p
364)817 K457A
under VRP1 promoter This study
pAM873 YCplac111 expressing C-Vrp1p
364)817 K485AR486A
under VRP1 promoter This study
pAM874 YCplac111 expressing C-Vrp1p
364)817 D502AK503A

under VRP1 promoter This study
pAM885 YCplac111 expressing C-Vrp1p
465)817
–GFP under VRP1 promoter This study
pAM886 YCplac111 expressing C-Vrp1p
493)817
–GFP under VRP1 promoter This study
pAM887 YCplac111 expressing C-Vrp1p
533)817
–GFP under VRP1 promoter This study
pAM888 YCplac111 expressing C-Vrp1p
614)817
–GFP under VRP1 promoter This study
pAM889 YCplac111 expressing C-Vrp1p
716)817
–GFP under VRP1 promoter This study
pAM890 YCplac111 expressing C-Vrp1p
760)817
–GFP under VRP1 promoter This study
pAM891 YCplac111 expressing C-Vrp1p
364)760
–GFP under VRP1 promoter This study
pAM892 YCplac111 expressing C-Vrp1p
364)760
–GST under VRP1 promoter This study
pAM895 YCplac111 expressing C-Vrp1p
760)817
-GST under VRP1 promoter This study
pAM896 YCplac111 expressing C-Vrp1p
364)760

This study
pAM912 pAS2-1 expressing N-Las17p
1)241
This study
pAM913 YCplac111 expressing C-Vrp1p
364)817K485AR486A
–GFP under VRP1 promoter This study
pAM914 YCplac111 expressing C-Vrp1p
364)817
–GST under VRP1 promoter This study
pAM915 YCplac111 expressing GST only under VRP1 promoter This study
pAM918 pACT2 expressing C-Vrp1p
465)533
This study
pAM919 pACT2 expressing C-Vrp1p
465)533K485AR486A
This study
pAM1001 pGEX-KG expressing C-Vrp1p
465)533
This study
pAM1002 pGEX-KG expressing C-Vrp1p
465)533K485AR486A
This study
pAM1003 YCplac111 expressing C-Vrp1p
364–760
–GFP–CAAX under VRP1 promoter This study
T. Thanabalu et al. Function of Vrp1p C-terminal module
FEBS Journal 274 (2007) 4103–4125 ª 2007 The Authors Journal compilation ª 2007 FEBS 4121
plasmid [67] under the control of the VRP1 promoter
(nucleotides )240 to the VRP1 ATG start codon).

NaCl ⁄ P
i
and the GST fusion protein was purified using
glutathione–agarose beads. The beads were washed exten-
sively with NaCl ⁄ P
i
. S. cerevisiae cells were lysed in G-actin
buffer (5 mm Tris ⁄ HCl, 0.2 mm CaCl
2
, 0.2 mm ATP,
0.2 mm dithiothreitol using glass beads [65] and incubated
with the purified GST fusion proteins at 4 °C for 2 h. The
beads were washed and the bound proteins eluted by boil-
ing in SDS sample buffer and analysed by SDS ⁄ PAGE and
immunoblot. We also incubated the GST fusion proteins
with purified yeast actin or purified rabbit skeletal muscle
actin in G-actin buffer and analysed the bound proteins.
Actin was purified from S. cerevisiae by affinity chromato-
graphy using DNase I–Sepharose. Wild-type S. cerevisiae
cells were lysed in G-actin buffer using a bead beater (Bio-
spec. Products, Bartlesville, OK). The cell lysate was clar-
ified by centrifugation and the supernatant incubated with
DNase I coupled to Affi-gel 10. The bound actin was eluted
with G-actin buffer containing 50% formamide and dia-
lysed against G-actin buffer overnight.
Protein extracts, PAGE, and immunoblotting
Yeast cells growing in exponential phase were harvested
and a cell pellet representing 7 D
600
units was resuspended

permeablised using 1% Triton X-100 in NaCl ⁄ P
i
, stained
with Alexa-488-conjugated phalloidin and F-actin visualized
using fluorescence microscopy and FITC-specific light filters.
Light microscopy
Light microscopy was performed using a Leica DMLB
fluorescence microscope (Leica, Singapore). Images were
captured using an Optronix DEI-470T cooled charge–
coupled device camera and qwin software (Leica).
Acknowledgements
We thank V. Boulton for reading and offering valuable
suggestions on the manuscript. We are grateful to
R. Tsien and the Howard Hughes Medical Institute,
University of California, San Diego, USA for permis-
sion to use the S65T mutant form of GFP. We thank
the IMA ⁄ TLL DNA Sequencing Facility for DNA
sequence analysis. Funding from BMRC (Singapore)
(A*STAR 03 ⁄ 1 ⁄ 22 ⁄ 19 ⁄ 262), A*STAR (Singapore)
(ALM), the National Health and Medical Research
Function of Vrp1p C-terminal module T. Thanabalu et al.
4122 FEBS Journal 274 (2007) 4103–4125 ª 2007 The Authors Journal compilation ª 2007 FEBS
Council (Australia) Project Grant 298921 (ALM), and
the Queensland State Government (ALM) is gratefully
acknowledged.
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online:
Fig. S1. C-Vrp1p–GFP fusion proteins are functional
for growth at elevated temperature.
Fig. S2. C-Vrp1p charged-cluster residues K485R486
are not essential for interaction with Las17p or localiza-
tion to cortical patches.
Fig. S3. C-Vrp1p residues 465–492 containing the
K485R486 charged cluster are not essential for interac-
tion with Las17p or localization to cortical patches.
Fig. S4. The 57-residue LBD localizes C-Vrp1p
364-817
to cortical patches in wild-type cells.
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FEBS Journal 274 (2007) 4103–4125 ª 2007 The Authors Journal compilation ª 2007 FEBS 4125