Tài liệu Báo cáo khoa học: Verprolin function in endocytosis and actin organization Roles of the Las17p (yeast WASP)-binding domain and a novel C-terminal actin-binding domain doc

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

have mammalian counterparts with analogous func-
tions. Therefore, S. cerevisiae represents a useful model
Keywords
actin patch; Arp2 ⁄ 3; Bee1p; cell polarity;
WH2 domain
Correspondence
A. Munn, Institute for Molecular Bioscience,
The University of Queensland, St Lucia,
Queensland, 4072, Australia
Fax: +61 7 3346 2101
Tel: +61 7 3346 2017
E-mail:
*Present address
Institute for Molecular Bioscience, The
University of Queensland, St Lucia, Australia
(Received 11 April 2007, revised 22 May
2007, accepted 12 June 2007)
doi:10.1111/j.1742-4658.2007.05936.x
Vrp1p (verprolin, End5p) is the yeast ortholog of human Wiskott–Aldrich
syndrome protein (WASP)-interacting protein (WIP). Vrp1p localizes to
the cortical actin cytoskeleton, is necessary for its polarization to sites of
growth and is also essential for endocytosis. At elevated temperature,
Vrp1p becomes essential for growth. A C-terminal Vrp1p fragment
(C-Vrp1p) retains the ability to localize to the cortical actin cytoskeleton
and function in actin-cytoskeleton polarization, endocytosis and growth.
Here, we demonstrate that two submodules in C-Vrp1p are required for
actin-cytoskeleton polarization: a novel C-terminal actin-binding submod-
ule (CABS) that contains a novel G-actin-binding domain, which we call a
verprolin homology 2 C-terminal (VH2-C) domain; and a second submod-
ule comprising the Las17p-binding domain (LBD) that binds Las17p (yeast

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].

A key regulator of cortical actin-patch distribution
and endocytosis in S. cerevisiae is Vrp1p (verprolin ⁄
End5p), a proline-rich protein related to mammalian
Wiskott–Aldrich syndrome protein (WASP)-interacting
protein (WIP) [16–21]. Vrp1p localizes to cortical pat-
ches that display a subcellular distribution polarized
towards sites of surface growth and partially colocaliz-
es with cortical actin patches. Vrp1p localization to
cortical patches is not abolished by depolymerization
of actin filaments [19,20]. Loss of Vrp1p (vrp1D) leads
to a partial loss of cortical actin-patch polarization
and severe defects in internalization of both receptor-
bound and fluid-phase endocytic cargo [16,17,19,20].
Vrp1p is nonessential for growth at normal growth
temperatures but becomes essential at elevated temper-
atures [16,17,20,22–24]. The relationships among actin-
patch polarization, endocytosis, and growth are still
not well understood.
Structure–function studies aimed at elucidating the
molecular basis of Vrp1p function have revealed that
Vrp1p comprises two functional modules: an N-ter-
minal module (residues 1–364, N-Vrp1p) and a C-ter-
minal module (residues 364–817, C-Vrp1p) [23].
Each Vrp1p module interacts with a distinct set of
partner proteins: N-Vrp1p
1)364
binds actin monomers
[19,21,23], whereas C-Vrp1p
364)817
binds WASP-family

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

machine highly conserved from yeast to mammals
that requires interaction with nucleation-promoting
factors for activity [39–41]. In yeast, the Arp2 ⁄ 3
complex localizes to cortical patches that partially
colocalize with cortical actin patches like Vrp1p,
Las17p, and type I myosins [42]. Vrp1p is essential for
activation of the Arp2 ⁄ 3 complex by type I myosins
in vitro [15].
In a previous study we showed that C-Vrp1p
364)817
functionally replaces full-length Vrp1p for growth at
elevated temperatures. Furthermore, like full-length
Vrp1p, C-Vrp1p
364)817
efficiently localizes to cortical
actin patches. Localization of C-Vrp1p
364)817
to these
patches is critically dependent on Las17p [23]. Also
like full-length Vrp1p, C-Vrp1p
364)817
efficiently
mediates cortical actin-patch polarization [23]. How
does C-Vrp1p
364)817
mediate cortical actin-patch
polarization? Does C-Vrp1p
364)817
interact with actin,
or is its ability to interact with Las17p sufficient for

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

was generated and seven charged residues or pairs of
charged residues predicted to be surface exposed and
potentially involved in intra- or intermolecular inter-
actions were chosen for substitution with alanine
(residues K457, K485R486, D502K503, K512D513,
D594K595, E692, and K740). Because of an earlier
study that highlighted the role of bulky hydrophobic
residues in interaction of mammalian WASP and WIP
family proteins [32], we also substituted the single tryp-
tophan residue in C-Vrp1p
364)817
(W782) with alanine.
These eight DNA fragments encoding mutant
C-Vrp1p
364)817
proteins were placed under the control
of the native VRP1 promoter on a centromeric plas-
mid and introduced into vrp1 D (AMY88) cells. The
ability of the mutated C-Vrp1p
364)817
proteins to
restore defects caused by loss of Vrp1p was examined
(Fig. 2A–E and data not shown). Cells were stained
with fluorophore-conjugated phalloidin to visualize
their actin cytoskeleton. Interestingly, substitution of
residues K485R486 slightly reduced the activity of
C-Vrp1p
364)817
in growth at elevated temperatures
(Fig. 2A,B) and abolished its activity in cortical actin-

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

a sequence encoding green fluorescent protein (GFP)
and expressed from the VRP1 promoter carried on a
1817
Vrp1p
1 364
N-Vrp1p
1-364
HOT domain
Las17p-binding domain
X
CAAX box (lipid anchor)
Glutathione S-transferase (GST)
actin-binding domain
1817
465
C-Vrp1p
465-817
1817716
C-Vrp1p
716-817
1817
493C-Vrp1p
493-817
817
533
1817
533C-Vrp1p
533-817
1817
C-Vrp1p

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
?
?
(CABS)
Fig. 1. Vrp1p domain structure. Schematic
of Vrp1p showing the Vrp1p truncations and
mutant proteins used in this study and their
various known domains: actin-binding
domains, Hof one trap (HOT) domain, and
LBD. The fragment C-Vrp1p
364)760
is also
known as CABS. The actin-binding domain
closest to the N-terminus is also known as
the WH2 domain (WH2-1 or D1). The predic-
ted WH2 domain (WH2-2 or D2) identified
by Paunola et al. [43] by homology is not
shown here because this putative domain
has not yet been shown to bind actin. The
actin-binding site within residues 270–364
[23] has not yet been precisely mapped and
arrows labeled with question marks denote
its position. NB, actin-binding may or may
not be mediated by the sequence VH2-N
(Fig. 4A).
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 4105
centromeric plasmid. As a control, we also made an
equivalent construct expressing GFP only. Both GFP-
tagged 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

growth and in no case did fusion to GFP appear to
affect in vivo function (Fig. S1, data not shown). We
expect that the relative expression level of the GFP-
tagged fusion proteins is indicative of that of the
equivalent untagged proteins.
We examined whether the various charged-to-alan-
ine substitutions affected the ability of full-length
Vrp1p to restore cortical actin-patch polarization,
fluid-phase endocytosis, or growth at elevated tempera-
tures to vrp1D cells (data not shown). None of the
mutations had an obvious effect on any of these func-
tions, including K485A R486A. N-terminal sequences
A
B
C
D
E
Fig. 2. C-Vrp1p charged-cluster residues K485R486 are essential
for cortical actin-patch polarization, but not for endocytosis or
growth at elevated temperatures. (A) The C-Vrp1p
364)817
charged-
cluster residues K485R486 are not essential for growth on solid
medium at elevated temperatures. Growth at 24 and 37 °Cof
vrp1D (AMY88) cells carrying YCplac111 vector (vect), pAM236
expressing C-Vrp1p
364)817
(C-Vrp1p
364)817
) and pAM873 expressing

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

charged-cluster residues
K485R486 are essential for cortical actin-patch polarization. Cortical
actin-patch polarization in vrp1D (AMY88) cells carrying YCplac111
vector (vect), pAM236 expressing C-Vrp1p
364)817
(C-Vrp1p
364)817
),
and pAM873 expressing C-Vrp1p
364)817K485AR486A
(C-Vrp1p
364)817
AA). Cells were grown in YPUAD to exponential
phase at 24 °C and 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 specifically chosen to compare the polar-
ization of cortical actin patches at this stage of the cell cycle.
Bar ¼ 5 lm. (D) C-Vrp1p
364)817
charged-cluster residues K485R486
are not essential for endocytosis. vrp1D (AMY88) cells carrying
YCplac111 vector (vect), pAM236 expressing C-Vrp1p
364)817
(C-Vrp1p
364)817
) or pAM873 expressing C-Vrp1p
364)817K485AR486A
(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
,

may compensate for loss of K485R486.
Localization of C-Vrp1p
364)817
to cortical patches is
dependent on Las17p [23]. The minimal Vrp1p
sequences required for interaction with Las17p have
been mapped to the C-terminal 36 residues [27].
Consistent with this, substitution of K485R486 with
alanine did not abolish two-hybrid interaction of
C-Vrp1p
364)817
with N-Las17p
1)241
(Fig. S2A). The
substitution of K485R486 with alanine also did
not abolish C-Vrp1p
364)817
–GFP localization to cor-
tical patches in vrp1D cells (Fig. S2B). However,
C-Vrp1p
364)817
–GFP patches were polarized to sites of
surface growth, whereas C-Vrp1p
364)817K485AR486A
–
GFP patches were depolarized (Fig. S2B). We conclude
that loss of function of C-Vrp1p
364)817K485AR486A
in
cortical actin-patch polarization is not due to an effect

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

C-Vrp1p
364)817
functions. Additional deletion of 28
residues from the N-terminus resulted in a protein
(C-Vrp1p
493)817
) unable to restore cortical actin-patch
polarization (Fig. 3C). This protein exhibited reduced
function in growth at elevated temperature, but did
retain some residual function (Fig. 3A,B).
Immunoblot analysis of total-cell extracts prepared
from vrp1D (AMY88) cells expressing the correspond-
ing GFP-tagged versions of each protein (Fig. 3D)
showed that deletion of residues 364–492 resulted in,
at most, a twofold reduction in protein expression
compared with C-Vrp1p
364)817
. We cannot formally
exclude the possibility that this slight reduction in
expression level is responsible for the loss of function
in growth at elevated temperatures. We consider it
unlikely that this slight reduction in expression level is
responsible for the loss of cortical actin-patch polariza-
tion because this deletion removes critical residues
K485 and R486. Substitution of K485 and R486 with
alanine is alone sufficient to abolish C-Vrp1p
364)817
function in actin-patch polarization and these muta-
tions (unlike deletion of residues 364–492) do not
cause a significant reduction in protein expression level

(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

contribute to endocytosis and may be necessary for
maximal endocytic efficiency.
We next examined the subcellular localization and
protein interactions of the various truncated forms
of C-Vrp1p
364)817
(Fig. S3A,B). Consistent with the
results observed for alanine substitution of K485R486,
none of the five deletions abolished interaction with
Table 1. Actin-patch polarization of vrp1D cells carrying vector, or
plasmids expressing Vrp1p, C-Vrp1p or its derivatives. Cells were
grown to exponential phase at 24 °C and either shifted to 37 °C for
2 h or left at 24 °C. Cells were then fixed with formaldehyde, perme-
abilized with Triton X-100, and the actin patches stained with Alexa-
488–phalloidin. FITC-fluorescence microscopy was used to visualize
the actin patches. The percentages of small budded cells with depo-
larized actin patches were estimated by scoring a total of 200 cells
from each sample. A mother cell with more than 10 actin patches
was counted as having a depolarized actin patch phenotype.
24 °C37°C
Polarized Depolarized Polarized Depolarized
Vector 2 98 0 100
Vrp1p 94 6 91 9
C-Vrp1p 80 20 60 40
C-Vrp1p AA 22 78 18 82
C-Vrp1p
364)760
1 99 0 100
C-Vrp1p
364)760

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

(KLK45K46AET) (sequence WH2-1 in Fig. 4A)
[19,21,23]. We therefore examined the ability of a wild-
type fragment comprising Vrp1p residues 465–533 and
the equivalent fragment containing the K485AR486A
mutations to interact with actin in the two-hybrid
system (Fig. 4B). Vrp1p
465)533
exhibited two-hybrid
interaction with actin. In contrast, the mutated frag-
ment in which K485R486 were substituted with alanine
did not exhibit detectable interaction with actin.
To test whether C-Vrp1p
465)533
associates with actin
in crude yeast lysates we expressed wild-type and
K485R486 mutant C-Vrp1p
465)533
fragments as gluta-
thione S-transferase (GST) fusion proteins as well as
GST only in Escherichia coli and incubated beads
bearing the purified GST only and GST fusion pro-
teins with crude yeast-cell lysate in G-actin buffer. The
proteins bound to the beads were eluted, resolved
by SDS ⁄ PAGE, and analysed by immunoblotting
with anti-actin serum. Although the wild-type
C-Vrp1p
465)533
fragment associated with actin in crude
yeast-cell lysate, the K485R486 mutant protein and
GST alone did not (Fig. 4C, upper).

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-

tosis or growth at elevated temperatures. (A) C-Vrp1p
364)817
residues 465–492 containing the K485R486 charged cluster contribute to, but
are not essential for, growth on solid medium at elevated temperatures. Growth at 24 and 37 °Cofvrp1D (AMY88) cells carrying YCplac111
vector (vect), 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
716)817
).
Each strain was streaked for single colonies on YPUAD solid medium, incubated at either 24 or 37 °C, and photographed after 3 days.
(B) C-Vrp1p
364)817
residues 465–492 containing the K485R486 charged cluster contribute to, but are not essential for, growth in liquid med-

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

was monitored at 1 h intervals. (C) C-Vrp1p
364)817
residues 465–492 containing the K485R486 charged cluster are essential for cortical
actin-patch polarization. Cortical actin-patch polarization in 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
716)817
).

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

716)817
) 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. The 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
fragments lacking residues 465–492 containing the K485R486 charged cluster are
stably expressed. Total extracts from vrp1D (AMY88) cells carrying pAM241 expressing C-Vrp1p
364)817
fused at its C-terminus
to GFP (C-Vrp1p
364)817
–GFP), pAM885 expressing C-Vrp1p
465)817
–GFP (C-Vrp1p
465)817
–GFP), pAM886 expressing C-Vrp1p
493)817
–GFP
(C-Vrp1p
493)817
–GFP), pAM887 expressing C-Vrp1p
533)817
–GFP (C-Vrp1p
533)817
–GFP), pAM888 expressing C-Vrp1p
614)817
–GFP
(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

for various Vrp1p-dependent functions (Fig. 5A–C).
To examine whether deletion of the LBD abolishes the
function of C-Vrp1p
364)817
in growth we expressed
C-Vrp1p
364)760
in vrp1D (AMY88) cells and examined
growth at elevated temperature (Fig. 5A,B). Loss of
the LBD abolished the ability of C-Vrp1p
364)817
to
restore growth of vrp1D cells at elevated temperature.
We next assessed the importance of the LBD for the
ability of C-Vrp1p
364)817
to restore cortical actin-patch
polarization and endocytosis to vrp1D cells. vrp1D
(AMY88) cells expressing either full-length
C-Vrp1p
364)817
or the truncated form lacking a LBD
(C-Vrp1p
364)760
) were stained with fluorophore-conju-
gated phalloidin to visualize their actin cytoskeleton
(Fig. 5C, Table 1). Deletion of the LBD abolished
the ability of C-Vrp1p
364)817
to mediate cortical

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

1)70
), pAM918 expressing Gal4-AD-C-Vrp1p
465)533
(AD-C-Vrp1p
465)533
), pAM919 expressing Gal4-AD-C-Vrp1p
465)533K485AR486A
(AD-C-Vrp1p
465)533
AA), pAM908 expressing Gal4-AD-C-Vrp1p
716)817
(AD-C-Vrp1p
716)817
), or pACT2 AD vector only expressing Gal4-AD
(AD-vect). Plasmids were introduced into the tester strain PJ69-4A and interaction was assessed by growth on medium lacking histidine and
containing 2 m
M 3-amino 1,2,4-triazole. Plates were photographed after 4 days. (C) The C-Vrp1p
364)817
charged cluster associates with
G-actin present in crude yeast lysates in vitro and residues K485R486 are essential. pGEX-KG expressing GST only (GST), pAM1001 expres-
sing GST–C-Vrp1p
465)533
(GST–C-Vrp1p
465)533
), or pAM1002 expressing GST-C-Vrp1p
465)533K485AR486A
(GST–C-Vrp1p
465)533
AA) were intro-
duced into E. coli, the encoded proteins were expressed and affinity purified, and beads bearing the purified proteins were incubated with

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

(Fig. 5E). The results show that (at least as a GFP
fusion protein) C-Vrp1p
364)760
is expressed at equival-
ent levels to C-Vrp1p
364)817
. We were unable to assay
the relative expression level of the untagged proteins,
but we expect they would also be similar.
The LBD is necessary and sufficient for
localization of C-Vrp1p to cortical patches
We next examined whether the LBD is necessary
and ⁄ or sufficient for localization of C-Vrp1p
364)817
to
cortical patches or interaction with Las17p (Fig. 6A–C).
The subcellular distribution of C-Vrp1p
364)760
–GFP
was analysed using live cell fluorescence imaging
A
AD-N-Vrp1p
1-70
AD-C-Vrp1p
465-533
AD-C-Vrp1p
465-533
AA
AD-C-Vrp1p
716-817

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

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

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

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

-
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
-

5
3
3
G
S
T
Bound
Load
G
S
T
-
C
-
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
-
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

–GFP displayed a diffuse
cytoplasmic localization similar to GFP alone. In con-
trast, C-Vrp1p
364)817
–GFP localized to cortical patches
(Fig. 6A) consistent with our previous report [23]. The
expression level of truncated C-Vrp1p
364)760
–GFP was
equivalent to that of C-Vrp1p
364)817
–GFP (Fig. 5E).
This suggests that loss of cortical-patch localization is
not an indirect consequence of lowered expression of the
truncated C-Vrp1p
364)760
–GFP fusion protein relative
to C-Vrp1p
364)817
–GFP. We expect that the relative
expression level of the GFP-tagged proteins is indicative
of that of the equivalent untagged proteins.
A
B
C
C-Vrp1p
364-817
vect
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

C-Vrp1p
364-760
-CAAX
E
C-Vrp1p
364-760
-GFP
C-Vrp1p
364-817
-GFP
-GFP
-Hex
D
Function of Vrp1p C-terminal module T. Thanabalu et al.
4112 FEBS Journal 274 (2007) 4103–4125 ª 2007 The Authors Journal compilation ª 2007 FEBS
Madania et al. [27] showed that the Vrp1p C-terminal
36 residues are sufficient for two-hybrid interaction with
Las17p. Interestingly, however, the 3D structure of the
equivalent mammalian N-WASP–WIP complex reveals
a major contact outside the equivalent C-terminal
36 residues of WIP [33]. To test whether the LBD is
essential for interaction of C-Vrp1p
364)817
with Las17p
we performed two-hybrid tests. C-Vrp1p
364)817
exhib-
ited two-hybrid interaction with an N-terminal
fragment of Las17p (N-Las17p
1)241

), 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

760)817
–GFP fusion pro-
tein in wild-type (RH1657) cells. The 57-residue LBD
was sufficient to mediate cortical patch localization
similar to that of C-Vrp1p
364)817
–GFP in cells expres-
sing full-length wild-type Vrp1p (Fig. S4).
The cortical patch localization of C-Vrp1p
760)817
–
GFP is dependent on Las17p. When expressed in las17D
(IDY166) cells, C-Vrp1p
760)817
–GFP did not localize to
cortical patches but rather displayed a diffuse cytoplas-
mic distribution (Fig. 6C, right). This is consistent with
what we reported previously for C-Vrp1p
364)817
when
expressed in las17D cells [23]. The expression level of
C-Vrp1p
760)817
–GFP was examined by SDS ⁄ PAGE
and immunoblot (Fig. 6D) and found to be easily
detectible but reduced compared with that of
C-Vrp1p
716)817
–GFP. We expect that the relative
expression level of the GFP-tagged proteins is indicative

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

with the CAAX box of S. cerevisiae Ras1p [45]
Fig. 5. The LBD of C-Vrp1p is essential for cortical actin-patch polarization, endocytosis, and growth at elevated temperature. (A) The 57-resi-
due LBD of C-Vrp1p
364)817
is essential for growth on solid medium at elevated temperature. Growth at 24 and 37 °Cofvrp1D (AMY88)
cells carrying YCplac111 vector (vect), pAM236 expressing C-Vrp1p
364)817
(C-Vrp1p
364)817
), or pAM896 expressing C-Vrp1p
364)760
(C-Vrp1p
364)760
). 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 57-residue LBD of C-Vrp1p
364)817
is essential for growth in liquid medium at elevated temperature. Growth rate of
vrp1D (AMY88) cells carrying YCplac111 vector (vect), pAM236 expressing C-Vrp1p
364)817
(C-Vrp1p
364)817
) and pAM896 expressing
C-Vrp1p
364)760
(C-Vrp1p
364)760
). 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 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

364)760
-CAAX) 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. The cells were washed and fluorescence was visualized using fluores-
cence microscopy. (Upper) Fluorescence optics. (Lower) DIC optics. Bar ¼ 5 lm. (E) C-Vrp1p
364)817
lacking the 57-residue LBD is stably
expressed. Total extracts from vrp1D (AMY88) cells carrying pAM241 expressing C-Vrp1p
364)817
fused at its C-terminus to GFP
(C-Vrp1p
364)817
–GFP) or pAM891 expressing C-Vrp1p
364)760
–GFP (C-Vrp1p
364)760
–GFP) resolved by SDS ⁄ PAGE, transferred to a PVDF mem-
brane, and immunoblotted with a polyclonal anti-GFP serum (a-GFP) and with a-hexokinase serum 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 4113
to yield C-Vrp1p
364)760
–CAAX. This protein was
expressed in vrp1D (AMY88) cells and its ability to
restore various Vrp1p-dependent functions was com-
pared with that of C-Vrp1p
364)817
containing the LBD
(Figs 5D and 7A–C). Strikingly, the presence of the

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

GFP
C-Vrp1p
364-817
-GFP
C-Vrp1p
364-760
-GFP-CAAX
D
α-Hex
α-GFP
GFP
C-Vrp1p
716-817
-GFP
C-Vrp1p
760-817
-GFP
C
GFP C-Vrp1p
760-817
-GFP
C-Vrp1p
760-817
-GFP
las17
Δ
vrp1
Δ
GFP
Fig. 6. The LBD of C-Vrp1p is necessary and sufficient for localization to cortical patches. (A) The 57-residue LBD of C-Vrp1p

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

(AD-C-Vrp1p
364)760
), pAM904 expressing Gal4-
AD-C-Vrp1p
465)817
(AD-C-Vrp1p
465)817
), pAM910 expressing Gal4-AD-C-Vrp1p
465)760
(AD-C-Vrp1p
465)760
), and pACT2 AD vector only expres-
sing Gal4-AD (AD-vect). Plasmids were introduced into the tester strain PJ69-4A and interaction was assessed by growth on medium lacking
histidine and containing 2 m
M 3-amino 1,2,4-triazole. Plates were photographed after 4 days. (C) The 57-residue LBD of C-Vrp1p
364)817
is
sufficient for localization to cortical patches. vrp1D (AMY88) or las17D (IDY166) cells carrying pAM237 expressing GFP (GFP) or pAM890
expressing C-Vrp1p
760)817
–GFP (C-Vrp1p
760)817
–GFP) were grown in YPUAD to exponential phase at 24 °C and GFP fluorescence was visu-
alized in living cells by fluorescence microscopy. (Upper) FITC-fluorescence optics. (Lower) DIC optics. Bar ¼ 5 lm. (D) The 57-residue LBD
of C-Vrp1p
364)817
is stably expressed as a fusion to GFP. Total extracts from vrp1D (AMY88) cells carrying pAM889 expressing
C-Vrp1p
716)817
–GFP (C-Vrp1p

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

600
vect
C-Vrp1p
364-817
C-Vrp1p
364-760
-CAAX
C-Vrp1p
364-760
A
B
C
C-Vrp1p
364-760
C-Vrp1p
364-817
C-Vrp1p
364-760
-CAAXvect
24°C
Fig. 7. Lipid-anchoring of C-Vrp1p bypasses the requirement for the LBD for endocytosis and growth at elevated temperature, but not corti-
cal actin-patch polarization. (A) Addition of a CAAX box to confer lipid anchoring bypasses the requirement for the LBD of C-Vrp1p
364)817
for
growth on solid media at elevated temperature. Growth at 24 and 37 °Cofvrp1D (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
), 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

was monitored at 1 h intervals. (C) Addition of a CAAX box to confer lipid anchoring
does not bypass the requirement for the LBD of C-Vrp1p
364)817
for cortical actin-patch polarization. Cortical actin-patch polarization in 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
–CAAX (C-Vrp1p
364)760
–CAAX). Cells were grown in YPUAD to expo-
nential phase at 24 °C. The cells were fixed with formaldehyde, permeabilized, and F-actin stained with Alexa-488-conjugated phalloidin.
Stained cells were viewed by fluorescence microscopy. Fields containing small-budded cells were specifically chosen to compare the polar-
ization of cortical actin patches at this stage of the cell cycle. Bar ¼ 5 lm.
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 4115
that another membrane-targeting sequence can bypass
the requirement for the LBD at least in growth at
elevated temperature and endocytosis.
We next examined whether C-Vrp1p
364)760
–CAAX
is able to restore cortical actin-patch polarization

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

364)760
–GFP–CAAX to cortical pat-
ches. C-Vrp1p
364)760
–GFP–CAAX-labeled membranes
evenly without concentration into puncta (Fig. 6A).
The LBD can functionally substitute for
full-length Vrp1p in endocytosis and growth
at elevated temperatures, but not in cortical
actin-patch polarization
To test whether the LBD alone retains some function
apart from the ability to localize to cortical patches,
we expressed two Vrp1p C-terminal fragments contain-
ing the LBD (C-Vrp1p
716)817
and C-Vrp1p
760)817
)in
vrp1D (AMY88) cells and tested their ability to restore
various Vrp1p-dependent functions. The longer frag-
ment, C-Vrp1p
716)817
, could rescue growth at elevated
temperature moderately well (Fig. 3A), but the shorter
fragment, C-Vrp1p
760)817
(which contains the LBD
only) could not (data not shown). Because the LBD is
rather short, comprising 57 residues, we then fused the
DNA encoding the LBD inframe to sequences enco-

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

Here, we have examined the in vivo function of
two submodules in the Vrp1p C-terminal module
(C-Vrp1p
364)817
) (Fig. 1). The first is the CABS (resi-
dues 364–760), of which residues 465–533 represent a
novel actin-binding domain featuring a charged cluster
KK485R486DDR. Thus, Vrp1p has at least three
experimentally verified actin-binding domains located in
Fig. 8. The 57-residue LBD of C-Vrp1p is sufficient for endocytosis and growth at elevated temperature, but not cortical actin-patch polariza-
tion. (A) The 57-residue LBD of C-Vrp1p
364)817
is sufficient for growth on solid medium at elevated temperature. Growth at 24 and 37 °Cof
vrp1D (AMY88) cells carrying pAM915 expressing GST only (GST), pAM914 expressing C-Vrp1p
364)817
fused at its C-terminus to GST
(C-Vrp1p
364)817
–GST), pAM895 expressing C-Vrp1p
760)817
–GST (C-Vrp1p
760)817
–GST), or pAM892 expressing C-Vrp1p
364)760
–GST
(C-Vrp1p
364)760
–GST). Each strain was streaked for single colonies on YPUAD solid medium, incubated at either 24 or 37 °C, and photo-
graphed after 3 days. (B) The 57-residue LBD of C-Vrp1p
364)817

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

were incubated with LY dye for 1 h at 24 °C. The cells were washed and fluorescence was visualized using fluorescence microscopy.
(Upper) Fluorescence optics. (lower) DIC optics. Bar ¼ 5 lm. (D) The 57-residue LBD of C-Vrp1p
364)817
is not sufficient for polarization of
cortical actin patches. Cortical actin-patch polarization in vrp1D (AMY88) cells carrying pAM915 expressing GST only (GST), pAM236 expres-
sing C-Vrp1p
364)817
(C-Vrp1p
364)817
), or pAM895 expressing C-Vrp1p
760)817
–GST (C-Vrp1p
760)817
–GST). Cells were grown in YPUAD to expo-
nential phase at 24 °C. The cells were fixed with formaldehyde, permeabilized, and F-actin stained with Alexa-488-conjugated phalloidin.
Stained cells were viewed by fluorescence microscopy. Fields containing small-budded cells were specifically chosen to compare the polar-
ization of cortical actin patches at this stage of the cell cycle. Bar, 5 lm.
Function of Vrp1p C-terminal module T. Thanabalu et al.
4116 FEBS Journal 274 (2007) 4103–4125 ª 2007 The Authors Journal compilation ª 2007 FEBS
fragments comprising residues 1–70 (KLK45K46AET –
the original WH2 domain), 270–364 (residues not yet
mapped), and 465–533 (KK485R486DDR). We have
named the novel C-terminal actin binding domain the
VH2-C domain to distinguish it from the two previously
reported N-terminal actin-binding domains [19,21,23].
The second submodule comprises the LBD (residues
760–817 ⁄ end). We show that the LBD is both necessary
and sufficient for localization of C-Vrp1p
364)817
to

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

364-817
C-Vrp1p
760-817
-GST
GST
C-Vrp1p
760-817
-GST
GST C-Vrp1p
364-817
C-Vrp1p
760-817
-GST
GST C-Vrp1p
364-817
24
o
C
D
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 4117
C-Vrp1p
364)817
to cortical patches [23]. It is also consis-
tent with a recent report demonstrating a role for
Las17p in patch localization of full-length Vrp1p [15].
We have shown that C-Vrp1p
364)817
exhibits
two-hybrid interaction with actin and that GST-

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

effects on C-Vrp1p
364)817
folding because the truncated
fragment retained the ability to support endocytosis
and growth at elevated temperature if lipid-anchored
(Figs 5D and 7A,B). Hence, the mutations we
introduced are unlikely to have grossly perturbed
C-Vrp1p
364)817
folding.
The VH2-C domain charged cluster and the LBD
are both essential for cortical actin-patch polarization
mediated by C-Vrp1p
364)817
. This suggests that inter-
actions mediated by these two domains are directly
implicated in polarization of the yeast actin cytoskele-
ton. The presence of the novel actin-binding VH2-C
domain in C-Vrp1p
364)817
may explain why mutations
in the N-terminal actin-binding WH2 domain (WH2-1)
or even deletion of the entire N-terminal module of
Vrp1p did not significantly perturb the activity of
Vrp1p in polarization of cortical actin patches [23].
One role of the LBD is to target C-Vrp1p
364)817
to
cortical patches where it interacts with Las17p and
type I myosins. Las17p and type I myosins activate the

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

latrunculin A [19]. Type I myosins localize to cortical
actin patches and interact via their Src homology 3
(SH3) domain with multiple Vrp1p fragments inclu-
ding those that overlap with CABS (C-Vrp1p
364)760
)
[11,15,28,34,49] (our unpublished data). If type I
myosins interact with CABS then these interactions are
also insufficient to confer cortical-patch localization.
This is consistent with our finding that N-Vrp1p
1)364
does not localize to cortical patches despite its ability
to interact with the type I myosin Myo3p [22–24].
An earlier study presented evidence that Vrp1p–
Las17p interaction is important for polarization of
Las17p, but not Vrp1p, cortical patches [29]. Deletion
of the Vrp1p LBD abolished polarization of Las17p–
GFP (Bee1p–GFP) cortical patches (although Las17p–
GFP was still localized to cortical patches). By
contrast, deletion of the WASP homology 1 (WH1)
domain of Las17p (Bee1p) that interacts with Vrp1p
did not abolish either localization of Vrp1p–GFP to
cortical patches or polarization of Vrp1p–GFP cortical
patches. Our data showing that Vrp1p–Las17p interac-
tion is essential for localization of C-Vrp1p
364)817
to
cortical patches (Fig. 6A,C) may appear inconsistent
with these earlier findings. However, we find that
full-length Vrp1p–GFP behaves differently from

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

760)817
is functional only
when fused to GST (Figs 3A,B and 8A–C). Perhaps
C-Vrp1p
760)817
is proteolytically unstable unless fused
to GST. An alternative possibility is that dimerization
mediated by GST enhances its in vivo activity.
The ability of both the CABS and LBD to restore
significant endocytosis and growth at elevated
temperature despite an inability to restore cortical actin-
patch polarization is further evidence that the functions
of Vrp1p in endocytosis, cytokinesis, and growth at
elevated temperature, on the one hand, and in cortical
actin-patch polarization, on the other hand, are at least
partially distinct [23]. However, there may still be a
functional link between endocytosis and actin-patch
polarization. A previous study showed that the polar-
ized distribution of surface-membrane proteins in yeast
can be achieved by efficient endocytosis [50]. Given that
the LY uptake assays used here are not quantitative it is
possible that the CABS and LBD individually only
partially restore endocytosis. If so, the reduction in
uptake efficiency may be sufficient to generally perturb
cell polarity and abolish actin-patch polarization.
Our data suggest a close relationship between the
function of Vrp1p in endocytosis and growth at eleva-
ted temperature. No mutated Vrp1p protein we tested
restored growth at elevated temperature without also
restoring endocytosis and vice versa. In a previous

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

with Las17p but not other proteins would be required
to address this question.
Our data further support the view that interaction
of the Vrp1p LBD with Las17p in yeast is physiologi-
cally important. Moreover, WIP interacts via a
C-terminal domain with WASP-family proteins in
mammalian cells suggesting that the physiological role
of this interaction is conserved [30,52]. Interactions of
Vrp1p and Las17p in yeast and WIP and WASP-fam-
ily proteins in mammalian cells are direct and result
in constitutive and apparently stable complexes
[28,29,31,32]. In yeast, Vrp1p and Las17p are believed
to exist predominantly within this complex rather than
as separate proteins [29]. The physiological importance
of WASP–WIP interaction in mammalian cells is sup-
ported by the observation that the vast majority of the
disease-causing missense mutations in human WASP
map to the N-terminal WH1 (EVH1) domain of
WASP which interacts with WIP and destabilize this
interaction [52–56]. The 3D structure of a complex
comprising the binding domains of WIP and the
WASP-family protein N-WASP has been characterized
using NMR [33]. Residues in N-WASP and WIP that
make key contacts in the NMR structure are con-
served in yeast Vrp1p and Las17p. Interestingly, the
Vrp1p residues equivalent to those at the contact site
in WIP are residues 701–730, which lie outside the
LBD. We have yet to observe an interaction between
Vrp1p residues 701–730 and Las17p in vivo.
The role of the WIP–WASP interaction, however, is

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

to fully understand the physiological roles of the Vrp1p–
Las17p and WIP–WASP-family protein interactions.
In yeast, Vrp1p is implicated in type I myosin-
dependent Arp2 ⁄ 3 activation [15,28,29,35,38]. Type I
myosins can interact via an acidic tail (A) domain with
the Arp2 ⁄ 3 complex like Las17p, but they lack an
actin-monomer-binding WH2 domain, which in
WASP-family proteins (including Las17p) is essential
for effective Arp2 ⁄ 3 activation [15,28,29,37,38,43,58].
Vrp1p possesses an N-terminal WH2 domain, but
lacks the ability to interact with the Arp2 ⁄ 3 complex
[15,19,21,23,48,59]. A complex of Vrp1p with type I
myosin has both actin monomer- and Arp2 ⁄ 3-binding
functions and can activate the Arp2 ⁄ 3 complex in a
manner analogous to WASP-family proteins such as
Las17p [15,29]. In this context, Vrp1p acts as a multi-
valent adaptor to recruit other proteins such as
Las17p, type I myosins, and actin monomers to sites
of actin-filament assembly.
One of the most novel and potentially significant
findings from our functional analysis of Vrp1p is that
small fragments of Vrp1p with limited ability to act as
multivalent adaptors still retain significant in vivo func-
tion. This suggests that interaction of short sequences
in Vrp1p with proteins such as actin, type I myosins,
and Las17p may be required individually to keep these
interacting proteins functional, especially under condi-
tions of temperature stress. Elevated temperature is a
condition known to promote protein aggregation. Fur-
thermore, proteins known to interact with Vrp1p (e.g.

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

uracil. SD minimal medium is described in
Adams et al. [65]. 3-Amino 1,2,4-triazole was from Sigma-
Aldrich (St Louis, MO, USA). The plasmids used in this
study are listed in Table 2. The rabbit polyclonal GFP-spe-
cific antiserum was a gift from J. Kahana and P. Silver
(Dana Farber Cancer Center, Boston, MA). The anti-actin
mAb was MAB1501 from Chemicon International (Teme-
cula, CA). The anti-hexokinase rabbit polyclonal serum
was 100–4159 from Rockland Inc. (Gilbertsville, PA).
Alexa-488-conjugated phalloidin was from Invitro-
gen ⁄ Molecular Probes (Eugene, OR, USA). Poly(vinylidene
fluoride) membranes (Immobilon-P
SQ
) were from Millipore
(Bedford, MA). Horseradish peroxidase-conjugated anti-
(rabbit IgG) secondary sera used for immunoblot were
from Sigma-Aldrich. Affi-gel 10 was from Bio-Rad (Hercu-
les, CA). ECL reagents were from GE Healthcare ⁄
Amersham Biosciences (Amersham, UK). Immobilized
glutathione was from Sigma-Aldrich. Purified rabbit muscle
actin was from Cytoskeleton, Inc (Denver, CO).
Yeast techniques
Plasmid DNA was introduced into yeast cells using a modi-
fication of the lithium acetate protocol [17]. Yeast two–
hybrid interactions were tested using PJ69-4A as described
previously [64].
Function of Vrp1p C-terminal module T. Thanabalu et al.
4120 FEBS Journal 274 (2007) 4103–4125 ª 2007 The Authors Journal compilation ª 2007 FEBS
DNA techniques and plasmid construction
Standard DNA techniques were performed as described

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
pAM875 YCplac111 expressing C-Vrp1p
364)817 K512AD513A
under VRP1 promoter This study
pAM876 YCplac111 expressing C-Vrp1p
364)817 D594AK595A
under VRP1 promoter This study
pAM877 YCplac111 expressing C-Vrp1p
364)817 E692A
under VRP1 promoter This study
pAM878 YCplac111 expressing C-Vrp1p
364)817 K740A
under VRP1 promoter This study
pAM879 YCplac111 expressing C-Vrp1p
364)817 W782A
under VRP1 promoter This study
pAM880 YCplac111 expressing C-Vrp1p
465)817
under VRP1 promoter This study
pAM881 YCplac111 expressing C-Vrp1p
493)817
under VRP1 promoter This study
pAM882 YCplac111 expressing C-Vrp1p
533)817
under VRP1 promoter This study
pAM883 YCplac111 expressing C-Vrp1p
614)817
under VRP1 promoter This study
pAM884 YCplac111 expressing C-Vrp1p
716)817

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

under VRP1 promoter This study
pAM899 YCplac111 expressing C-Vrp1p
364)760
-CAAX under VRP1 promoter This study
pAM902 pACT2 expressing C-Vrp1p
364)817
This study
pAM903 pACT2 expressing C-Vrp1p
364)817K485AR486A
This study
pAM904 pACT2 expressing C-Vrp1p
465)817
This study
pAM905 pACT2 expressing C-Vrp1p
493)817
This study
pAM906 pACT2 expressing C-Vrp1p
533)817
This study
pAM907 pACT2 expressing C-Vrp1p
614)817
This study
pAM908 pACT2 expressing C-Vrp1p
716)817
This study
pAM909 pACT2 expressing C-Vrp1p
364)760
This study
pAM910 pACT2 expressing C-Vrp1p
465)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).

Sequences encoding various fragments of C-Vrp1p
364)817
were amplified by PCR and inserted into this plasmid. In
constructs expressing GFP-tagged Vrp1p fragments, a
sequence encoding yeast codon-bias GFP (yEGFP) (a gift
of B. Winsor, IBMC, Universite
´
Louis Pasteur, Strasbourg,
France) was fused inframe downstream of the VRP1 coding
sequence. The C-Vrp1p
364)817
– CAAX construct was made
by inframe fusion of a sequence encoding the Ras1p CAAX
box (GCCIIC) downstream of the VRP1 coding sequence
[45]. Site-directed mutagenesis was carried out using a
Unique Site Elimination kit (GE Healthcare ⁄ Amersham
Biosciences) and the mutated VRP1 sequences were used to
replace wild-type sequences in the plasmids described
above. The sequences of all constructs were confirmed by
automated DNA sequencing. Further details of the con-
structions are available upon request.
GST pull-down assay
A DNA fragment encoding Vrp1p residues 465–533 (wild-
type and K485AR486A mutant) were ligated with DNA of
the plasmid pGEX-KG encoding GST. E. coli transform-
ants were grown to exponential phase (D
600
¼ 0.5) and
induced with 0.1 mm isopropyl thio-b-d-galactoside for 3 h
at 37 °C. A cell lysate was prepared by sonication in

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

in 240 lL lysis solution (1.85 m NaOH ⁄ 1.06 m b-mercapto-
ethanol) and incubated on ice for 10 min. The protein was
precipitated with an equal volume of 20% trichloroacetic
acid on ice for 10 min. The pellet was collected by centrifu-
gation, resuspended in 100 lL of SDS ⁄ PAGE loading buf-
fer, and heated to 95 °C for 2 min. Proteins were resolved
on a 10% SDS ⁄ PAGE gel, electroblotted onto a poly(viny-
lidene difluoride) (PVDF) membrane, probed with appro-
priate primary antibodies and HRP-conjugated secondary
antibodies and detected with an enhanced chemilumines-
cence (ECL) kit [66].
LY uptake assay
Assays for measuring fluid phase endocytosis of LY were
performed as described previously [20]. All the transform-
ants were grown to exponential phase in YPUAD and LY
uptake was performed at either 24 or 37 °C as specified for
1 h. The cells were washed and then visualized using a
LY-specific light filter.
Visualization of Vrp1p subcellular localization
To visualize the subcellular distribution of Vrp1p–GFP,
cells carrying Vrp1p–GFP-expressing constructs growing
exponentially in SD selective medium at 24 °C were applied
to a microscope slide and the GFP signal in living cells was
visualized by fluorescence microscopy using a fluorescein
isothiocyanate (FITC)-specific light filter.
Visualization of F-actin
Yeast cells were grown in YPUAD to exponential phase at
24 °C and then fixed by direct addition of 3.7% formalde-
hyde to the culture and incubation for 30 min at the tem-
perature of growth as described [68]. Fixed cells were

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.
References
1 Adams AE & Pringle JR (1984) Relationship of actin
and tubulin distribution to bud growth in wild-type and
morphogenetic–mutant Saccharomyces cerevisiae. J Cell
Biol 98, 934–945.

2 Lew DJ & Reed SI (1993) Morphogenesis in the yeast
cell cycle: regulation by Cdc28 and cyclins. J Cell Biol
120, 1305–1320.
3 Amberg DC (1998) Three-dimensional imaging of the
yeast actin cytoskeleton through the budding cell cycle.
Mol Biol Cell 9, 3259–3262.
4 Winsor B & Schiebel E (1997) Review: an overview of
the Saccharomyces cerevisiae microtubule and microfila-
ment cytoskeleton. Yeast 13, 399–434.
5 Engqvist-Goldstein AE & Drubin DG (2003) Actin
assembly and endocytosis: from yeast to mammals.
Annu Rev Cell Dev Biol 19, 287–332.
6 Waddle JA, Karpova TS, Waterston RH & Cooper JA
(1996) Movement of cortical actin patches in yeast.
J Cell Biol 132, 861–870.
7 Doyle T & Botstein D (1996) Movement of yeast corti-
cal actin cytoskeleton visualized in vivo. Proc Natl Acad
Sci USA 93, 3886–3891.
8 Smith MG, Swamy SR & Pon LA (2001) The life cycle
of actin patches in mating yeast. J Cell Sci 114, 1505–
1513.
9 Pelham RJ Jr & Chang F (2001) Role of actin polymer-
ization and actin cables in actin-patch movement in
Schizosaccharomyces pombe. Nat Cell Biol 3, 235–244.
10 Kaksonen M, Sun Y & Drubin DG (2003) A pathway
for association of receptors, adaptors, and actin during
endocytic internalization. Cell 115, 475–487.
11 Jonsdottir GA & Li R (2004) Dynamics of yeast Myo-
sin I: evidence for a possible role in scission of endocy-
tic vesicles. Curr Biol 14, 1604–1609.

12 Huckaba TM, Gay AC, Pantalena LF, Yang HC &
Pon LA (2004) Live cell imaging of the assembly, disas-
sembly, and actin cable-dependent movement of endo-
somes and actin patches in the budding yeast,
Saccharomyces cerevisiae. J Cell Biol 167, 519–530.
13 Kaksonen M, Toret CP & Drubin DG (2005) A modu-
lar design for the clathrin- and actin-mediated endocyto-
sis machinery. Cell 123, 305–320.
14 Newpher TM, Smith RP, Lemmon V & Lemmon SK
(2005) In vivo dynamics of clathrin and its adaptor-
dependent recruitment to the actin-based endocytic
machinery in yeast. Dev Cell 9, 87–98.
15 Sun Y, Martin AC & Drubin DG (2006) Endocytic
internalization in budding yeast requires coordinated
actin nucleation and myosin motor activity. Dev Cell
11, 33–46.
16 Donnelly SF, Pocklington MJ, Pallotta D & Orr E
(1993) A proline-rich protein, verprolin, involved in
cytoskeletal organization and cellular growth in the
yeast Saccharomyces cerevisiae. Mol Microbiol 10,
585–596.
17 Munn AL, Stevenson BJ, Geli MI & Riezman H (1995)
end5,
end6, and end7: mutations that cause actin
delocalization and block the internalization step of
endocytosis in Saccharomyces cerevisiae. Mol Biol Cell
6, 1721–1742.
18 Zoladek T, Vaduva G, Hunter LA, Boguta M, Go BD,
Martin NC & Hopper AK (1995) Mutations altering
the mitochondrial–cytoplasmic distribution of Mod5p

implicate the actin cytoskeleton and mRNA 3¢ ends
and ⁄ or protein synthesis in mitochondrial delivery. Mol
Cell Biol 15, 6884–6894.
19 Vaduva G, Martin NC & Hopper AK (1997) Actin-
binding verprolin is a polarity development protein
required for the morphogenesis and function of the
yeast actin cytoskeleton. J Cell Biol 139, 1821–1833.
20 Naqvi SN, Zahn R, Mitchell DA, Stevenson BJ &
Munn AL (1998) The WASp homologue Las17p func-
tions with the WIP homologue End5p ⁄ verprolin and is
essential for endocytosis in yeast. Curr Biol 8, 959–962.
21 Vaduva G, Martinez-Quiles N, Anton IM, Martin NC,
Geha RS, Hopper AK & Ramesh N (1999) The human
WASP-interacting protein, WIP, activates the cell polar-
ity pathway in yeast. J Biol Chem 274, 17103–17108.
22 Naqvi SN, Feng Q, Boulton VJ, Zahn R & Munn AL
(2001) Vrp1p functions in both actomyosin ring-depend-
ent and Hof1p-dependent pathways of cytokinesis.
Traffic 2, 189–201.
23 Thanabalu T & Munn AL (2001) Functions of Vrp1p in
cytokinesis and actin patches are distinct and neither
requires a WH2 ⁄ V domain. EMBO J 20, 6979–6989.
24 Ren G, Wang J, Brinkworth R, Winsor B, Kobe B &
Munn AL (2005) Verprolin cytokinesis function medi-
ated by the Hof one trap domain. Traffic 6, 575–593.
25 Li R (1997) Bee1, a yeast protein with homology to
Wiscott–Aldrich syndrome protein, is critical for the
assembly of cortical actin cytoskeleton. J Cell Biol 136,
649–658.
26 Karpova TS, McNally JG, Moltz SL & Cooper JA

(1998) Assembly and function of the actin cytoskeleton
of yeast: relationships between cables and patches.
J Cell Biol 142, 1501–1517.
27 Madania A, Dumoulin P, Grava S, Kitamoto H, Scha-
rer-Brodbeck C, Soulard A, Moreau V & Winsor B
(1999) The Saccharomyces cerevisiae homologue of
human Wiskott–Aldrich syndrome protein Las17p inter-
acts with the Arp2 ⁄ 3 complex. Mol Biol Cell 10, 3521–
3538.
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 4123
28 Evangelista M, Klebl BM, Tong AH, Webb BA,
Leeuw T, Leberer E, Whiteway M, Thomas DY &
Boone C (2000) A role for myosin-I in actin assembly
through interactions with Vrp1p, Bee1p, and the
Arp2 ⁄ 3 complex. J Cell Biol 148, 353–362.
29 Lechler T, Jonsdottir GA, Klee SK, Pellman D & Li R
(2001) A two-tiered mechanism by which Cdc42 con-
trols the localization and activation of an Arp2 ⁄ 3-acti-
vating motor complex in yeast. J Cell Biol 155, 261–
270.
30 Ramesh N, Anton IM, Hartwig JH & Geha RS (1997)
WIP, a protein associated with Wiskott–Aldrich syn-
drome protein, induces actin polymerization and redis-
tribution in lymphoid cells. Proc Natl Acad Sci USA 94,
14671–14676.
31 Martinez-Quiles N, Rohatgi R, Anton IM, Medina M,
Saville SP, Miki H, Yamaguchi H, Takenawa T,
Hartwig JH, Geha RS et al. (2001) WIP regulates
N-WASP-mediated actin polymerization and filopodium

formation. Nat Cell Biol 3, 484–491.
32 Zettl M & Way M (2002) The WH1 and EVH1
domains of WASP and Ena ⁄ VASP family members
bind distinct sequence motifs. Curr Biol 12, 1617–1622.
33 Volkman BF, Prehoda KE, Scott JA, Peterson FC &
Lim WA (2002) Structure of the N-WASP EVH1
domain–WIP complex: insight into the molecular basis
of Wiskott–Aldrich syndrome. Cell 111, 565–576.
34 Anderson BL, Boldogh I, Evangelista M, Boone C,
Greene LA & Pon LA (1998) The Src homology
domain 3 (SH3) of a yeast type I myosin, Myo5p, binds
to verprolin and is required for targeting to sites of
actin polarization. J Cell Biol 141, 1357–1370.
35 Geli MI, Lombardi R, Schmelzl B & Riezman H (2000)
An intact SH3 domain is required for myosin I-induced
actin polymerization. EMBO J 19, 4281–4291.
36 Geli MI & Riezman H (1996) Role of type I myosins in
receptor-mediated endocytosis in yeast. Science 272,
533–535.
37 Winter D, Lechler T & Li R (1999) Activation of the
yeast Arp2 ⁄ 3 complex by Bee1p, a WASP-family pro-
tein. Curr Biol 9, 501–504.
38 Lechler T, Shevchenko A, Shevchenko A & Li R (2000)
Direct involvement of yeast type I myosins in Cdc42-
dependent actin polymerization. J Cell Biol 148, 363–373.
39 Winter D, Podtelejnikov AV, Mann M & Li R (1997)
The complex containing actin-related proteins Arp2 and
Arp3 is required for the motility and integrity of yeast
actin patches. Curr Biol 7, 519–529.
40 Welch MD (1999) The world according to Arp: regula-

tion of actin nucleation by the Arp2 ⁄ 3 complex. Trends
Cell Biol 9, 423–427.
41 Higgs HN & Pollard TD (2001) Regulation of actin fil-
ament network formation through Arp2 ⁄ 3 complex:
activation by a diverse array of proteins. Annu Rev
Biochem 70, 649–676.
42 Moreau V, Madania A, Martin RP & Winsor B (1996)
The Saccharomyces cerevisiae actin-related protein Arp2
is involved in the actin cytoskeleton.
J Cell Biol 134,
117–132.
43 Paunola E, Mattila PK & Lappalainen P (2002) WH2
domain: a small, versatile adapter for actin monomers.
FEBS Lett 513, 92–97.
44 Golsteyn RM, Beckerle MC, Koay T & Friederich E
(1997) Structural and functional similarities between
the human cytoskeletal protein zyxin and the ActA
protein of Listeria monocytogenes. J Cell Sci 110,
1893–1906.
45 Powers S, Kataoka T, Fasano O, Goldfarb M, Strath-
ern J, Broach J & Wigler M (1984) Genes in S. cerevisi-
ae encoding proteins with domains homologous to the
mammalian ras proteins. Cell 36 , 607–612.
46 Yang J, Ishimori K & O’Brian MR (2005) Two heme
binding sites are involved in the regulated degradation
of the bacterial iron response regulator (Irr) protein.
J Biol Chem 280, 7671–7676.
47 Walker J, Crowley P, Moreman AD & Barrett J (1993)
Biochemical properties of cloned glutathione S-trans-
ferases from Schistosoma mansoni and Schistosoma

japonicum. Mol Biochem Parasitol 61, 255–264.
48 Sirotkin V, Beltzner CC, Marchand JB & Pollard TD
(2005) Interactions of WASp, myosin-I, and verprolin
with Arp2 ⁄ 3 complex during actin patch assembly in
fission yeast. J Cell Biol 170, 637–648.
49 Goodson HV, Anderson BL, Warrick HM, Pon LA &
Spudich JA (1996) Synthetic lethality screen identifies a
novel yeast myosin I gene (MYO5): myosin I proteins
are required for polarization of the actin cytoskeleton.
J Cell Biol 133, 1277–1291.
50 Valdez-Taubas J & Pelham HR (2003) Slow diffusion of
proteins in the yeast plasma membrane allows polarity
to be maintained by endocytic cycling. Curr Biol 13,
1636–1640.
51 Vallen EA, Caviston J & Bi E (2000) Roles of Hof1p,
Bni1p, Bnr1p, and Myo1p in cytokinesis in Saccharomy-
ces cerevisiae. Mol Biol Cell 11, 593–611.
52 Moreau V, Frischknecht F, Reckmann I, Vincentelli R,
Rabut G, Stewart D & Way M (2000) A complex of
N-WASP and WIP integrates signalling cascades that
lead to actin polymerization. Nat Cell Biol 2, 441–448.
53 Schindelhauer D, Weiss M, Hellebrand H, Golla A,
Hergersberg M, Seger R, Belohradsky BH & Meindl A
(1996) Wiskott–Aldrich syndrome: no strict genotype–
phenotype correlations but clustering of missense muta-
tions in the amino-terminal part of the WASP gene
product. Hum Genet 98, 68–76.
54 Stewart DM, Tian L & Nelson DL (1999) Mutations
that cause the Wiskott–Aldrich syndrome impair the
interaction of Wiskott–Aldrich syndrome protein

(WASP) with WASP interacting protein. J Immunol
162, 5019–5024.
Function of Vrp1p C-terminal module T. Thanabalu et al.
4124 FEBS Journal 274 (2007) 4103–4125 ª 2007 The Authors Journal compilation ª 2007 FEBS
55 Rajmohan R, Meng L, Yu S & Thanabalu T (2006)
WASP suppresses the growth defect of Saccharomyces
cerevisiae las17D strain in the presence of WIP. Biochem
Biophys Res Commun 342, 529–536.
56 de la Fuente MA, Sasahara Y, Calamito M, Anton IM,
Elkhal A, Gallego MD, Suresh K, Siminovitch K, Ochs
HD, Anderson KC et al. (2007) WIP is a chaperone for
Wiskott–Aldrich syndrome protein (WASP). Proc Natl
Acad Sci USA 104, 926–931.
57 Sawa M & Takenawa T (2006) Caenorhabditis elegans
WASP-interacting protein homologue WIP-1 is
involved in morphogenesis through maintenance of
WSP-1 protein levels. Biochem Biophys Res Commun
340, 709–717.
58 Machesky LM & Insall RH (1998) Scar1 and the related
Wiskott–Aldrich syndrome protein, WASP, regulate the
actin cytoskeleton through the Arp2 ⁄ 3 complex. Curr
Biol 8, 1347–1356.
59 Aspenstrom P (2005) The verprolin family of proteins:
regulators of cell morphogenesis and endocytosis. FEBS
Lett 579, 5253–5259.
60 Liu H, Krizek J & Bretscher A (1992) Construction of a
GAL1-regulated yeast cDNA expression library and its
application to the identification of genes whose overex-
pression causes lethality in yeast. Genetics 132, 665–673.
61 Lippincott J & Li R (1998) Dual function of Cyk2, a

cdc15 ⁄ PSTPIP family protein, in regulating actomyosin
ring dynamics and septin distribution. J Cell Biol 143,
1947–1960.
62 Colwill K, Field D, Moore L, Friesen J & Andrews B
(1999) In vivo analysis of the domains of yeast Rvs167p
suggests Rvs167p function is mediated through multiple
protein interactions. Genetics 152, 881–893.
63 Norden C, Liakopoulos D & Barral Y (2004) Dissection
of septin actin interactions using actin overexpression in
Saccharomyces cerevisiae. Mol Microbiol 53, 469–483.
64 James P, Halladay J & Craig EA (1996) Genomic
libraries and a host strain designed for highly efficient
two-hybrid selection in yeast. Genetics 144, 1425–1436.
65 Adams A, Gottschling DE, Kaiser KA & Stearns T
(1997) Methods in Yeast Genetics. A Cold Spring Harbor
Laboratory Course Manual. Cold Spring Harbor Labor-
atory Press, Cold Spring Harbor, NY.
66 Sambrook J, Fritsch EF & Maniatis T (1989) Molecular
Cloning: A Laboratory Manual, 2nd edn. Cold Spring.
Harbor Laboratory Press, Cold Spring Harbor, NY.
67 Gietz RD & Sugino A (1988) New yeast–Escherichia
coli shuttle vectors constructed with in vitro mutagen-
ized yeast genes lacking six-base pair restriction sites.
Gene 74, 527–534.
68 Chowdhury S, Smith KW & Gustin MC (1992) Osmotic
stress and the yeast cytoskeleton: phenotype-specific
suppression of an actin mutation. J Cell Biol 118, 561–
571.
Supplementary material
The following supplementary material is available

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.
This material is available as part of the online article
from
Please note: Blackwell Publishing is not responsible
for the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corres-
ponding author for the article.
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 4125