Verprolin function in endocytosis and actin organization

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Verprolinfunctioninendocytosisandactinorganization—rolesoftheLas17p(yeastWASP)-bindingdomainandanovelC-terminalactin-bindingdomain.FEBSJ

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6AUTHORS,INCLUDING:

ThirumaranThanabalu

NanyangTechnologicalUniversity

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RajmohanRajamuthiah

MassachusettsGeneralHospital

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LeiMeng

NanyangTechnologicalUniversity

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AlanLMunn

GriffithUniversity

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Verprolin function in endocytosis and actin organization

Roles of the Las17p (yeast WASP)-binding domain and a novelC-terminal actin-binding domain

Thirumaran Thanabalu1,2, Rajamuthiah Rajmohan2, Lei Meng2, Gang Ren4,5, Parimala R. Vajjhala4

and Alan L. Munn1,3,4,6*

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: a.munn@imb.uq.edu.au

*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) leadsto 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-Vrp1p1)364 binds actin monomers

[19,21,23], whereas C-Vrp1p364)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 ⁄ 3complex 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-Vrp1p364)817functionally replaces full-length Vrp1p for growth at

elevated temperatures. Furthermore, like full-length

Vrp1p, C-Vrp1p364)817 efficiently localizes to cortical

actin patches. Localization of C-Vrp1p364)817 to these

patches is critically dependent on Las17p [23]. Also

like full-length Vrp1p, C-Vrp1p364)817 efficiently

mediates cortical actin-patch polarization [23]. How

does C-Vrp1p364)817 mediate cortical actin-patch

polarization? Does C-Vrp1p364)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-Vrp1p364)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-Vrp1p364)817was 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-Vrp1p364)817 (W782) with alanine.

These eight DNA fragments encoding mutant

C-Vrp1p364)817 proteins were placed under the control

of the native VRP1 promoter on a centromeric plas-

mid and introduced into vrp1D (AMY88) cells. The

ability of the mutated C-Vrp1p364)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-Vrp1p364)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-Vrp1p364)817, we meas-

ured uptake of the membrane-impermeant fluid-phase

endocytic dye Lucifer yellow (LY). vrp1D cells expres-

sing C-Vrp1p364)817 or C-Vrp1p364–817K485AR486A 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-Vrp1p364)817 and

C-Vrp1p364)817K485AR486A were both fused inframe to

a sequence encoding green fluorescent protein (GFP)

and expressed from the VRP1 promoter carried on a

1 817Vrp1p

1 364N-Vrp1p1-364

HOT domain

Las17p-binding domain

X CAAX box (lipid anchor)

Glutathione S-transferase (GST)

actin-binding domain

1 817465C-Vrp1p465-817

1 817716C-Vrp1p716-817

1 817493C-Vrp1p493-817

5331 817533C-Vrp1p533-817

1 817C-Vrp1p614-817 614

364 760C-Vrp1p364-760

1 817760C-Vrp1p760-817

364 760C-Vrp1p364-760-CAAX X

1 760C-Vrp1p760-817-GST

1 817364

364 760C-Vrp1p364-760-GST

C-Vrp1p364-817 K485A, R486A

1 817364C-Vrp1p364-817-GST

1 817364C-Vrp1p364-817

(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-Vrp1p364)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

centromeric plasmid. As a control, we also made an

equivalent construct expressing GFP only. Both GFP-

tagged C-Vrp1p364)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-Vrp1p364)817function (Fig. S1). Total-cell extracts were prepared

from vrp1D cells expressing C-Vrp1p364)817–GFP or

C-Vrp1p364)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-Vrp1p364)817–GFP and

C-Vrp1p364)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-Vrp1p364)817and C-Vrp1p364)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

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-Vrp1p364)817 charged-

cluster residues K485R486 are not essential for growth on solid

medium at elevated temperatures. Growth at 24 and 37 �C of

vrp1D (AMY88) cells carrying YCplac111 vector (vect), pAM236

expressing C-Vrp1p364)817 (C-Vrp1p364)817) and pAM873 expressing

C-Vrp1p364)817K485AR486A (C-Vrp1p364)817AA). 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-Vrp1p364)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-Vrp1p364)817 (C-Vrp1p364)817), and pAM873 expres-

sing C-Vrp1p364)817K485AR486A (C-Vrp1p364)817AA). A YPUAD culture

of each strain was grown at 24 �C, diluted to D600 ¼ 0.05 in fresh

YPUAD medium, and incubated at 37 �C. D600 was monitored

at 1 h intervals. (C) The C-Vrp1p364)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-Vrp1p364)817 (C-Vrp1p364)817),

and pAM873 expressing C-Vrp1p364)817K485AR486A

(C-Vrp1p364)817AA). 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-Vrp1p364)817 charged-cluster residues K485R486

are not essential for endocytosis. vrp1D (AMY88) cells carrying

YCplac111 vector (vect), pAM236 expressing C-Vrp1p364)817

(C-Vrp1p364)817) or pAM873 expressing C-Vrp1p364)817K485AR486A

(C-Vrp1p364)817AA) were grown in YPUAD to exponential phase at

24 �C and 1 · 107 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-Vrp1p364)817 with charged-cluster resi-

dues K485R486 substituted with alanine is stably expressed. Total

extracts from vrp1D (AMY88) cells carrying pAM241 expres-

sing C-Vrp1p364)817 fused at its C-terminus to green fluorescent

protein (GFP) (C-Vrp1p364)817–GFP) or pAM913 expressing

C-Vrp1p364)817K485AR486A–GFP (C-Vrp1p364)817AA–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).

present in full-length Vrp1p, but not C-Vrp1p364)817,

may compensate for loss of K485R486.

Localization of C-Vrp1p364)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-Vrp1p364)817 with N-Las17p1)241 (Fig. S2A). The

substitution of K485R486 with alanine also did

not abolish C-Vrp1p364)817–GFP localization to cor-

tical patches in vrp1D cells (Fig. S2B). However,

C-Vrp1p364)817–GFP patches were polarized to sites of

surface growth, whereas C-Vrp1p364)817K485AR486A–

GFP patches were depolarized (Fig. S2B). We conclude

that loss of function of C-Vrp1p364)817K485AR486A in

cortical actin-patch polarization is not due to an effect

of these mutations on localization of C-Vrp1p364)817 to

cortical patches, but may be due to inefficient polariza-

tion of C-Vrp1p364)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-Vrp1p364)817 important for function we

constructed deletions initiating at the N-terminus of

C-Vrp1p364)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-Vrp1p364)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-Vrp1p364)817 functions. Additional deletion of 28

residues from the N-terminus resulted in a protein

(C-Vrp1p493)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-Vrp1p364)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-Vrp1p364)817function 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-Vrp1p493)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-Vrp1p465)817,

C-Vrp1p493)817, C-Vrp1p533)817, C-Vrp1p614)817 or

C-Vrp1p716)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-Vrp1p364)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 �C 37 �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-Vrp1p364)760 1 99 0 100

C-Vrp1p364)760CAAX 5 95 3 97

N-Las17p1)241 (Fig. S3A). Furthermore, none of the

five deletions (including deletion of residues 364–492)

abolished localization of C-Vrp1p364)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-

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). Vrp1p465)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-Vrp1p465)533 associates with actin

in crude yeast lysates we expressed wild-type and

K485R486 mutant C-Vrp1p465)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-Vrp1p465)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-Vrp1p465)533–GST fusion pro-

teins with purified Saccharomyces cerevisiae actin in

G-actin buffer. Bound proteins were analysed as

above. The wild-type C-Vrp1p465–533 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–Vrp1p465)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-Vrp1p364)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 �C of vrp1D (AMY88) cells carrying YCplac111

vector (vect), pAM880 expressing C-Vrp1p465)817 (C-Vrp1p465)817), pAM881 expressing C-Vrp1p493)817 (C-Vrp1p493)817), pAM882 expressing

C-Vrp1p533)817 (C-Vrp1p533)817), pAM883 expressing C-Vrp1p614)817 (C-Vrp1p614)817), or pAM884 expressing C-Vrp1p716)817 (C-Vrp1p716)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-Vrp1p364)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-Vrp1p364)817

(C-Vrp1p364)817), pAM880 expressing C-Vrp1p465)817 (C-Vrp1p465)817), pAM881 expressing C-Vrp1p493)817 (C-Vrp1p493)817), pAM882 expres-

sing C-Vrp1p533)817 (C-Vrp1p533)817), pAM883 expressing C-Vrp1p614)817 (C-Vrp1p614)817), or pAM884 expressing C-Vrp1p716)817

(C-Vrp1p716)817). A YPUAD culture of each strain was grown at 24 �C, diluted to D600 ¼ 0.05 in fresh YPUAD medium and shifted to 37 �C.

D600 was monitored at 1 h intervals. (C) C-Vrp1p364)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-Vrp1p364)817 (C-Vrp1p364)817),

pAM880 expressing C-Vrp1p465)817 (C-Vrp1p465)817), pAM881 expressing C-Vrp1p493)817 (C-Vrp1p493)817), pAM882 expressing

C-Vrp1p533)817 (C-Vrp1p533)817), pAM883 expressing C-Vrp1p614)817 (C-Vrp1p614)817), or pAM884 expressing C-Vrp1p716)817 (C-Vrp1p716)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-Vrp1p364)817 residues

465–492 containing the K485R486 charged cluster are not essential for fluid-phase endocytosis. vrp1D (AMY88) cells carrying pAM236

expressing C-Vrp1p364)817 (C-Vrp1p364)817), pAM880 expressing C-Vrp1p465)817 (C-Vrp1p465)817), pAM881 expressing C-Vrp1p493)817

(C-Vrp1p493)817), pAM882 expressing C-Vrp1p533)817 (C-Vrp1p533)817), pAM883 expressing C-Vrp1p614)817 (C-Vrp1p614)817), or pAM884

expressing C-Vrp1p716)817 (C-Vrp1p716)817) were grown in YPUAD to exponential phase at 24 �C and 1 · 107 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-Vrp1p364)817 fragments lacking residues 465–492 containing the K485R486 charged cluster are

stably expressed. Total extracts from vrp1D (AMY88) cells carrying pAM241 expressing C-Vrp1p364)817 fused at its C-terminus

to GFP (C-Vrp1p364)817–GFP), pAM885 expressing C-Vrp1p465)817–GFP (C-Vrp1p465)817–GFP), pAM886 expressing C-Vrp1p493)817–GFP

(C-Vrp1p493)817–GFP), pAM887 expressing C-Vrp1p533)817–GFP (C-Vrp1p533)817–GFP), pAM888 expressing C-Vrp1p614)817–GFP

(C-Vrp1p614)817–GFP), or pAM889 expressing C-Vrp1p716)817–GFP (C-Vrp1p716)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).

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-Vrp1p364)817 in growth we expressed

C-Vrp1p364)760 in vrp1D (AMY88) cells and examined

growth at elevated temperature (Fig. 5A,B). Loss of

the LBD abolished the ability of C-Vrp1p364)817 to

restore growth of vrp1D cells at elevated temperature.

We next assessed the importance of the LBD for the

ability of C-Vrp1p364)817 to restore cortical actin-patch

polarization and endocytosis to vrp1D cells. vrp1D(AMY88) cells expressing either full-length

C-Vrp1p364)817 or the truncated form lacking a LBD

(C-Vrp1p364)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-Vrp1p364)817 to mediate cortical

actin-patch polarization. The truncated form of

C-Vrp1p364)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-Vrp1p364)817expression or stability the C-terminus of a trun-

cated form of C-Vrp1p364)817 lacking the LBD

(C-Vrp1p364)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-Vrp1p465)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-Vrp1p1)70

(AD-N-Vrp1p1)70), pAM918 expressing Gal4-AD-C-Vrp1p465)533 (AD-C-Vrp1p465)533), pAM919 expressing Gal4-AD-C-Vrp1p465)533K485AR486A

(AD-C-Vrp1p465)533AA), pAM908 expressing Gal4-AD-C-Vrp1p716)817 (AD-C-Vrp1p716)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 mM 3-amino 1,2,4-triazole. Plates were photographed after 4 days. (C) The C-Vrp1p364)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-Vrp1p465)533 (GST–C-Vrp1p465)533), or pAM1002 expressing GST-C-Vrp1p465)533K485AR486A (GST–C-Vrp1p465)533AA) 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-Vrp1p364)817 charged cluster directly binds yeast G-actin in vitro and residues K485R486 are essential. Beads bearing GST (GST),

GST–C-Vrp1p465)533 (GST–C-Vrp1p465)533), and GST–C-Vrp1p465)533K485AR486A (GST-C-Vrp1p465)533AA), 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.

C-Vrp1p364)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-Vrp1p364)760 is expressed at equival-

ent levels to C-Vrp1p364)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-Vrp1p364)817 to

cortical patches or interaction with Las17p (Fig. 6A–C).

The subcellular distribution of C-Vrp1p364)760–GFP

was analysed using live cell fluorescence imaging

AD-N-Vrp1p1-70

AD-C-Vrp1p465-533

AD-C-Vrp1p465-533 AA

AD-C-Vrp1p716-817

AD-vect

+His -His

-actin

-Actin

Bound Load

Purified

GST fusion

(Fig. 6A). C-Vrp1p364)760–GFP displayed a diffuse

cytoplasmic localization similar to GFP alone. In con-

trast, C-Vrp1p364)817–GFP localized to cortical patches

(Fig. 6A) consistent with our previous report [23]. The

expression level of truncated C-Vrp1p364)760–GFP was

equivalent to that of C-Vrp1p364)817–GFP (Fig. 5E).

This suggests that loss of cortical-patch localization is

not an indirect consequence of lowered expression of the

truncated C-Vrp1p364)760–GFP fusion protein relative

to C-Vrp1p364)817–GFP. We expect that the relative

expression level of the GFP-tagged proteins is indicative

of that of the equivalent untagged proteins.

C-Vrp1p364-817vect

C-Vrp1p364-760

Time (h)

1 2 3 4 5 6 7 8

C-Vrp1p364-817

C-Vrp1p364-760

37°C24°C

C-Vrp1p364-760C-Vrp1p364-817vect

C-Vrp1p364-760C-Vrp1p364-817vect C-Vrp1p

364-760-CAAX

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-Vrp1p364)817 with Las17p

we performed two-hybrid tests. C-Vrp1p364)817 exhib-

ited two-hybrid interaction with an N-terminal

fragment of Las17p (N-Las17p1)241), however, in

contrast, the truncated form C-Vrp1p364)760 was unable

to interact with N-Las17p1)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-Vrp1p364)817 to cortical patches? To address

this question we tagged the isolated LBD of

Vrp1p with GFP to create C-Vrp1p760)817–GFP.

C-Vrp1p760)817–GFP was expressed in vrp1D (AMY88)

cells and its subcellular distribution examined by live

cell fluorescence imaging (Fig. 6C, left). Strikingly,

C-Vrp1p760)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-Vrp1p760)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-Vrp1p364)817–GFP in cells expres-

sing full-length wild-type Vrp1p (Fig. S4).

The cortical patch localization of C-Vrp1p760)817–

GFP is dependent on Las17p. When expressed in las17D

(IDY166) cells, C-Vrp1p760)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-Vrp1p364)817 when

expressed in las17D cells [23]. The expression level of

C-Vrp1p760)817–GFP was examined by SDS ⁄PAGE

and immunoblot (Fig. 6D) and found to be easily

detectible but reduced compared with that of

C-Vrp1p716)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-Vrp1p1)364 fragment [23]. Addition of the CAAX

box also enhances the function of N-Vrp1p1)364 in

growth at elevated temperature such that it rescues the

temperature-sensitive growth defect of vrp1D with an

efficiency approaching that of C-Vrp1p364)817 or full-

length Vrp1p [23]. We therefore asked whether using

the same technique to anchor C-Vrp1p364)760 (CABS)

to membranes would restore function in the absence of

the LBD. C-Vrp1p364)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-Vrp1p364)817 is essential for growth on solid medium at elevated temperature. Growth at 24 and 37 �C of vrp1D (AMY88)

cells carrying YCplac111 vector (vect), pAM236 expressing C-Vrp1p364)817 (C-Vrp1p364)817), or pAM896 expressing C-Vrp1p364)760

(C-Vrp1p364)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-Vrp1p364)817 is essential for growth in liquid medium at elevated temperature. Growth rate of

vrp1D (AMY88) cells carrying YCplac111 vector (vect), pAM236 expressing C-Vrp1p364)817 (C-Vrp1p364)817) and pAM896 expressing

C-Vrp1p364)760 (C-Vrp1p364)760). A YPUAD culture of each strain was grown at 24 �C, diluted to D600 ¼ 0.05 in fresh YPUAD medium, and

incubated at 37 �C. D600 was monitored at 1 h intervals. (C) The 57-residue LBD of C-Vrp1p364)817 is essential for polarization of cortical actin

patches. Cortical actin-patch polarization in vrp1D (AMY88) cells carrying YCplac111 vector (vect), pAM236 expressing C-Vrp1p364)817

(C-Vrp1p364)817), pAM896 expressing C-Vrp1p364)760 (C-Vrp1p364)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-Vrp1p364)817 LBD is essential for endocytosis. vrp1D (AMY88) cells carrying YCplac111 vector only (vect),

pAM236 expressing C-Vrp1p364)817 (C-Vrp1p364)817), pAM896 expressing C-Vrp1p364)760 (C-Vrp1p364)760) or pAM899 expressing

C-Vrp1p364)760 fused at its C-terminus to the CAAX box of Ras1p (C-Vrp1p364)760-CAAX) were grown in YPUAD to exponential phase at

24 �C and 1 · 107 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-Vrp1p364)817 lacking the 57-residue LBD is stably

expressed. Total extracts from vrp1D (AMY88) cells carrying pAM241 expressing C-Vrp1p364)817 fused at its C-terminus to GFP

(C-Vrp1p364)817–GFP) or pAM891 expressing C-Vrp1p364)760–GFP (C-Vrp1p364)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).

to yield C-Vrp1p364)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-Vrp1p364)817 containing the LBD

(Figs 5D and 7A–C). Strikingly, the presence of the

CAAX box allowed the truncated C-Vrp1p364)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

+His -His

p 1-24

AD-C-Vrp1p364-817

AD-C-Vrp1p364-760

AD-C-Vrp1p465-817

AD-C-Vrp1p465-760

AD-vect

p 1-24

1C-Vrp1p

364-760-GFPGFP C-Vrp1p

364-817-GFPC-Vrp1p

364-760-GFP-CAAX

α-Hex

α-GFP

C GFP C-Vrp1p

760-817-GFPC-Vrp1p

760-817-GFP

las17Δvrp1Δ

Fig. 6. The LBD of C-Vrp1p is necessary and sufficient for localization to cortical patches. (A) The 57-residue LBD of C-Vrp1p364)817 is essen-

tial for localization to cortical patches. vrp1D (AMY88) cells carrying pAM237 expressing GFP, pAM241 expressing C-Vrp1p364)817–GFP

(C-Vrp1p364)817–GFP), pAM891 expressing C-Vrp1p364)760–GFP (C-Vrp1p364)760–GFP) or pAM1003 expressing C-Vrp1p364)760–GFP fused at

its C-terminus to the CAAX box of Ras1p (C-Vrp1p364)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-Vrp1p364)817 interaction with Las17p. pAM912 expressing Gal4-BD-N-Las17p1)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-Vrp1p364)817 (AD-C-Vrp1p364)817), pAM909 expressing Gal4-AD-C-Vrp1p364)760 (AD-C-Vrp1p364)760), pAM904 expressing Gal4-

AD-C-Vrp1p465)817 (AD-C-Vrp1p465)817), pAM910 expressing Gal4-AD-C-Vrp1p465)760 (AD-C-Vrp1p465)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 mM 3-amino 1,2,4-triazole. Plates were photographed after 4 days. (C) The 57-residue LBD of C-Vrp1p364)817 is

sufficient for localization to cortical patches. vrp1D (AMY88) or las17D (IDY166) cells carrying pAM237 expressing GFP (GFP) or pAM890

expressing C-Vrp1p760)817–GFP (C-Vrp1p760)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-Vrp1p364)817 is stably expressed as a fusion to GFP. Total extracts from vrp1D (AMY88) cells carrying pAM889 expressing

C-Vrp1p716)817–GFP (C-Vrp1p716)817–GFP), pAM237 expressing GFP (GFP) or pAM890 expressing C-Vrp1p760)817 – GFP (C-Vrp1p760)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).

vect C-Vrp1p364-817

C-Vrp1p364-760

24°C 37°C

1 2 3 4 5 6 7 8Time (h)

C-Vrp1p364-817

C-Vrp1p364-760-CAAX

C-Vrp1p364-760

C C-Vrp1p 364-760C-Vrp1p 364-817 C-Vrp1p 364-760-CAAXvect

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-Vrp1p364)817 for

growth on solid media at elevated temperature. Growth at 24 and 37 �C of vrp1D (AMY88) cells carrying YCplac111 vector (vect), pAM236

expressing C-Vrp1p364)817 (C-Vrp1p364)817), pAM896 expressing C-Vrp1p364)760 (C-Vrp1p364)760), or pAM899 expressing C-Vrp1p364)760

fused at its C-terminus to the CAAX box of Ras1p (C-Vrp1p364)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-Vrp1p364)817 for growth in liquid medium at elevated temperature. Growth rate of vrp1D (AMY88) cells carry-

ing YCplac111 vector (vect), pAM236 expressing C-Vrp1p364)817 (C-Vrp1p364)817), pAM896 expressing C-Vrp1p364)760 (C-Vrp1p364)760), or

pAM899 expressing C-Vrp1p364)760–CAAX (C-Vrp1p364)760–CAAX). A YPUAD culture of each strain was grown at 24 �C, diluted to D600 ¼0.05 in fresh YPUAD medium and shifted to 37 �C. D600 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-Vrp1p364)817 for cortical actin-patch polarization. Cortical actin-patch polarization in vrp1D

(AMY88) cells carrying YCplac111 vector only (vect), pAM236 expressing C-Vrp1p364)817 (C-Vrp1p364)817), pAM896 expressing

C-Vrp1p364)760 (C-Vrp1p364)760), or pAM899 expressing C-Vrp1p364)760–CAAX (C-Vrp1p364)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.

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-Vrp1p364)760–CAAX

is able to restore cortical actin-patch polarization

to vrp1D cells. vrp1D (AMY88) cells expressing

C-Vrp1p364)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-Vrp1p364)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-Vrp1p364)760 restores localization to cortical patches.

The DNA sequence encoding C-Vrp1p364)760 was fused

inframe to sequences encoding GFP–CAAX and

expressed in vrp1D cells (AMY88) (Fig. 6A). The

CAAX sequence targeted C-Vrp1p364)760 to membranes

including the plasma membrane consistent with

our earlier findings with N-Vrp1p1)364 [23]. Signifi-

cantly, however, the CAAX sequence failed to

target C-Vrp1p364)760–GFP–CAAX to cortical pat-

ches. C-Vrp1p364)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-Vrp1p716)817 and C-Vrp1p760)817) in

vrp1D (AMY88) cells and tested their ability to restore

various Vrp1p-dependent functions. The longer frag-

ment, C-Vrp1p716)817, could rescue growth at elevated

temperature moderately well (Fig. 3A), but the shorter

fragment, C-Vrp1p760)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-Vrp1p760)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-Vrp1p760)817–GST, but not GST alone or

C-Vrp1p364)760–GST, restored growth at elevated tem-

perature to vrp1D cells moderately well (Fig. 8A,B).

This result demonstrates that C-Vrp1p760)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-Vrp1p760)817–GST also restored endocytosis

to vrp1D cells (Fig. 8C). However, in contrast,

C-Vrp1p760)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-Vrp1p364)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-Vrp1p364)817 is sufficient for growth on solid medium at elevated temperature. Growth at 24 and 37 �C of

vrp1D (AMY88) cells carrying pAM915 expressing GST only (GST), pAM914 expressing C-Vrp1p364)817 fused at its C-terminus to GST

(C-Vrp1p364)817–GST), pAM895 expressing C-Vrp1p760)817–GST (C-Vrp1p760)817–GST), or pAM892 expressing C-Vrp1p364)760–GST

(C-Vrp1p364)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-Vrp1p364)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-Vrp1p364)817 (C-Vrp1p364)817), or pAM895

expressing C-Vrp1p760)817–GST (C-Vrp1p760)817–GST). A YPUAD culture of each strain was grown at 24 �C, diluted to D600 ¼ 0.05 in fresh

YPUAD medium and shifted to 37 �C. D600 was monitored at 1 h intervals. (C) The 57-residue LBD of C-Vrp1p364)817 is sufficient for fluid-

phase endocytosis. vrp1D (AMY88) cells carrying pAM915 expressing GST only (GST), pAM236 expressing C-Vrp1p364)817 (C-Vrp1p364)817),

or pAM895 expressing C-Vrp1p760)817–GST (C-Vrp1p760)817–GST) were grown in YPUAD to exponential phase at 24 �C and 1 · 107 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-Vrp1p364)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-Vrp1p364)817 (C-Vrp1p364)817), or pAM895 expressing C-Vrp1p760)817–GST (C-Vrp1p760)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.

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-Vrp1p364)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

C-Vrp1p364-760-GST

C-Vrp1p364-817-GST

C-Vrp1p760-817-GST

24°C 37°C

Time (h)

1 2 3 4 5 6 7 8 9

0 C-Vrp1p364-817

C-Vrp1p760-817-GST

C-Vrp1p

760-817-GSTGST C-Vrp1p 364-817

C-Vrp1p760-817-GSTGST C-Vrp1p 364-817

C-Vrp1p364)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-Vrp1p364)817 exhibits

two-hybrid interaction with actin and that GST-

C-Vrp1p465)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-Vrp1p364)817 (Figs 2E and 5E).

Moreover, mutation of the VH2-C domain charged

cluster did not appear to have global effects on

C-Vrp1p364)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-Vrp1p364)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-Vrp1p364)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-Vrp1p364)817 folding.

The VH2-C domain charged cluster and the LBD

are both essential for cortical actin-patch polarization

mediated by C-Vrp1p364)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-Vrp1p364)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-Vrp1p364)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-Vrp1p364)817localization to cortical patches demonstrates that the

VH2-C domain alone is not sufficient to confer cortical

patch localization on C-Vrp1p364)817 despite its ability

to interact with actin. Moreover, C-Vrp1p760)817comprising 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-Vrp1p364)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-Vrp1p364)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-Vrp1p1)364does 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-Vrp1p364)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-Vrp1p364)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

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-Vrp1p716)817 retains some ability

to function without any additional sequences, whereas

the shorter fragment C-Vrp1p760)817 is functional only

when fused to GST (Figs 3A,B and 8A–C). Perhaps

C-Vrp1p760)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-

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 stabilizeLas17p 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-bindingfunctions 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 gal80Dmet2::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-PSQ) 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].

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-Vrp1p364)817 under VRP1 promoter [23]

pAM237 YCplac111 expressing GFP under VRP1 promoter [23]

pAM241 YCplac111 expressing C-Vrp1p364)817–GFP under VRP1 promoter [23]

pAM252 YCplac22 with GAL4BD-ACT1 [23]

pAM253 pACT2 expressing N-Vrp1p1)70 [23]

pAM872 YCplac111 expressing C-Vrp1p364)817 K457A under VRP1 promoter This study

pAM873 YCplac111 expressing C-Vrp1p364)817 K485AR486A under VRP1 promoter This study

pAM874 YCplac111 expressing C-Vrp1p364)817 D502AK503A under VRP1 promoter This study

pAM875 YCplac111 expressing C-Vrp1p364)817 K512AD513A under VRP1 promoter This study

pAM876 YCplac111 expressing C-Vrp1p364)817 D594AK595A under VRP1 promoter This study

pAM877 YCplac111 expressing C-Vrp1p364)817 E692A under VRP1 promoter This study

pAM878 YCplac111 expressing C-Vrp1p364)817 K740A under VRP1 promoter This study

pAM879 YCplac111 expressing C-Vrp1p364)817 W782A under VRP1 promoter This study

pAM880 YCplac111 expressing C-Vrp1p465)817 under VRP1 promoter This study

pAM885 YCplac111 expressing C-Vrp1p465)817–GFP under VRP1 promoter This study

pAM892 YCplac111 expressing C-Vrp1p364)760–GST under VRP1 promoter This study

pAM895 YCplac111 expressing C-Vrp1p760)817-GST under VRP1 promoter This study

pAM899 YCplac111 expressing C-Vrp1p364)760-CAAX under VRP1 promoter This study

pAM902 pACT2 expressing C-Vrp1p364)817 This study

pAM903 pACT2 expressing C-Vrp1p364)817K485AR486A This study

pAM912 pAS2-1 expressing N-Las17p1)241 This study

pAM913 YCplac111 expressing C-Vrp1p364)817K485AR486A–GFP under VRP1 promoter This study

pAM914 YCplac111 expressing C-Vrp1p364)817–GST under VRP1 promoter This study

pAM915 YCplac111 expressing GST only under VRP1 promoter This study

pAM919 pACT2 expressing C-Vrp1p465)533K485AR486A This study

pAM1001 pGEX-KG expressing C-Vrp1p465)533 This study

pAM1002 pGEX-KG expressing C-Vrp1p465)533K485AR486A This study

pAM1003 YCplac111 expressing C-Vrp1p364–760–GFP–CAAX under VRP1 promoter This study

plasmid [67] under the control of the VRP1 promoter

(nucleotides )240 to the VRP1 ATG start codon).

Sequences encoding various fragments of C-Vrp1p364)817were 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-Vrp1p364)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 (D600 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 ⁄Pi and the GST fusion protein was purified using

glutathione–agarose beads. The beads were washed exten-

sively with NaCl ⁄Pi. S. cerevisiae cells were lysed in G-actin

buffer (5 mm Tris ⁄HCl, 0.2 mm CaCl2, 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 D600 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 ⁄Pi, 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

Council (Australia) Project Grant 298921 (ALM), and

the Queensland State Government (ALM) is gratefully

acknowledged.

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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-Vrp1p364-817to cortical patches in wild-type cells.

This material is available as part of the online article

from http://www.blackwell-synergy.com

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.

Verprolin function in endocytosis and actin organization

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