Vrp1p localizes tothe cortical actin cytoskeleton, is necessary for its polarization to sites ofgrowth and is also essential for endocytosis.. A C-terminal Vrp1p fragmentC-Vrp1p retains
Trang 1Roles of the Las17p (yeast WASP)-binding domain and a novel
C-terminal actin-binding domain
Thirumaran Thanabalu1,2, Rajamuthiah Rajmohan2, Lei Meng2, Gang Ren4,5, Parimala R Vajjhala4and 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 skeleton components and regulators first discovered in
cyto-S cerevisiae have often subsequently been found tohave 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,
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–Aldrichsyndrome protein (WASP)-interacting protein (WIP) Vrp1p localizes tothe cortical actin cytoskeleton, is necessary for its polarization to sites ofgrowth 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 cytoskeletonand function in actin-cytoskeleton polarization, endocytosis and growth.Here, we demonstrate that two submodules in C-Vrp1p are required foractin-cytoskeleton polarization: a novel C-terminal actin-binding submod-ule (CABS) that contains a novel G-actin-binding domain, which we call averprolin homology 2 C-terminal (VH2-C) domain; and a second submod-ule comprising the Las17p-binding domain (LBD) that binds Las17p (yeastWASP) The LBD localizes C-Vrp1p to membranes and the cortical actincytoskeleton Intriguingly, the LBD is sufficient to restore endocytosis andgrowth at elevated temperature to Vrp1p-deficient cells The CABS alsorestores these functions, but only if modified by a lipid anchor to providemembrane association Our findings highlight the role of Las17p bindingfor Vrp1p membrane association, suggest general membrane associationmay be more important than specific targeting to the cortical actin cytoske-leton for Vrp1p function in endocytosis and cell growth, and suggest thatVrp1p 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.
Trang 2organism 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 movemove-ments
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 patchcolocaliz-es 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-Vrp1p1)364 binds actin monomers
[19,21,23], whereas C-Vrp1p364)817binds 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 forboth fluid-phase and receptor-mediated endocytosis[20,27,36] Like Vrp1p, localization of Las17p to corti-cal patches is not perturbed by depolymerization ofactin filaments, however, polarization of Las17p pat-ches requires F-actin [27,29] Similarly, Las17p local-ization to cortical patches is not dependent on Vrp1pbut Vrp1p is required for polarization of Las17p pat-ches [29] (our unpublished data) The localization oftype I myosins to cortical patches is also not depend-ent on Vrp1p, however, polarization of type I myosinpatches is dependent on Vrp1p [34] This is consistentwith a role of F-actin and⁄ or actin polymerization inthe generation or maintenance of a polarized distribu-tion of cortical patches
Las17p and type I myosins promote the assembly ofactin monomers into short actin filaments by bindingand stimulating the Arp2⁄ 3 complex [28,29,35,37,38].The Arp2⁄ 3 complex is an actin filament nucleationmachine highly conserved from yeast to mammalsthat requires interaction with nucleation-promotingfactors for activity [39–41] In yeast, the Arp2⁄ 3complex localizes to cortical patches that partiallycolocalize with cortical actin patches like Vrp1p,Las17p, and type I myosins [42] Vrp1p is essential foractivation 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 atelevated temperatures Furthermore, like full-lengthVrp1p, C-Vrp1p364)817 efficiently localizes to corticalactin patches Localization of C-Vrp1p364)817 to thesepatches is critically dependent on Las17p [23] Alsolike full-length Vrp1p, C-Vrp1p364)817 efficientlymediates cortical actin-patch polarization [23] Howdoes C-Vrp1p364)817 mediate cortical actin-patchpolarization? Does C-Vrp1p364)817 interact with actin,
or is its ability to interact with Las17p sufficient forcortical 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 verprolinhomology 2 C-terminal (VH2-C) domain and a sec-ond submodule comprising the previously character-ized LBD are essential for cortical actin-patchpolarization Intriguingly, however, we find that each
of these submodules has the potential to at least tially support endocytosis and growth at elevatedtemperatures We revise the model for Vrp1p func-tion in the actin cytoskeleton based on these newfindings
Trang 3C-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)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-Vrp1p364)817(W782) with alanine
These eight DNA fragments encoding mutant
C-Vrp1p364)817proteins 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 stainedwith fluorophore-conjugated phalloidin to visualizetheir actin cytoskeleton Interestingly, substitution ofresidues K485R486 slightly reduced the activity ofC-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 theother seven substitutions had any apparent effect ongrowth at elevated temperatures or cortical actin-patchpolarization (data not shown) This result highlightsthe importance of residues K485R486, especially forcortical actin-patch polarization
To determine whether K485R486 are required forendocytosis in the context of C-Vrp1p364)817, we meas-ured uptake of the membrane-impermeant fluid-phaseendocytic dye Lucifer yellow (LY) vrp1D cells expres-sing C-Vrp1p364)817 or C-Vrp1p364–817K485AR486A took
up LY at 24C (Fig 2D) and 37 C (data not shown).Hence, these charged residues are not essential forendocytosis As LY uptake is only a qualitative indica-tor of endocytosis and not quantitative, it is possiblethat the charged residues nevertheless increase the effi-ciency of endocytosis
To examine the expression level of each mutantprotein, the genes encoding C-Vrp1p364)817 andC-Vrp1p364)817K485AR486A were both fused inframe to
a sequence encoding green fluorescent protein (GFP)and expressed from the VRP1 promoter carried on a
X CAAX box (lipid anchor) Glutathione S-transferase (GST) actin-binding domain
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)760is 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).
Trang 4centromeric 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)817
function (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)817
and 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) Weexpect that the relative expression level of the GFP-tagged fusion proteins is indicative of that of theequivalent untagged proteins
We examined whether the various ine substitutions affected the ability of full-lengthVrp1p to restore cortical actin-patch polarization,fluid-phase endocytosis, or growth at elevated tempera-tures to vrp1D cells (data not shown) None of themutations had an obvious effect on any of these func-tions, including K485A R486A N-terminal sequences
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 )817charged-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-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 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-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)817charged-cluster residues K485R486 are not essential for endocytosis vrp1D (AMY88) cells carrying YCplac111 vector (vect), pAM236 expressing C-Vrp1p 364 )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 · 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-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).
Trang 5present in full-length Vrp1p, but not C-Vrp1p364)817,
may compensate for loss of K485R486
Localization of C-Vrp1p364)817to 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)817to
cortical patches, but may be due to inefficient
polariza-tion of C-Vrp1p364)817cortical 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 ofC-Vrp1p364)817had no obvious effect on cortical actin-patch polarization (Fig 3C) or on growth at elevatedtemperatures (Fig 3A,B), thus demonstrating thatthis region is not essential for either of theseC-Vrp1p364)817 functions Additional deletion of 28residues from the N-terminus resulted in a protein(C-Vrp1p493)817) unable to restore cortical actin-patchpolarization (Fig 3C) This protein exhibited reducedfunction in growth at elevated temperature, but didretain some residual function (Fig 3A,B)
Immunoblot analysis of total-cell extracts preparedfrom 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 expressioncompared with C-Vrp1p364)817 We cannot formallyexclude the possibility that this slight reduction inexpression level is responsible for the loss of function
in growth at elevated temperatures We consider itunlikely that this slight reduction in expression level isresponsible for the loss of cortical actin-patch polariza-tion because this deletion removes critical residuesK485 and R486 Substitution of K485 and R486 withalanine is alone sufficient to abolish C-Vrp1p364)817function in actin-patch polarization and these muta-tions (unlike deletion of residues 364–492) do notcause 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 residues465–492 rather than an indirect consequence ofreduced C-Vrp1p493)817expression levels We were notable to assay the expression level of the untagged pro-teins, but we expect that the relative expression level ofthe tagged proteins is indicative of that of the equival-ent untagged proteins
To assess the function of these proteins in cytosis we carried out LY uptake assays onvrp1D (AMY88) cells expressing C-Vrp1p465)817,C-Vrp1p493)817, C-Vrp1p533)817, C-Vrp1p614)817 orC-Vrp1p716)817 All five proteins rescued the endocyto-sis defect at both 24C (Fig 3E) and 37 C (data notshown) This suggests that residues 465–492 are notessential for endocytosis This is consistent with ourfinding that K485 and R486 are not essential for endo-cytosis (Fig 2D) Residues 465–492 may neverthelesscontribute to endocytosis and may be necessary formaximal endocytic efficiency
endo-We next examined the subcellular localization andprotein interactions of the various truncated forms
of C-Vrp1p364)817 (Fig S3A,B) Consistent with theresults 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.
Trang 6N-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 465–533including the charged cluster KK485R486DDRinteracts with actin
Inspection of the amino acid sequence in the regionbordered by residues 465 and 492 revealed the exist-
Trang 7ence 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)533associates 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)533fragment associated with actin in crude
yeast-cell lysate, the K485R486 mutant protein andGST alone did not (Fig 4C, upper)
To further test if binding is direct, we incubated thebeads bearing GST only or the wild-type andK485R486 mutant C-Vrp1p465)533–GST fusion pro-teins with purified Saccharomyces cerevisiae actin inG-actin buffer Bound proteins were analysed asabove The wild-type C-Vrp1p465–533 fragment bound
to purified yeast G-actin, however, the K485R486mutant protein as well as GST alone did not (Fig 4D,left) The wild-type Vrp1p fragment also boundpurified G-actin from rabbit skeletal muscle (data notshown) The wild-type GST–Vrp1p465)533fragment didnot cosediment with F-actin from rabbit skeletalmuscle in an F-actin-pelleting assay (data not shown).Thus the biochemical data are consistent with ouryeast two-hybrid data and suggests that the chargedcluster interacts with G-actin, but not F-actin
An alignment of the various known and putativeactin-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 mediateinteraction 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 tosis or growth at elevated temperatures (A) C-Vrp1p 364 )817residues 465–492 containing the K485R486 charged cluster contribute to, but
endocy-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-Vrp1p 533 )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)817residues 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-Vrp1p364)817), pAM880 expressing C-Vrp1p465)817(C-Vrp1p465)817), pAM881 expressing C-Vrp1p493)817(C-Vrp1p493)817), pAM882 sing C-Vrp1p533)817 (C-Vrp1p533)817), pAM883 expressing C-Vrp1p614)817 (C-Vrp1p614)817), or pAM884 expressing C-Vrp1p716)817(C-Vrp1p 716 )817) A YPUAD culture of each strain was grown at 24C, diluted to D 600 ¼ 0.05 in fresh YPUAD medium and shifted to 37 C.
expres-D 600 was monitored at 1 h intervals (C) C-Vrp1p364)817residues 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-Vrp1p 533 )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-Vrp1p 364 )817residues
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-Vrp1p 493 )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 · 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 )817fragments 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-Vrp1p 493 )817–GFP), pAM887 expressing C-Vrp1p533 )817–GFP (C-Vrp1p533 )817–GFP), pAM888 expressing C-Vrp1p614 )817–GFP
(C-Vrp1p 614 )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).
Trang 8names 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-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 thefunction of C-Vrp1p364)817 in growth we expressedC-Vrp1p364)760 in vrp1D (AMY88) cells and examinedgrowth at elevated temperature (Fig 5A,B) Loss ofthe LBD abolished the ability of C-Vrp1p364)817 torestore growth of vrp1D cells at elevated temperature
We next assessed the importance of the LBD for theability of C-Vrp1p364)817to restore cortical actin-patchpolarization and endocytosis to vrp1D cells vrp1D(AMY88) cells expressing either full-lengthC-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 abolishedthe ability of C-Vrp1p364)817 to mediate corticalactin-patch polarization The truncated form ofC-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)533and 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-Vrp1p 465 )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 m M 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.
Trang 9C-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)760is 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 forlocalization of C-Vrp1p to cortical patches
We next examined whether the LBD is necessaryand⁄ or sufficient for localization of C-Vrp1p364)817 tocortical patches or interaction with Las17p (Fig 6A–C).The subcellular distribution of C-Vrp1p364)760–GFPwas 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
rp 1p
46
53 3
G ST -C -V
GST fusion
GST
*
*
Trang 10(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 isnot an indirect consequence of lowered expression of thetruncated C-Vrp1p364)760–GFP fusion protein relative
to C-Vrp1p364)817–GFP We expect that the relativeexpression level of the GFP-tagged proteins is indicative
of that of the equivalent untagged proteins
37°C 24°C
C-Vrp1p 364-760 C-Vrp1p 364-817
vect
24°C
C-Vrp1p 364-760 C-Vrp1p 364-817
D
Trang 11Madania 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)817with 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)760was 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 tocortical patches but rather displayed a diffuse cytoplas-mic distribution (Fig 6C, right) This is consistent withwhat we reported previously for C-Vrp1p364)817 whenexpressed in las17D cells [23] The expression level ofC-Vrp1p760)817–GFP was examined by SDS⁄ PAGEand immunoblot (Fig 6D) and found to be easilydetectible but reduced compared with that ofC-Vrp1p716)817–GFP We expect that the relativeexpression level of the GFP-tagged proteins is indicative
of that of the equivalent untagged proteins
Lipid anchoring of C-Vrp1p bypasses therequirement for the LBD for endocytosis andgrowth at elevated temperatures, but not forcortical actin-patch polarization
Addition of a CAAX box to the C-terminus of teins confers covalent lipid attachment and efficientmembrane anchoring to proteins that do not normallyassociate with membranes [44] We have previouslyshown that the Ras1p CAAX box confers efficientmembrane anchoring on the otherwise cytoplasmicN-Vrp1p1)364 fragment [23] Addition of the CAAXbox also enhances the function of N-Vrp1p1)364 ingrowth at elevated temperature such that it rescues thetemperature-sensitive growth defect of vrp1D with anefficiency approaching that of C-Vrp1p364)817 or full-length Vrp1p [23] We therefore asked whether usingthe same technique to anchor C-Vrp1p364)760 (CABS)
pro-to membranes would respro-tore function in the absence ofthe LBD C-Vrp1p364)760was tagged at the C-terminuswith 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 due LBD of C-Vrp1p 364 )817is essential for growth on solid medium at elevated temperature Growth at 24 and 37C 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-Vrp1p 364 )817is essential for growth in liquid medium at elevated temperature Growth rate of
57-resi-vrp1D (AMY88) cells carrying YCplac111 vector (vect), pAM236 expressing C-Vrp1p 364 )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 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-Vrp1p364)817is 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-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-Vrp1p 364 )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 · 10 7
cells were incubated with LY dye for 1 h at 24 C The cells were washed and fluorescence was visualized using 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-Vrp1p 364 )817 fused at its C-terminus to GFP
fluores-(C-Vrp1p364)817–GFP) or pAM891 expressing C-Vrp1p364)760–GFP (C-Vrp1p364)760–GFP) resolved by SDS ⁄ PAGE, transferred to a PVDF brane, and immunoblotted with a polyclonal anti-GFP serum (a-GFP) and with a-hexokinase serum as a loading control (a-Hex).