Verprolin function in endocytosis and actin organization Roles of the Las17p (yeast WASP)-binding domain and a novel C-terminal actin-binding domain Thirumaran Thanabalu 1,2 , Rajamuthiah Rajmohan 2 , Lei Meng 2 , Gang Ren 4,5 , Parimala R. Vajjhala 4 and Alan L. Munn 1,3,4,6 * 1 Institute of Molecular and Cell Biology, A*STAR Biomedical Science Institutes, Singapore 2 School of Biological Sciences, Nanyang Technological University, Singapore 3 Department of Biochemistry, Yong Loo Lin School of Medicine, The National University of Singapore, Singapore 4 Institute for Molecular Bioscience and ARC Special Research Centre for Functional and Applied Genomics, The University of Queensland, St Lucia, Australia 5 UMR7156, CNRS, Universite Louis Pasteur, Strasbourg, France 6 School of Biomedical Sciences, The University of Queensland, St Lucia, Australia The actin cytoskeleton is a complex and highly dynamic intracellular protein network with essential roles in cell polarity and morphogenesis. Much of our under- standing of the actin cytoskeleton has come from genetic studies using the unicellular eukaryote Saccharomyces cerevisiae (budding yeast). Actin cyto- skeleton components and regulators first discovered in S. cerevisiae have often subsequently been found to have mammalian counterparts with analogous func- tions. Therefore, S. cerevisiae represents a useful model Keywords actin patch; Arp2 ⁄ 3; Bee1p; cell polarity; WH2 domain Correspondence A. Munn, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Queensland, 4072, Australia Fax: +61 7 3346 2101 Tel: +61 7 3346 2017 E-mail: 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) leads to a partial loss of cortical actin-patch polarization and severe defects in internalization of both receptor- bound and fluid-phase endocytic cargo [16,17,19,20]. Vrp1p is nonessential for growth at normal growth temperatures but becomes essential at elevated temper- atures [16,17,20,22–24]. The relationships among actin- patch polarization, endocytosis, and growth are still not well understood. Structure–function studies aimed at elucidating the molecular basis of Vrp1p function have revealed that Vrp1p comprises two functional modules: an N-ter- minal module (residues 1–364, N-Vrp1p) and a C-ter- minal module (residues 364–817, C-Vrp1p) [23]. Each Vrp1p module interacts with a distinct set of partner proteins: N-Vrp1p 1)364 binds actin monomers [19,21,23], whereas C-Vrp1p 364)817 binds WASP-family proteins (the sole yeast member is Las17p ⁄ Bee1p) [20,25–29]. Interactions with actin monomers and WASP-family proteins are key features shared with human WIP [30–33]. Both N- and C-terminal Vrp1p modules also bind type I myosins [22,28,34,35]. Eluci- dating the physiological role of these interactions is essential to understand the molecular basis of Vrp1p function. Like Vrp1p, Las17p and type I myosins localize to cortical patches with a polarized distribution and parti- ally colocalize with cortical actin patches [11,25,27,34]. Las17p and type I myosins are also essential for both fluid-phase and receptor-mediated endocytosis [20,27,36]. Like Vrp1p, localization of Las17p to corti- cal patches is not perturbed by depolymerization of actin filaments, however, polarization of Las17p pat- ches requires F-actin [27,29]. Similarly, Las17p local- ization to cortical patches is not dependent on Vrp1p but Vrp1p is required for polarization of Las17p pat- ches [29] (our unpublished data). The localization of type I myosins to cortical patches is also not depend- ent on Vrp1p, however, polarization of type I myosin patches is dependent on Vrp1p [34]. This is consistent with a role of F-actin and ⁄ or actin polymerization in the generation or maintenance of a polarized distribu- tion of cortical patches. Las17p and type I myosins promote the assembly of actin monomers into short actin filaments by binding and stimulating the Arp2 ⁄ 3 complex [28,29,35,37,38]. The Arp2 ⁄ 3 complex is an actin filament nucleation machine highly conserved from yeast to mammals that requires interaction with nucleation-promoting factors for activity [39–41]. In yeast, the Arp2 ⁄ 3 complex localizes to cortical patches that partially colocalize with cortical actin patches like Vrp1p, Las17p, and type I myosins [42]. Vrp1p is essential for activation of the Arp2 ⁄ 3 complex by type I myosins in vitro [15]. In a previous study we showed that C-Vrp1p 364)817 functionally replaces full-length Vrp1p for growth at elevated temperatures. Furthermore, like full-length Vrp1p, C-Vrp1p 364)817 efficiently localizes to cortical actin patches. Localization of C-Vrp1p 364)817 to these patches is critically dependent on Las17p [23]. Also like full-length Vrp1p, C-Vrp1p 364)817 efficiently mediates cortical actin-patch polarization [23]. How does C-Vrp1p 364)817 mediate cortical actin-patch polarization? Does C-Vrp1p 364)817 interact with actin, or is its ability to interact with Las17p sufficient for cortical actin-patch polarization? What is the rela- tionship among cortical actin-patch polarization, endocytosis, and growth at elevated temperatures? Here we address these questions and show that both a novel C-terminal actin-binding submodule (CABS) containing a novel actin monomer binding verprolin homology 2 C-terminal (VH2-C) domain and a sec- ond submodule comprising the previously character- ized LBD are essential for cortical actin-patch polarization. Intriguingly, however, we find that each of these submodules has the potential to at least par- tially support endocytosis and growth at elevated temperatures. We revise the model for Vrp1p func- tion in the actin cytoskeleton based on these new findings. Function of Vrp1p C-terminal module T. Thanabalu et al. 4104 FEBS Journal 274 (2007) 4103–4125 ª 2007 The Authors Journal compilation ª 2007 FEBS Results C-Vrp1p residues K485 and R486 are essential for cortical actin-patch polarization, but not for localization to patches, endocytosis, or growth at elevated temperature To delineate the domains of C-Vrp1p 364)817 (Fig. 1) responsible for restoration of endocytosis, growth at elevated temperatures, and full cortical actin-patch polarization we performed charged-to-alanine scanning mutagenesis. A hydrophilicity profile of C-Vrp1p 364)817 was generated and seven charged residues or pairs of charged residues predicted to be surface exposed and potentially involved in intra- or intermolecular inter- actions were chosen for substitution with alanine (residues K457, K485R486, D502K503, K512D513, D594K595, E692, and K740). Because of an earlier study that highlighted the role of bulky hydrophobic residues in interaction of mammalian WASP and WIP family proteins [32], we also substituted the single tryp- tophan residue in C-Vrp1p 364)817 (W782) with alanine. These eight DNA fragments encoding mutant C-Vrp1p 364)817 proteins were placed under the control of the native VRP1 promoter on a centromeric plas- mid and introduced into vrp1 D (AMY88) cells. The ability of the mutated C-Vrp1p 364)817 proteins to restore defects caused by loss of Vrp1p was examined (Fig. 2A–E and data not shown). Cells were stained with fluorophore-conjugated phalloidin to visualize their actin cytoskeleton. Interestingly, substitution of residues K485R486 slightly reduced the activity of C-Vrp1p 364)817 in growth at elevated temperatures (Fig. 2A,B) and abolished its activity in cortical actin- patch polarization (Fig. 2C, Table 1). None of the other seven substitutions had any apparent effect on growth at elevated temperatures or cortical actin-patch polarization (data not shown). This result highlights the importance of residues K485R486, especially for cortical actin-patch polarization. To determine whether K485R486 are required for endocytosis in the context of C-Vrp1p 364)817 , we meas- ured uptake of the membrane-impermeant fluid-phase endocytic dye Lucifer yellow (LY). vrp1D cells expres- sing C-Vrp1p 364)817 or C-Vrp1p 364–817K485AR486A took up LY at 24 °C (Fig. 2D) and 37 °C (data not shown). Hence, these charged residues are not essential for endocytosis. As LY uptake is only a qualitative indica- tor of endocytosis and not quantitative, it is possible that the charged residues nevertheless increase the effi- ciency of endocytosis. To examine the expression level of each mutant protein, the genes encoding C-Vrp1p 364)817 and C-Vrp1p 364)817K485AR486A were both fused inframe to a sequence encoding green fluorescent protein (GFP) and expressed from the VRP1 promoter carried on a 1817 Vrp1p 1 364 N-Vrp1p 1-364 HOT domain Las17p-binding domain X CAAX box (lipid anchor) Glutathione S-transferase (GST) actin-binding domain 1817 465 C-Vrp1p 465-817 1817716 C-Vrp1p 716-817 1817 493C-Vrp1p 493-817 817 533 1817 533C-Vrp1p 533-817 1817 C-Vrp1p 614-817 614 364 760 C-Vrp1p 364-760 1 817760 C-Vrp1p 760-817 364 760 C-Vrp1p 364-760 -CAAX X 1 760 C-Vrp1p 760-817 -GST 1817364 364 760 C-Vrp1p 364-760 -GST C-Vrp1p 364-817 K485A, R486A 1817364 C-Vrp1p 364-817 -GST 1817364 C-Vrp1p 364-817 ? ? (CABS) Fig. 1. Vrp1p domain structure. Schematic of Vrp1p showing the Vrp1p truncations and mutant proteins used in this study and their various known domains: actin-binding domains, Hof one trap (HOT) domain, and LBD. The fragment C-Vrp1p 364)760 is also known as CABS. The actin-binding domain closest to the N-terminus is also known as the WH2 domain (WH2-1 or D1). The predic- ted WH2 domain (WH2-2 or D2) identified by Paunola et al. [43] by homology is not shown here because this putative domain has not yet been shown to bind actin. The actin-binding site within residues 270–364 [23] has not yet been precisely mapped and arrows labeled with question marks denote its position. NB, actin-binding may or may not be mediated by the sequence VH2-N (Fig. 4A). T. Thanabalu et al. Function of Vrp1p C-terminal module FEBS Journal 274 (2007) 4103–4125 ª 2007 The Authors Journal compilation ª 2007 FEBS 4105 centromeric plasmid. As a control, we also made an equivalent construct expressing GFP only. Both GFP- tagged C-Vrp1p 364)817 proteins, but not GFP only, were functional in restoring growth at elevated temper- atures when introduced into vrp1D cells, indicating that addition of the GFP did not perturb C-Vrp1p 364)817 function (Fig. S1). Total-cell extracts were prepared from vrp1D cells expressing C-Vrp1p 364)817 –GFP or C-Vrp1p 364)817K485AR486A –GFP, the proteins were resolved by SDS ⁄ PAGE, and immunoblotted with a polyclonal anti–GFP serum (Fig. 2E). This ana- lysis revealed that both C-Vrp1p 364)817 –GFP and C-Vrp1p 364)817K485AR486A –GFP are expressed at equiv- alent levels. We were unable to raise a Vrp1p-specific polyclonal antiserum and therefore could not assess the expression level of the untagged C-Vrp1p 364)817 and C-Vrp1p 364)817K485AR486A proteins. However, we have tested all C-Vrp1p–GFP fusion proteins used in this study for rescue of vrp1D temperature-sensitive growth and in no case did fusion to GFP appear to affect in vivo function (Fig. S1, data not shown). We expect that the relative expression level of the GFP- tagged fusion proteins is indicative of that of the equivalent untagged proteins. We examined whether the various charged-to-alan- ine substitutions affected the ability of full-length Vrp1p to restore cortical actin-patch polarization, fluid-phase endocytosis, or growth at elevated tempera- tures to vrp1D cells (data not shown). None of the mutations had an obvious effect on any of these func- tions, including K485A R486A. N-terminal sequences A B C D E Fig. 2. C-Vrp1p charged-cluster residues K485R486 are essential for cortical actin-patch polarization, but not for endocytosis or growth at elevated temperatures. (A) The C-Vrp1p 364)817 charged- cluster residues K485R486 are not essential for growth on solid medium at elevated temperatures. Growth at 24 and 37 °Cof vrp1D (AMY88) cells carrying YCplac111 vector (vect), pAM236 expressing C-Vrp1p 364)817 (C-Vrp1p 364)817 ) and pAM873 expressing C-Vrp1p 364)817K485AR486A (C-Vrp1p 364)817 AA). Each strain was streaked for single colonies on YPUAD solid medium, incubated at either 24 or 37 °C, and photographed after 3 days. (B) The C-Vrp1p 364)817 charged-cluster residues K485R486 are not essential for growth in liquid medium at elevated temperatures. Growth rate of vrp1D (AMY88) cells carrying YCplac111 vector (vect), pAM236 expressing C-Vrp1p 364)817 (C-Vrp1p 364)817 ), and pAM873 expres- sing C-Vrp1p 364)817K485AR486A (C-Vrp1p 364)817 AA). A YPUAD culture of each strain was grown at 24 °C, diluted to D 600 ¼ 0.05 in fresh YPUAD medium, and incubated at 37 °C. D 600 was monitored at 1 h intervals. (C) The C-Vrp1p 364)817 charged-cluster residues K485R486 are essential for cortical actin-patch polarization. Cortical actin-patch polarization in vrp1D (AMY88) cells carrying YCplac111 vector (vect), pAM236 expressing C-Vrp1p 364)817 (C-Vrp1p 364)817 ), and pAM873 expressing C-Vrp1p 364)817K485AR486A (C-Vrp1p 364)817 AA). Cells were grown in YPUAD to exponential phase at 24 °C and fixed with formaldehyde, permeabilized, and F-actin stained with Alexa-488-conjugated phalloidin. Stained cells were viewed using fluorescence microscopy. Fields containing small-budded cells were specifically chosen to compare the polar- ization of cortical actin patches at this stage of the cell cycle. Bar ¼ 5 lm. (D) C-Vrp1p 364)817 charged-cluster residues K485R486 are not essential for endocytosis. vrp1D (AMY88) cells carrying YCplac111 vector (vect), pAM236 expressing C-Vrp1p 364)817 (C-Vrp1p 364)817 ) or pAM873 expressing C-Vrp1p 364)817K485AR486A (C-Vrp1p 364)817 AA) were grown in YPUAD to exponential phase at 24 °C and 1 · 10 7 cells were incubated with LY dye for 1 h at 24 °C. Cells were washed and fluorescence was visualized using fluorescence microscopy. (Upper) Fluorescence optics. (Lower) DIC optics. Bar ¼ 5 lm. (E) C-Vrp1p 364)817 with charged-cluster resi- dues K485R486 substituted with alanine is stably expressed. Total extracts from vrp1D (AMY88) cells carrying pAM241 expres- sing C-Vrp1p 364)817 fused at its C-terminus to green fluorescent protein (GFP) (C-Vrp1p 364)817 –GFP) or pAM913 expressing C-Vrp1p 364)817K485AR486A –GFP (C-Vrp1p 364)817 AA–GFP) resolved by SDS ⁄ PAGE, transferred to a PVDF membrane, and immunoblotted with a polyclonal anti-GFP serum (a-GFP) and with anti-hexokinase serum as a loading control (a-Hex). Function of Vrp1p C-terminal module T. Thanabalu et al. 4106 FEBS Journal 274 (2007) 4103–4125 ª 2007 The Authors Journal compilation ª 2007 FEBS present in full-length Vrp1p, but not C-Vrp1p 364)817 , may compensate for loss of K485R486. Localization of C-Vrp1p 364)817 to cortical patches is dependent on Las17p [23]. The minimal Vrp1p sequences required for interaction with Las17p have been mapped to the C-terminal 36 residues [27]. Consistent with this, substitution of K485R486 with alanine did not abolish two-hybrid interaction of C-Vrp1p 364)817 with N-Las17p 1)241 (Fig. S2A). The substitution of K485R486 with alanine also did not abolish C-Vrp1p 364)817 –GFP localization to cor- tical patches in vrp1D cells (Fig. S2B). However, C-Vrp1p 364)817 –GFP patches were polarized to sites of surface growth, whereas C-Vrp1p 364)817K485AR486A – GFP patches were depolarized (Fig. S2B). We conclude that loss of function of C-Vrp1p 364)817K485AR486A in cortical actin-patch polarization is not due to an effect of these mutations on localization of C-Vrp1p 364)817 to cortical patches, but may be due to inefficient polariza- tion of C-Vrp1p 364)817 cortical patches. C-Vrp1p residues 465–492 are essential for cortical actin-patch polarization, but nonessential for endocytosis and growth at elevated temperatures As an independent approach to identify domains within C-Vrp1p 364)817 important for function we constructed deletions initiating at the N-terminus of C-Vrp1p 364)817 (Fig. 1). Five deletion constructs were introduced into vrp1D (AMY88) cells and its ability to functionally substitute for full-length Vrp1p was assessed (Fig. 3A–C). Cells were stained with fluoro- phore-conjugated phalloidin to visualize their actin cytoskeleton. Deletion of residues 364–464 of C-Vrp1p 364)817 had no obvious effect on cortical actin- patch polarization (Fig. 3C) or on growth at elevated temperatures (Fig. 3A,B), thus demonstrating that this region is not essential for either of these C-Vrp1p 364)817 functions. Additional deletion of 28 residues from the N-terminus resulted in a protein (C-Vrp1p 493)817 ) unable to restore cortical actin-patch polarization (Fig. 3C). This protein exhibited reduced function in growth at elevated temperature, but did retain some residual function (Fig. 3A,B). Immunoblot analysis of total-cell extracts prepared from vrp1D (AMY88) cells expressing the correspond- ing GFP-tagged versions of each protein (Fig. 3D) showed that deletion of residues 364–492 resulted in, at most, a twofold reduction in protein expression compared with C-Vrp1p 364)817 . We cannot formally exclude the possibility that this slight reduction in expression level is responsible for the loss of function in growth at elevated temperatures. We consider it unlikely that this slight reduction in expression level is responsible for the loss of cortical actin-patch polariza- tion because this deletion removes critical residues K485 and R486. Substitution of K485 and R486 with alanine is alone sufficient to abolish C-Vrp1p 364)817 function in actin-patch polarization and these muta- tions (unlike deletion of residues 364–492) do not cause a significant reduction in protein expression level (Fig. 2E). Thus, loss of cortical actin-patch polariza- tion is likely to be a direct effect of the loss of residues 465–492 rather than an indirect consequence of reduced C-Vrp1p 493)817 expression levels. We were not able to assay the expression level of the untagged pro- teins, but we expect that the relative expression level of the tagged proteins is indicative of that of the equival- ent untagged proteins. To assess the function of these proteins in endo- cytosis we carried out LY uptake assays on vrp1D (AMY88) cells expressing C-Vrp1p 465)817 , C-Vrp1p 493)817 , C-Vrp1p 533)817 , C-Vrp1p 614)817 or C-Vrp1p 716)817 . All five proteins rescued the endocyto- sis defect at both 24 °C (Fig. 3E) and 37 °C (data not shown). This suggests that residues 465–492 are not essential for endocytosis. This is consistent with our finding that K485 and R486 are not essential for endo- cytosis (Fig. 2D). Residues 465–492 may nevertheless contribute to endocytosis and may be necessary for maximal endocytic efficiency. We next examined the subcellular localization and protein interactions of the various truncated forms of C-Vrp1p 364)817 (Fig. S3A,B). Consistent with the results observed for alanine substitution of K485R486, none of the five deletions abolished interaction with Table 1. Actin-patch polarization of vrp1D cells carrying vector, or plasmids expressing Vrp1p, C-Vrp1p or its derivatives. Cells were grown to exponential phase at 24 °C and either shifted to 37 °C for 2 h or left at 24 °C. Cells were then fixed with formaldehyde, perme- abilized with Triton X-100, and the actin patches stained with Alexa- 488–phalloidin. FITC-fluorescence microscopy was used to visualize the actin patches. The percentages of small budded cells with depo- larized actin patches were estimated by scoring a total of 200 cells from each sample. A mother cell with more than 10 actin patches was counted as having a depolarized actin patch phenotype. 24 °C37°C Polarized Depolarized Polarized Depolarized Vector 2 98 0 100 Vrp1p 94 6 91 9 C-Vrp1p 80 20 60 40 C-Vrp1p AA 22 78 18 82 C-Vrp1p 364)760 1 99 0 100 C-Vrp1p 364)760 CAAX 5 95 3 97 T. Thanabalu et al. Function of Vrp1p C-terminal module FEBS Journal 274 (2007) 4103–4125 ª 2007 The Authors Journal compilation ª 2007 FEBS 4107 N-Las17p 1)241 (Fig. S3A). Furthermore, none of the five deletions (including deletion of residues 364–492) abolished localization of C-Vrp1p 364)817 to cortical patches, although all except deletion of residues 364– 464 affected polarization of the cortical patches (Fig. S3B). A C-Vrp1p fragment comprising residues 465–533 including the charged cluster KK485R486DDR interacts with actin Inspection of the amino acid sequence in the region bordered by residues 465 and 492 revealed the exist- C B A E D Function of Vrp1p C-terminal module T. Thanabalu et al. 4108 FEBS Journal 274 (2007) 4103–4125 ª 2007 The Authors Journal compilation ª 2007 FEBS ence of a charged cluster surrounding K485 and R486: KK485R486DDR (see Vrp1p-VH2-C sequence in Fig. 4A). This charged cluster has some features in common with the charged cluster in the N-terminal WH2 domain of Vrp1p, which is known to bind actin (KLK45K46AET) (sequence WH2-1 in Fig. 4A) [19,21,23]. We therefore examined the ability of a wild- type fragment comprising Vrp1p residues 465–533 and the equivalent fragment containing the K485AR486A mutations to interact with actin in the two-hybrid system (Fig. 4B). Vrp1p 465)533 exhibited two-hybrid interaction with actin. In contrast, the mutated frag- ment in which K485R486 were substituted with alanine did not exhibit detectable interaction with actin. To test whether C-Vrp1p 465)533 associates with actin in crude yeast lysates we expressed wild-type and K485R486 mutant C-Vrp1p 465)533 fragments as gluta- thione S-transferase (GST) fusion proteins as well as GST only in Escherichia coli and incubated beads bearing the purified GST only and GST fusion pro- teins with crude yeast-cell lysate in G-actin buffer. The proteins bound to the beads were eluted, resolved by SDS ⁄ PAGE, and analysed by immunoblotting with anti-actin serum. Although the wild-type C-Vrp1p 465)533 fragment associated with actin in crude yeast-cell lysate, the K485R486 mutant protein and GST alone did not (Fig. 4C, upper). To further test if binding is direct, we incubated the beads bearing GST only or the wild-type and K485R486 mutant C-Vrp1p 465)533 –GST fusion pro- teins with purified Saccharomyces cerevisiae actin in G-actin buffer. Bound proteins were analysed as above. The wild-type C-Vrp1p 465–533 fragment bound to purified yeast G-actin, however, the K485R486 mutant protein as well as GST alone did not (Fig. 4D, left). The wild-type Vrp1p fragment also bound purified G-actin from rabbit skeletal muscle (data not shown). The wild-type GST–Vrp1p 465)533 fragment did not cosediment with F-actin from rabbit skeletal muscle in an F-actin-pelleting assay (data not shown). Thus the biochemical data are consistent with our yeast two-hybrid data and suggests that the charged cluster interacts with G-actin, but not F-actin. An alignment of the various known and putative actin-binding sequences in Vrp1p is shown in Fig. 4A. Vrp1p-WH2-1 is the WH2 domain at the N-terminus of Vrp1p that has previously been shown to mediate interaction with G-actin [19]. Vrp1p-WH2-2 is a puta- tive second WH2 domain identified by sequence align- ment with other WH2 domains [43]. Note that the Fig. 3. C-Vrp1p residues 465–492 containing the K485R486 charged cluster are essential for cortical actin-patch polarization, but not endocy- tosis or growth at elevated temperatures. (A) C-Vrp1p 364)817 residues 465–492 containing the K485R486 charged cluster contribute to, but are not essential for, growth on solid medium at elevated temperatures. Growth at 24 and 37 °Cofvrp1D (AMY88) cells carrying YCplac111 vector (vect), pAM880 expressing C-Vrp1p 465)817 (C-Vrp1p 465)817 ), pAM881 expressing C-Vrp1p 493)817 (C-Vrp1p 493)817 ), pAM882 expressing C-Vrp1p 533)817 (C-Vrp1p 533)817 ), pAM883 expressing C-Vrp1p 614)817 (C-Vrp1p 614)817 ), or pAM884 expressing C-Vrp1p 716)817 (C-Vrp1p 716)817 ). Each strain was streaked for single colonies on YPUAD solid medium, incubated at either 24 or 37 °C, and photographed after 3 days. (B) C-Vrp1p 364)817 residues 465–492 containing the K485R486 charged cluster contribute to, but are not essential for, growth in liquid med- ium at elevated temperatures. Growth rate of vrp1D (AMY88) cells carrying YCplac111 vector (vect), pAM236 expressing C-Vrp1p 364)817 (C-Vrp1p 364)817 ), pAM880 expressing C-Vrp1p 465)817 (C-Vrp1p 465)817 ), pAM881 expressing C-Vrp1p 493)817 (C-Vrp1p 493)817 ), pAM882 expres- sing C-Vrp1p 533)817 (C-Vrp1p 533)817 ), pAM883 expressing C-Vrp1p 614)817 (C-Vrp1p 614)817 ), or pAM884 expressing C-Vrp1p 716)817 (C-Vrp1p 716)817 ). A YPUAD culture of each strain was grown at 24 °C, diluted to D 600 ¼ 0.05 in fresh YPUAD medium and shifted to 37 °C. D 600 was monitored at 1 h intervals. (C) C-Vrp1p 364)817 residues 465–492 containing the K485R486 charged cluster are essential for cortical actin-patch polarization. Cortical actin-patch polarization in vrp1D (AMY88) cells carrying pAM236 expressing C-Vrp1p 364)817 (C-Vrp1p 364)817 ), pAM880 expressing C-Vrp1p 465)817 (C-Vrp1p 465)817 ), pAM881 expressing C-Vrp1p 493)817 (C-Vrp1p 493)817 ), pAM882 expressing C-Vrp1p 533)817 (C-Vrp1p 533)817 ), pAM883 expressing C-Vrp1p 614)817 (C-Vrp1p 614)817 ), or pAM884 expressing C-Vrp1p 716)817 (C-Vrp1p 716)817 ). Cells were grown in YPUAD to exponential phase at 24 °C. Cells were fixed with formaldehyde, permeabilized, and F-actin stained with Alexa-488-conjugated phalloidin. Stained cells were viewed using fluorescence microscopy. Fields containing small-budded cells were specif- ically chosen to compare the polarization of cortical actin patches at this stage of the cell cycle. Bar ¼ 5 lm. (D) C-Vrp1p 364)817 residues 465–492 containing the K485R486 charged cluster are not essential for fluid-phase endocytosis. vrp1D (AMY88) cells carrying pAM236 expressing C-Vrp1p 364)817 (C-Vrp1p 364)817 ), pAM880 expressing C-Vrp1p 465)817 (C-Vrp1p 465)817 ), pAM881 expressing C-Vrp1p 493)817 (C-Vrp1p 493)817 ), pAM882 expressing C-Vrp1p 533)817 (C-Vrp1p 533)817 ), pAM883 expressing C-Vrp1p 614)817 (C-Vrp1p 614)817 ), or pAM884 expressing C-Vrp1p 716)817 (C-Vrp1p 716)817 ) were grown in YPUAD to exponential phase at 24 °C and 1 · 10 7 cells were incubated with LY dye for 1 h at 24 °C. The cells were washed and fluorescence was visualized using fluorescence microscopy. (upper) Fluorescence optics. (Lower) DIC optics. Bar ¼ 5 lm. (E) C-Vrp1p 364)817 fragments lacking residues 465–492 containing the K485R486 charged cluster are stably expressed. Total extracts from vrp1D (AMY88) cells carrying pAM241 expressing C-Vrp1p 364)817 fused at its C-terminus to GFP (C-Vrp1p 364)817 –GFP), pAM885 expressing C-Vrp1p 465)817 –GFP (C-Vrp1p 465)817 –GFP), pAM886 expressing C-Vrp1p 493)817 –GFP (C-Vrp1p 493)817 –GFP), pAM887 expressing C-Vrp1p 533)817 –GFP (C-Vrp1p 533)817 –GFP), pAM888 expressing C-Vrp1p 614)817 –GFP (C-Vrp1p 614)817 –GFP), or pAM889 expressing C-Vrp1p 716)817 –GFP (C-Vrp1p 716)817 –GFP), resolved by SDS ⁄ PAGE, transferred to a PVDF membrane, and immunoblotted with a polyclonal anti-GFP serum (a-GFP) and with a-hexokinase as a loading control (a-Hex). T. Thanabalu et al. Function of Vrp1p C-terminal module FEBS Journal 274 (2007) 4103–4125 ª 2007 The Authors Journal compilation ª 2007 FEBS 4109 names D1 and D2 are used by Paunola et al. [43] to refer to WH2-1 and WH2-2, respectively. Vrp1p-WH2- 2 has not yet been shown to bind actin experimentally and a fragment comprising Vrp1p residues 70–270 that includes Vrp1p-WH2-2 does not exhibit two-hybrid interaction with actin [23]. Vrp1p-VH2-C is the actin- binding domain identified here containing K485R486. We have aligned Vrp1p-VH2-C with a sequence within the fragment comprising residues 270–364 of Vrp1p (Vrp1p-verprolin homology 2 N-terminal or VH2-N) which we previously showed does contain an actin- binding domain (this actin-binding domain has not yet been mapped) [23]. We name the actin-binding domain that we have identified VH2-C and VH2-N because it is not yet clear how closely these domains resemble the WH2 (also known as VH) domain. Function of C-Vrp1p in cortical actin-patch polarization, endocytosis, and growth at elevated temperatures requires the LBD Residues 760–817 ⁄ end comprise the LBD of Vrp1p [20,27–29]. We therefore tested if the LBD is important for various Vrp1p-dependent functions (Fig. 5A–C). To examine whether deletion of the LBD abolishes the function of C-Vrp1p 364)817 in growth we expressed C-Vrp1p 364)760 in vrp1D (AMY88) cells and examined growth at elevated temperature (Fig. 5A,B). Loss of the LBD abolished the ability of C-Vrp1p 364)817 to restore growth of vrp1D cells at elevated temperature. We next assessed the importance of the LBD for the ability of C-Vrp1p 364)817 to restore cortical actin-patch polarization and endocytosis to vrp1D cells. vrp1D (AMY88) cells expressing either full-length C-Vrp1p 364)817 or the truncated form lacking a LBD (C-Vrp1p 364)760 ) were stained with fluorophore-conju- gated phalloidin to visualize their actin cytoskeleton (Fig. 5C, Table 1). Deletion of the LBD abolished the ability of C-Vrp1p 364)817 to mediate cortical actin-patch polarization. The truncated form of C-Vrp1p 364)817 lacking the LBD also did not comple- ment the LY uptake defect of vrp1D cells (Fig. 5D). Hence, the LBD is essential for both cortical actin- patch polarization and endocytosis. To test if the LBD is essential for C-Vrp1p 364)817 expression or stability the C-terminus of a trun- cated form of C-Vrp1p 364)817 lacking the LBD (C-Vrp1p 364)760 ) was tagged with GFP to create Fig. 4. C-Vrp1p residues 465–533 containing the K485R486 charged cluster directly binds G-actin and residues K485R486 are critical. (A) Amino acid sequence alignment of actin-binding sequences in Vrp1p. Vrp1p-WH2-1 (D1 in Paunola et al. [43]) is the original WH2 domain shown to bind actin monomers by [19]. Vrp1p-WH2-2 (D2 in Paunola et al. [43]) is a sequence identified by Paunola et al. [43] as homolog- ous to a WH2 domain (but whether it binds actin is not yet known). Vrp1p-VH2-C is the actin-binding domain within the longer CABS frag- ment identified in this study that contains the K485R486 charged cluster. Vrp1p-VH2-N is a sequence within residues 270–364 of Vrp1p, which we have previously shown contains an actin-binding domain. Note that the domain within residues 270–364 that binds actin has not been mapped and may be distinct from the sequence VH2-N [23]. We use the nomenclature VH2 rather than WH2 because the sequence of VH2-C and VH2-N is different from a WH2 domain and it is not yet clear they adopt a structure similar to a WH2 domain. (B) K485R486 are essential for yeast two–hybrid interaction between C-Vrp1p 465)533 and actin. pAM252 expressing Gal4-BD-Act1p (BD-Act1p ) and pAS2-1 BD vector only expressing Gal4-BD (BD-vect) were tested for two–hybrid interaction with pAM253 expressing Gal4-AD-N-Vrp1p 1)70 (AD-N-Vrp1p 1)70 ), pAM918 expressing Gal4-AD-C-Vrp1p 465)533 (AD-C-Vrp1p 465)533 ), pAM919 expressing Gal4-AD-C-Vrp1p 465)533K485AR486A (AD-C-Vrp1p 465)533 AA), pAM908 expressing Gal4-AD-C-Vrp1p 716)817 (AD-C-Vrp1p 716)817 ), or pACT2 AD vector only expressing Gal4-AD (AD-vect). Plasmids were introduced into the tester strain PJ69-4A and interaction was assessed by growth on medium lacking histidine and containing 2 m M 3-amino 1,2,4-triazole. Plates were photographed after 4 days. (C) The C-Vrp1p 364)817 charged cluster associates with G-actin present in crude yeast lysates in vitro and residues K485R486 are essential. pGEX-KG expressing GST only (GST), pAM1001 expres- sing GST–C-Vrp1p 465)533 (GST–C-Vrp1p 465)533 ), or pAM1002 expressing GST-C-Vrp1p 465)533K485AR486A (GST–C-Vrp1p 465)533 AA) were intro- duced into E. coli, the encoded proteins were expressed and affinity purified, and beads bearing the purified proteins were incubated with crude yeast cell lysate. The beads were washed extensively and the bound proteins were eluted. The eluted proteins were resolved by SDS ⁄ PAGE and the proteins were transferred to a PVDF membrane and immunoblotted with an anti-actin mAb. Equivalent amounts of crude yeast cell lysate were used in each binding assay. The lower panel shows GST only and the GST fusion proteins used to coat the beads used for binding assays subjected to SDS ⁄ PAGE and stained with Coomassie Brilliant Blue. The full-length GST and GST fusion pro- teins are indicated (arrows). * indicates a protein that copurified with the fusion proteins and is likely to be a degradation product. (D) The C-Vrp1p 364)817 charged cluster directly binds yeast G-actin in vitro and residues K485R486 are essential. Beads bearing GST (GST), GST–C-Vrp1p 465)533 (GST–C-Vrp1p 465)533 ), and GST–C-Vrp1p 465)533K485AR486A (GST-C-Vrp1p 465)533 AA), prepared as in (C), were incubated with purified yeast actin in G buffer. The beads were washed extensively and the bound proteins were eluted. The eluted proteins were resolved by SDS ⁄ PAGE and the proteins were transferred to a PVDF membrane and immunoblotted with an anti-actin mAb (left). Equivalent amounts of purified yeast actin were used in each binding assay and an amount representing 10% of the load used in each binding assay is shown (right). The lower panel shows GST only and the GST fusion proteins used to coat the beads used for binding assays subjected to SDS ⁄ PAGE and stained with Coomassie Brilliant Blue. The full-length GST and GST fusion proteins are indicated (arrows). * indicates a protein that copurified with the fusion proteins and is likely to be a degradation product. Function of Vrp1p C-terminal module T. Thanabalu et al. 4110 FEBS Journal 274 (2007) 4103–4125 ª 2007 The Authors Journal compilation ª 2007 FEBS C-Vrp1p 364)760 –GFP. This protein was expressed in vrp1D (AMY88) cells and its steady-state expression level examined by SDS ⁄ PAGE and immunoblot (Fig. 5E). The results show that (at least as a GFP fusion protein) C-Vrp1p 364)760 is expressed at equival- ent levels to C-Vrp1p 364)817 . We were unable to assay the relative expression level of the untagged proteins, but we expect they would also be similar. The LBD is necessary and sufficient for localization of C-Vrp1p to cortical patches We next examined whether the LBD is necessary and ⁄ or sufficient for localization of C-Vrp1p 364)817 to cortical patches or interaction with Las17p (Fig. 6A–C). The subcellular distribution of C-Vrp1p 364)760 –GFP was analysed using live cell fluorescence imaging A AD-N-Vrp1p 1-70 AD-C-Vrp1p 465-533 AD-C-Vrp1p 465-533 AA AD-C-Vrp1p 716-817 AD-vect B D- Ac t 1 p B D- v e c t B B D - Ac t 1 p B D- v e c t +His -His C -actin G S T - C - V r p 1 p 4 6 5 - 5 3 3 G S T - C - V r p 1 p 4 6 5 - 5 3 3 A A G S T D G S T - C - V r p 1 p 4 6 5 - 5 3 3 A A G S T - C - V r p 1 p 4 6 5 - 5 3 3 G S T -Actin G S T - C - V r p 1 p 4 6 5 - 5 3 3 A A G S T - C - V r p 1 p 4 6 5 - 5 3 3 G S T Bound Load G S T - C - V r p 1 p 4 6 5 - 5 3 3 A A G S T - C - V r p 1 p 4 6 5 - 5 3 3 G S T Purified GST fusion GST GST fusion GST * * T. Thanabalu et al. Function of Vrp1p C-terminal module FEBS Journal 274 (2007) 4103–4125 ª 2007 The Authors Journal compilation ª 2007 FEBS 4111 (Fig. 6A). C-Vrp1p 364)760 –GFP displayed a diffuse cytoplasmic localization similar to GFP alone. In con- trast, C-Vrp1p 364)817 –GFP localized to cortical patches (Fig. 6A) consistent with our previous report [23]. The expression level of truncated C-Vrp1p 364)760 –GFP was equivalent to that of C-Vrp1p 364)817 –GFP (Fig. 5E). This suggests that loss of cortical-patch localization is not an indirect consequence of lowered expression of the truncated C-Vrp1p 364)760 –GFP fusion protein relative to C-Vrp1p 364)817 –GFP. We expect that the relative expression level of the GFP-tagged proteins is indicative of that of the equivalent untagged proteins. A B C C-Vrp1p 364-817 vect C-Vrp1p 364-760 Time (h) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1234567 8 D 600 vect C-Vrp1p 364-817 C-Vrp1p 364-760 37°C24°C C-Vrp1p 364-760 C-Vrp1p 364-817 vect 24°C C-Vrp1p 364-760 C-Vrp1p 364-817 vect C-Vrp1p 364-760 -CAAX E C-Vrp1p 364-760 -GFP C-Vrp1p 364-817 -GFP -GFP -Hex D Function of Vrp1p C-terminal module T. Thanabalu et al. 4112 FEBS Journal 274 (2007) 4103–4125 ª 2007 The Authors Journal compilation ª 2007 FEBS [...]... 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 lament assembly [11,15,35,37,48] The Vrp1p VH2-C domain, by supplying actin monomers for polymerization 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... implicated in polarization of the yeast actin cytoskeleton 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... 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 polarized distribution of surface-membrane proteins in yeast can be achieved by efficient endocytosis [50] Given that the LY uptake assays used here are... 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... [30,52] Interactions of Vrp1p and Las17p in yeast and WIP and WASP-family 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 supported by the observation... GSTC-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. .. the Arp2 ⁄ 3 complex [15,19,21,23,48,59] A complex of Vrp1p with type I myosin has both actin monomer- and Arp2 ⁄ 3-binding functions and can activate the Arp2 ⁄ 3 complex in a manner analogous to WASP-family proteins such as Las17p [15,29] In this context, Vrp1p acts as a multivalent adaptor to recruit other proteins such as Las17p, type I myosins, and actin monomers to sites of actin- filament assembly... myosindependent 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. .. MO, USA) The plasmids used in this study are listed in Table 2 The rabbit polyclonal GFP-specific 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 (Temecula, CA) The anti-hexokinase rabbit polyclonal serum was 100–4159 from Rockland Inc (Gilbertsville, PA) Alexa-488-conjugated phalloidin was from Invitrogen... 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 function 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, . Verprolin function in endocytosis and actin organization Roles of the Las17p (yeast WASP)-binding domain and a novel C-terminal actin- binding domain Thirumaran. known domains: actin- binding domains, Hof one trap (HOT) domain, and LBD. The fragment C-Vrp1p 364)760 is also known as CABS. The actin- binding domain closest