Verprolin function in endocytosis and actin organization

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1 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 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: Tel: 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: /j 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 () 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 are required for actin-cytoskeleton polarization: a novel C-terminal actin-binding submodule (CABS) that contains a novel G-actin-binding domain, which we call a verprolin homology 2 C-terminal (VH2-C) domain; and a second submodule comprising the Las17p-binding domain (LBD) that binds Las17p (yeast WASP). The LBD localizes 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 cytoskeleton for Vrp1p function in endocytosis and cell growth, and suggest that Vrp1p binding to individual effectors may alter their physiological activity. The actin cytoskeleton is a complex and highly dynamic intracellular protein network with essential roles in cell polarity and morphogenesis. Much of our understanding of the actin cytoskeleton has come from genetic studies using the unicellular eukaryote Saccharomyces cerevisiae (budding yeast). Actin cytoskeleton components and regulators first discovered in S. cerevisiae have often subsequently been found to have mammalian counterparts with analogous functions. Therefore, S. cerevisiae represents a useful model 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) ª 2007 The Authors Journal compilation ª 2007 FEBS 4103

2 Function of Vrp1p C-terminal module T. Thanabalu et al. 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 distribution 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 movement at the cortex [6 9]. Some of these movements correlate with endocytic cargo internalization, consistent with a role for cortical actin patches in endocytosis [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 patches that display a subcellular distribution polarized towards sites of surface growth and partially colocalizes 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 receptorbound and fluid-phase endocytic cargo [16,17,19,20]. Vrp1p is nonessential for growth at normal growth temperatures but becomes essential at elevated temperatures [16,17,20,22 24]. The relationships among actinpatch 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-terminal module (residues 1 364, N-Vrp1p) and a C-terminal module (residues , ) [23]. Each Vrp1p module interacts with a distinct set of partner proteins: N-Vrp1p 1)364 binds actin monomers [19,21,23], whereas 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]. Elucidating 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 partially 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 cortical patches is not perturbed by depolymerization of actin filaments, however, polarization of Las17p patches requires F-actin [27,29]. Similarly, Las17p localization to cortical patches is not dependent on Vrp1p but Vrp1p is required for polarization of Las17p patches [29] (our unpublished data). The localization of type I myosins to cortical patches is also not dependent 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 distribution 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 364)817 functionally replaces full-length Vrp1p for growth at elevated temperatures. Furthermore, like full-length Vrp1p, 364)817 efficiently localizes to cortical actin patches. Localization of 364)817 to these patches is critically dependent on Las17p [23]. Also like full-length Vrp1p, 364)817 efficiently mediates cortical actin-patch polarization [23]. How does 364)817 mediate cortical actin-patch polarization? Does 364)817 interact with actin, or is its ability to interact with Las17p sufficient for cortical actin-patch polarization? What is the relationship 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 second submodule comprising the previously characterized LBD are essential for cortical actin-patch polarization. Intriguingly, however, we find that each of these submodules has the potential to at least partially support endocytosis and growth at elevated temperatures. We revise the model for Vrp1p function in the actin cytoskeleton based on these new findings FEBS Journal 274 (2007) ª 2007 The Authors Journal compilation ª 2007 FEBS

3 T. Thanabalu et al. Function of Vrp1p C-terminal module Results 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 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 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 interactions 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 tryptophan residue in 364)817 (W782) with alanine. These eight DNA fragments encoding mutant 364)817 proteins were placed under the control of the native VRP1 promoter on a centromeric plasmid and introduced into vrp1d (AMY88) cells. The ability of the mutated 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 364)817 in growth at elevated temperatures (Fig. 2A,B) and abolished its activity in cortical actinpatch 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 364)817, we measured uptake of the membrane-impermeant fluid-phase endocytic dye Lucifer yellow (LY). vrp1d cells expressing 364)817 or K485AR486A 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 indicator of endocytosis and not quantitative, it is possible that the charged residues nevertheless increase the efficiency of endocytosis. To examine the expression level of each mutant protein, the genes encoding 364)817 and 364)817K485AR486A were both fused inframe to a sequence encoding green fluorescent protein (GFP) and expressed from the VRP1 promoter carried on a 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 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 predicted 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 [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). Vrp1p N-Vrp1p ? 1 817? GST K485A, 1 R486A CAAX GST (CABS) GST actin-binding domain X HOT domain Las17p-binding domain X CAAX box (lipid anchor) Glutathione S-transferase (GST) 817 FEBS Journal 274 (2007) ª 2007 The Authors Journal compilation ª 2007 FEBS 4105

4 Function of Vrp1p C-terminal module T. Thanabalu et al. A B C D centromeric plasmid. As a control, we also made an equivalent construct expressing GFP only. Both GFPtagged 364)817 proteins, but not GFP only, were functional in restoring growth at elevated temperatures when introduced into vrp1d cells, indicating that addition of the GFP did not perturb 364)817 function (Fig. S1). Total-cell extracts were prepared from vrp1d cells expressing 364)817 GFP or 364)817K485AR486A GFP, the proteins were resolved by SDS PAGE, and immunoblotted with a polyclonal anti GFP serum (Fig. 2E). This analysis revealed that both 364)817 GFP and 364)817K485AR486A GFP are expressed at equivalent levels. We were unable to raise a Vrp1p-specific polyclonal antiserum and therefore could not assess the expression level of the untagged 364)817 and 364)817K485AR486A proteins. However, we have tested all GFP fusion proteins used in this study for rescue of vrp1d temperature-sensitive growth and in no case did fusion to GFP appear to E Fig. 2. charged-cluster residues K485R486 are essential for cortical actin-patch polarization, but not for endocytosis or growth at elevated temperatures. (A) The 364)817 chargedcluster 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 364)817 ( 364)817 ) and pam873 expressing 364)817K485AR486A ( 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 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 364)817 ( 364)817 ), and pam873 expressing 364)817K485AR486A ( 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 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 364)817 ( 364)817 ), and pam873 expressing 364)817K485AR486A ( 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 polarization of cortical actin patches at this stage of the cell cycle. Bar ¼ 5 lm. (D) 364)817 charged-cluster residues K485R486 are not essential for endocytosis. vrp1d (AMY88) cells carrying YCplac111 vector (vect), pam236 expressing 364)817 ( 364)817 ) or pam873 expressing 364)817K485AR486A ( 364)817 AA) were grown in YPUAD to exponential phase at 24 C and 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) 364)817 with charged-cluster residues K485R486 substituted with alanine is stably expressed. Total extracts from vrp1d (AMY88) cells carrying pam241 expressing 364)817 fused at its C-terminus to green fluorescent protein (GFP) ( 364)817 GFP) or pam913 expressing 364)817K485AR486A GFP ( 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). affect in vivo function (Fig. S1, data not shown). We expect that the relative expression level of the GFPtagged fusion proteins is indicative of that of the equivalent untagged proteins. We examined whether the various charged-to-alanine substitutions affected the ability of full-length Vrp1p to restore cortical actin-patch polarization, fluid-phase endocytosis, or growth at elevated temperatures to vrp1d cells (data not shown). None of the mutations had an obvious effect on any of these functions, including K485A R486A. N-terminal sequences 4106 FEBS Journal 274 (2007) ª 2007 The Authors Journal compilation ª 2007 FEBS

5 T. Thanabalu et al. Function of Vrp1p C-terminal module Table 1. Actin-patch polarization of vrp1d cells carrying vector, or plasmids expressing 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, permeabilized 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 depolarized 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 Vrp1p AA ) )760 CAAX present in full-length Vrp1p, but not 364)817, may compensate for loss of K485R486. Localization of 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 364)817 with N-Las17p 1)241 (Fig. S2A). The substitution of K485R486 with alanine also did not abolish 364)817 GFP localization to cortical patches in vrp1d cells (Fig. S2B). However, 364)817 GFP patches were polarized to sites of surface growth, whereas 364)817K485AR486A GFP patches were depolarized (Fig. S2B). We conclude that loss of function of 364)817K485AR486A in cortical actin-patch polarization is not due to an effect of these mutations on localization of 364)817 to cortical patches, but may be due to inefficient polarization of 364)817 cortical patches. residues are essential for cortical actin-patch polarization, but nonessential for endocytosis and growth at elevated temperatures As an independent approach to identify domains within 364)817 important for function we constructed deletions initiating at the N-terminus of 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 fluorophore-conjugated phalloidin to visualize their actin cytoskeleton. Deletion of residues of 364)817 had no obvious effect on cortical actinpatch polarization (Fig. 3C) or on growth at elevated temperatures (Fig. 3A,B), thus demonstrating that this region is not essential for either of these 364)817 functions. Additional deletion of 28 residues from the N-terminus resulted in a protein ( 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 corresponding GFP-tagged versions of each protein (Fig. 3D) showed that deletion of residues resulted in, at most, a twofold reduction in protein expression compared with 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 polarization because this deletion removes critical residues K485 and R486. Substitution of K485 and R486 with alanine is alone sufficient to abolish 364)817 function in actin-patch polarization and these mutations (unlike deletion of residues ) do not cause a significant reduction in protein expression level (Fig. 2E). Thus, loss of cortical actin-patch polarization is likely to be a direct effect of the loss of residues rather than an indirect consequence of reduced 493)817 expression levels. We were not able to assay the expression level of the untagged proteins, but we expect that the relative expression level of the tagged proteins is indicative of that of the equivalent untagged proteins. To assess the function of these proteins in endocytosis we carried out LY uptake assays on vrp1d (AMY88) cells expressing 465)817, 493)817, 533)817, 614)817 or 716)817. All five proteins rescued the endocytosis defect at both 24 C (Fig. 3E) and 37 C (data not shown). This suggests that residues are not essential for endocytosis. This is consistent with our finding that K485 and R486 are not essential for endocytosis (Fig. 2D). Residues 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 364)817 (Fig. S3A,B). Consistent with the results observed for alanine substitution of K485R486, none of the five deletions abolished interaction with FEBS Journal 274 (2007) ª 2007 The Authors Journal compilation ª 2007 FEBS 4107

6 Function of Vrp1p C-terminal module T. Thanabalu et al. N-Las17p 1)241 (Fig. S3A). Furthermore, none of the five deletions (including deletion of residues ) abolished localization of 364)817 to cortical patches, although all except deletion of residues affected polarization of the cortical patches (Fig. S3B). A fragment comprising residues 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- A B C E D 4108 FEBS Journal 274 (2007) ª 2007 The Authors Journal compilation ª 2007 FEBS

7 T. Thanabalu et al. Function of Vrp1p C-terminal module 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 wildtype fragment comprising Vrp1p residues 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 fragment in which K485R486 were substituted with alanine did not exhibit detectable interaction with actin. To test whether 465)533 associates with actin in crude yeast lysates we expressed wild-type and K485R486 mutant 465)533 fragments as glutathione S-transferase (GST) fusion proteins as well as GST only in Escherichia coli and incubated beads bearing the purified GST only and GST fusion proteins 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 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 465)533 GST fusion proteins with purified Saccharomyces cerevisiae actin in G-actin buffer. Bound proteins were analysed as above. The wild-type 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 putative second WH2 domain identified by sequence alignment with other WH2 domains [43]. Note that the Fig. 3. residues containing the K485R486 charged cluster are essential for cortical actin-patch polarization, but not endocytosis or growth at elevated temperatures. (A) 364)817 residues 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 465)817 ( 465)817 ), pam881 expressing 493)817 ( 493)817 ), pam882 expressing 533)817 ( 533)817 ), pam883 expressing 614)817 ( 614)817 ), or pam884 expressing 716)817 ( 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) 364)817 residues containing the K485R486 charged cluster contribute to, but are not essential for, growth in liquid medium at elevated temperatures. Growth rate of vrp1d (AMY88) cells carrying YCplac111 vector (vect), pam236 expressing 364)817 ( 364)817 ), pam880 expressing 465)817 ( 465)817 ), pam881 expressing 493)817 ( 493)817 ), pam882 expressing 533)817 ( 533)817 ), pam883 expressing 614)817 ( 614)817 ), or pam884 expressing 716)817 ( 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) 364)817 residues containing the K485R486 charged cluster are essential for cortical actin-patch polarization. Cortical actin-patch polarization in vrp1d (AMY88) cells carrying pam236 expressing 364)817 ( 364)817 ), pam880 expressing 465)817 ( 465)817 ), pam881 expressing 493)817 ( 493)817 ), pam882 expressing 533)817 ( 533)817 ), pam883 expressing 614)817 ( 614)817 ), or pam884 expressing 716)817 ( 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 specifically chosen to compare the polarization of cortical actin patches at this stage of the cell cycle. Bar ¼ 5 lm. (D) 364)817 residues containing the K485R486 charged cluster are not essential for fluid-phase endocytosis. vrp1d (AMY88) cells carrying pam236 expressing 364)817 ( 364)817 ), pam880 expressing 465)817 ( 465)817 ), pam881 expressing 493)817 ( 493)817 ), pam882 expressing 533)817 ( 533)817 ), pam883 expressing 614)817 ( 614)817 ), or pam884 expressing 716)817 ( 716)817 ) were grown in YPUAD to exponential phase at 24 C and 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) 364)817 fragments lacking residues containing the K485R486 charged cluster are stably expressed. Total extracts from vrp1d (AMY88) cells carrying pam241 expressing 364)817 fused at its C-terminus to GFP ( 364)817 GFP), pam885 expressing 465)817 GFP ( 465)817 GFP), pam886 expressing 493)817 GFP ( 493)817 GFP), pam887 expressing 533)817 GFP ( 533)817 GFP), pam888 expressing 614)817 GFP ( 614)817 GFP), or pam889 expressing 716)817 GFP ( 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). FEBS Journal 274 (2007) ª 2007 The Authors Journal compilation ª 2007 FEBS 4109

8 Function of Vrp1p C-terminal module T. Thanabalu et al. 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 that includes Vrp1p-WH2-2 does not exhibit two-hybrid interaction with actin [23]. Vrp1p-VH2-C is the actinbinding domain identified here containing K485R486. We have aligned Vrp1p-VH2-C with a sequence within the fragment comprising residues of Vrp1p (Vrp1p-verprolin homology 2 N-terminal or VH2-N) which we previously showed does contain an actinbinding 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 in cortical actin-patch polarization, endocytosis, and growth at elevated temperatures requires the LBD Residues 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 364)817 in growth we expressed 364)760 in vrp1d (AMY88) cells and examined growth at elevated temperature (Fig. 5A,B). Loss of the LBD abolished the ability of 364)817 to restore growth of vrp1d cells at elevated temperature. We next assessed the importance of the LBD for the ability of 364)817 to restore cortical actin-patch polarization and endocytosis to vrp1d cells. vrp1d (AMY88) cells expressing either full-length 364)817 or the truncated form lacking a LBD ( 364)760 ) were stained with fluorophore-conjugated phalloidin to visualize their actin cytoskeleton (Fig. 5C, Table 1). Deletion of the LBD abolished the ability of 364)817 to mediate cortical actin-patch polarization. The truncated form of 364)817 lacking the LBD also did not complement the LY uptake defect of vrp1d cells (Fig. 5D). Hence, the LBD is essential for both cortical actinpatch polarization and endocytosis. To test if the LBD is essential for 364)817 expression or stability the C-terminus of a truncated form of 364)817 lacking the LBD ( 364)760 ) was tagged with GFP to create Fig. 4. residues 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 homologous 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 fragment identified in this study that contains the K485R486 charged cluster. Vrp1p-VH2-N is a sequence within residues of Vrp1p, which we have previously shown contains an actin-binding domain. Note that the domain within residues 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 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- 465)533 (AD- 465)533 ), pam919 expressing Gal4-AD- 465)533K485AR486A (AD- 465)533 AA), pam908 expressing Gal4-AD- 716)817 (AD- 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 mm 3-amino 1,2,4-triazole. Plates were photographed after 4 days. (C) The 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 expressing GST 465)533 (GST 465)533 ), or pam1002 expressing GST- 465)533K485AR486A (GST 465)533 AA) were introduced 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 proteins are indicated (arrows). * indicates a protein that copurified with the fusion proteins and is likely to be a degradation product. (D) The 364)817 charged cluster directly binds yeast G-actin in vitro and residues K485R486 are essential. Beads bearing GST (GST), GST 465)533 (GST 465)533 ), and GST 465)533K485AR486A (GST- 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 FEBS Journal 274 (2007) ª 2007 The Authors Journal compilation ª 2007 FEBS

9 T. Thanabalu et al. Function of Vrp1p C-terminal module 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) 364)760 is expressed at equivalent levels to 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 to cortical patches We next examined whether the LBD is necessary and or sufficient for localization of 364)817 to cortical patches or interaction with Las17p (Fig. 6A C). The subcellular distribution of 364)760 GFP was analysed using live cell fluorescence imaging A B BD-Act1p BD-vect BD-Act1p BD-vect AD-N-Vrp1p 1-70 AD C GST GST AA GST -actin AD AA AD GST fusion D -Actin +His GST -His - - AD-vect GST GST AA * GST GST GST AA GST Bound GST Load GST GST AA GST * GST fusion Purified FEBS Journal 274 (2007) ª 2007 The Authors Journal compilation ª 2007 FEBS 4111

10 Function of Vrp1p C-terminal module T. Thanabalu et al. (Fig. 6A). 364)760 GFP displayed a diffuse cytoplasmic localization similar to GFP alone. In contrast, 364)817 GFP localized to cortical patches (Fig. 6A) consistent with our previous report [23]. The expression level of truncated 364)760 GFP was equivalent to that of 364)817 GFP (Fig. 5E). This suggests that loss of cortical-patch localization is not an indirect consequence of lowered expression of the truncated 364)760 GFP fusion protein relative to 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 24 C 37 C vect B D vect Time (h) C vect C D vect CAAX E GFP GFP -GFP -Hex 4112 FEBS Journal 274 (2007) ª 2007 The Authors Journal compilation ª 2007 FEBS

11 T. Thanabalu et al. Function of Vrp1p C-terminal module 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 364)817 with Las17p we performed two-hybrid tests. 364)817 exhibited two-hybrid interaction with an N-terminal fragment of Las17p (N-Las17p 1)241 ), however, in contrast, the truncated form 364)760 was unable to interact with N-Las17p 1)241 (Fig. 6B). Hence, in yeast the Vrp1p C-terminal 56 residues are essential for interaction with Las17p. Is the LBD (residues ) sufficient for localization of 364)817 to cortical patches? To address this question we tagged the isolated LBD of Vrp1p with GFP to create 760)817 GFP. 760)817 GFP was expressed in vrp1d (AMY88) cells and its subcellular distribution examined by live cell fluorescence imaging (Fig. 6C, left). Strikingly, 760)817 GFP has the ability to localize to cortical patches. In contrast, GFP alone exhibited only a diffuse cytoplasmic localization. We also examined whether the LBD can mediate cortical patch localization in the presence of full-length Vrp1p by examining the localization of a 760)817 GFP fusion protein in wild-type (RH1657) cells. The 57-residue LBD was sufficient to mediate cortical patch localization similar to that of 364)817 GFP in cells expressing full-length wild-type Vrp1p (Fig. S4). The cortical patch localization of 760)817 GFP is dependent on Las17p. When expressed in las17d (IDY166) cells, 760)817 GFP did not localize to cortical patches but rather displayed a diffuse cytoplasmic distribution (Fig. 6C, right). This is consistent with what we reported previously for 364)817 when expressed in las17d cells [23]. The expression level of 760)817 GFP was examined by SDS PAGE and immunoblot (Fig. 6D) and found to be easily detectible but reduced compared with that of 716)817 GFP. We expect that the relative expression level of the GFP-tagged proteins is indicative of that of the equivalent untagged proteins. Lipid anchoring of 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 proteins confers covalent lipid attachment and efficient membrane anchoring to proteins that do not normally associate with membranes [44]. We have previously shown that the Ras1p CAAX box confers efficient membrane anchoring on the otherwise cytoplasmic N-Vrp1p 1)364 fragment [23]. Addition of the CAAX box also enhances the function of N-Vrp1p 1)364 in growth at elevated temperature such that it rescues the temperature-sensitive growth defect of vrp1d with an efficiency approaching that of 364)817 or fulllength Vrp1p [23]. We therefore asked whether using the same technique to anchor 364)760 (CABS) to membranes would restore function in the absence of the LBD. 364)760 was tagged at the C-terminus with the CAAX box of S. cerevisiae Ras1p [45] Fig. 5. The LBD of is essential for cortical actin-patch polarization, endocytosis, and growth at elevated temperature. (A) The 57-residue LBD of 364)817 is essential for growth on solid medium at elevated temperature. Growth at 24 and 37 C ofvrp1d (AMY88) cells carrying YCplac111 vector (vect), pam236 expressing 364)817 ( 364)817 ), or pam896 expressing 364)760 ( 364)760 ). Each strain was streaked for single colonies on YPUAD solid medium, incubated at either 24 or 37 C, and photographed after 3 days. (B) The 57-residue LBD of 364)817 is essential for growth in liquid medium at elevated temperature. Growth rate of vrp1d (AMY88) cells carrying YCplac111 vector (vect), pam236 expressing 364)817 ( 364)817 ) and pam896 expressing 364)760 ( 364)760 ). A YPUAD culture of each strain was grown at 24 C, diluted to D 600 ¼ 0.05 in fresh YPUAD medium, and incubated at 37 C. D 600 was monitored at 1 h intervals. (C) The 57-residue LBD of 364)817 is essential for polarization of cortical actin patches. Cortical actin-patch polarization in vrp1d (AMY88) cells carrying YCplac111 vector (vect), pam236 expressing 364)817 ( 364)817 ), pam896 expressing 364)760 ( 364)760 ). Cells were grown in YPUAD to exponential phase at 24 C, fixed with formaldehyde, permeabilized, and F-actin stained with Alexa-488-conjugated phalloidin. Stained cells were viewed by fluorescence microscopy. 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 364)817 LBD is essential for endocytosis. vrp1d (AMY88) cells carrying YCplac111 vector only (vect), pam236 expressing 364)817 ( 364)817 ), pam896 expressing 364)760 ( 364)760 ) or pam899 expressing 364)760 fused at its C-terminus to the CAAX box of Ras1p ( 364)760 -CAAX) were grown in YPUAD to exponential phase at 24 C and 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) 364)817 lacking the 57-residue LBD is stably expressed. Total extracts from vrp1d (AMY88) cells carrying pam241 expressing 364)817 fused at its C-terminus to GFP ( 364)817 GFP) or pam891 expressing 364)760 GFP ( 364)760 GFP) 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). FEBS Journal 274 (2007) ª 2007 The Authors Journal compilation ª 2007 FEBS 4113

12 Function of Vrp1p C-terminal module T. Thanabalu et al. A GFP GFP GFP GFP-CAAX B BD-N-Las17p BD-vect BD-N-Las17p BD-vect AD AD AD AD AD-vect +His -His C GFP GFP GFP GFP D GFP GFP GFP α-gfp α-hex vrp1δ las17δ Fig. 6. The LBD of is necessary and sufficient for localization to cortical patches. (A) The 57-residue LBD of 364)817 is essential for localization to cortical patches. vrp1d (AMY88) cells carrying pam237 expressing GFP, pam241 expressing 364)817 GFP ( 364)817 GFP), pam891 expressing 364)760 GFP ( 364)760 GFP) or pam1003 expressing 364)760 GFP fused at its C-terminus to the CAAX box of Ras1p ( 364)760 GFP-CAAX) were grown in YPUAD to exponential phase at 24 C and GFP fluorescence 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 364)817 interaction with Las17p. pam912 expressing Gal4-BD-N-Las17p 1)241 (BD-N-Las17p1 241) and pas2-1 BD vector only expressing Gal4-BD (BD-vect) were tested for two hybrid interaction with pam902 expressing Gal4- AD- 364)817 (AD- 364)817 ), pam909 expressing Gal4-AD- 364)760 (AD- 364)760 ), pam904 expressing Gal4- AD- 465)817 (AD- 465)817 ), pam910 expressing Gal4-AD- 465)760 (AD- 465)760 ), and 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 57-residue LBD of 364)817 is sufficient for localization to cortical patches. vrp1d (AMY88) or las17d (IDY166) cells carrying pam237 expressing GFP (GFP) or pam890 expressing 760)817 GFP ( 760)817 GFP) were grown in YPUAD to exponential phase at 24 C and GFP fluorescence was visualized in living cells by fluorescence microscopy. (Upper) FITC-fluorescence optics. (Lower) DIC optics. Bar ¼ 5 lm. (D) The 57-residue LBD of 364)817 is stably expressed as a fusion to GFP. Total extracts from vrp1d (AMY88) cells carrying pam889 expressing 716)817 GFP ( 716)817 GFP), pam237 expressing GFP (GFP) or pam890 expressing 760)817 GFP ( 760)817 GFP) were resolved by SDS PAGE, transferred to a PVDF membrane, and immunoblotted with a polyclonal anti-gfp serum (a-gfp) and with a-hexokinase serum as a loading control (a-hex). to yield 364)760 CAAX. This protein was expressed in vrp1d (AMY88) cells and its ability to restore various Vrp1p-dependent functions was compared with that of 364)817 containing the LBD (Figs 5D and 7A C). Strikingly, the presence of the CAAX box allowed the truncated 364)760 - CAAX that lacks a LBD to restore growth at elevated temperature to vrp1d cells (Fig. 7A,B) and endocytosis (Fig. 5D). This suggests that one of the functions of the LBD is to target Vrp1p to cortical patches and 4114 FEBS Journal 274 (2007) ª 2007 The Authors Journal compilation ª 2007 FEBS

13 T. Thanabalu et al. Function of Vrp1p C-terminal module A 24 C 37 C vect CAAX B D CAAX vect Time (h) C vect CAAX 24 C Fig. 7. Lipid-anchoring of bypasses the requirement for the LBD for endocytosis and growth at elevated temperature, but not cortical actin-patch polarization. (A) Addition of a CAAX box to confer lipid anchoring bypasses the requirement for the LBD of 364)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 364)817 ( 364)817 ), pam896 expressing 364)760 ( 364)760 ), or pam899 expressing 364)760 fused at its C-terminus to the CAAX box of Ras1p ( 364)760 CAAX). Each strain was streaked for single colonies on YPUAD solid medium, 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 364)817 for growth in liquid medium at elevated temperature. Growth rate of vrp1d (AMY88) cells carrying YCplac111 vector (vect), pam236 expressing 364)817 ( 364)817 ), pam896 expressing 364)760 ( 364)760 ), or pam899 expressing 364)760 CAAX ( 364)760 CAAX). A YPUAD culture of each strain was grown at 24 C, diluted to D 600 ¼ 0.05 in fresh YPUAD medium and shifted to 37 C. D 600 was monitored at 1 h intervals. (C) Addition of a CAAX box to confer lipid anchoring does not bypass the requirement for the LBD of 364)817 for cortical actin-patch polarization. Cortical actin-patch polarization in vrp1d (AMY88) cells carrying YCplac111 vector only (vect), pam236 expressing 364)817 ( 364)817 ), pam896 expressing 364)760 ( 364)760 ), or pam899 expressing 364)760 CAAX ( 364)760 CAAX). Cells were grown in YPUAD to exponential 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 polarization of cortical actin patches at this stage of the cell cycle. Bar ¼ 5 lm. FEBS Journal 274 (2007) ª 2007 The Authors Journal compilation ª 2007 FEBS 4115

14 Function of Vrp1p C-terminal module T. Thanabalu et al. 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 364)760 CAAX is able to restore cortical actin-patch polarization to vrp1d cells. vrp1d (AMY88) cells expressing 364)760 CAAX were stained with fluorochrome-conjugated phalloidin and their cortical actinpatch polarization was examined (Fig. 7C and Table 1). Perhaps not surprisingly (given the absence of the LBD), 364)760 CAAX was not able to restore a polarized cortical actin-patch distribution to vrp1d cells. We next asked whether addition of a CAAX box to 364)760 restores localization to cortical patches. The DNA sequence encoding 364)760 was fused inframe to sequences encoding GFP CAAX and expressed in vrp1d cells (AMY88) (Fig. 6A). The CAAX sequence targeted 364)760 to membranes including the plasma membrane consistent with our earlier findings with N-Vrp1p 1)364 [23]. Significantly, however, the CAAX sequence failed to target 364)760 GFP CAAX to cortical patches. 364)760 GFP CAAX-labeled membranes evenly without concentration into puncta (Fig. 6A). vrp1d (AMY88) cells and tested their ability to restore various Vrp1p-dependent functions. The longer fragment, 716)817, could rescue growth at elevated temperature moderately well (Fig. 3A), but the shorter fragment, 760)817 (which contains the LBD only) could not (data not shown). Because the LBD is rather short, comprising 57 residues, we then fused the DNA encoding the LBD inframe to sequences encoding GST ( 760)817 GST). Fusion to GST has been shown to stabilize some proteins to proteolysis [46]. GST is also known to dimerize [47]. This could potentially enhance the activity of some proteins it is fused to. We then expressed this fusion protein in vrp1d (AMY88) cells. Interestingly, expression of 760)817 GST, but not GST alone or 364)760 GST, restored growth at elevated temperature to vrp1d cells moderately well (Fig. 8A,B). This result demonstrates that 760)817 does possess sufficient information to mediate the growth function of Vrp1p, but that to perform this function it must be either stabilized or dimerized by fusion to GST. 760)817 GST also restored endocytosis to vrp1d cells (Fig. 8C). However, in contrast, 760)817 GST lacked the ability to restore cortical actin-patch polarization (Fig. 8D). 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 containing the LBD ( 716)817 and 760)817 ) in Discussion Here, we have examined the in vivo function of two submodules in the Vrp1p C-terminal module ( 364)817 ) (Fig. 1). The first is the CABS (residues ), of which residues 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 is sufficient for endocytosis and growth at elevated temperature, but not cortical actin-patch polarization. (A) The 57-residue LBD of 364)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 364)817 fused at its C-terminus to GST ( 364)817 GST), pam895 expressing 760)817 GST ( 760)817 GST), or pam892 expressing 364)760 GST ( 364)760 GST). 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 364)817 is sufficient for growth in liquid medium at elevated temperature. Growth rate of vrp1d (AMY88) cells carrying pam915 expressing GST only (GST), pam236 expressing 364)817 ( 364)817 ), or pam895 expressing 760)817 GST ( 760)817 GST). A YPUAD culture of each strain was grown at 24 C, diluted to D 600 ¼ 0.05 in fresh YPUAD medium and shifted to 37 C. D 600 was monitored at 1 h intervals. (C) The 57-residue LBD of 364)817 is sufficient for fluidphase endocytosis. vrp1d (AMY88) cells carrying pam915 expressing GST only (GST), pam236 expressing 364)817 ( 364)817 ), or pam895 expressing 760)817 GST ( 760)817 GST) were grown in YPUAD to exponential phase at 24 C and 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 364)817 is not sufficient for polarization of cortical actin patches. Cortical actin-patch polarization in vrp1d (AMY88) cells carrying pam915 expressing GST only (GST), pam236 expressing 364)817 ( 364)817 ), or pam895 expressing 760)817 GST ( 760)817 GST). Cells were grown in YPUAD to exponential 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 polarization of cortical actin patches at this stage of the cell cycle. Bar, 5 lm FEBS Journal 274 (2007) ª 2007 The Authors Journal compilation ª 2007 FEBS

15 T. Thanabalu et al. Function of Vrp1p C-terminal module fragments comprising residues 1 70 (KLK45K46AET the original WH2 domain), (residues not yet mapped), and (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 end). We show that the LBD is both necessary and sufficient for localization of 364)817 to cortical patches. A role for the LBD in cortical-patch localization is consistent with our previous results showing that Las17p is required for localization of A 24 C 37 C GST GST GST GST B D GST GST Time (h) C GST GST D GST GST 24 o C FEBS Journal 274 (2007) ª 2007 The Authors Journal compilation ª 2007 FEBS 4117