Vrp1p functions in both actomyosin ring-dependent and Hof1p-dependent pathways of cytokinesis

Vrp1p/verprolin/End5p is a Saccharomyces cerevisiae proline-rich protein, structurally and functionally related to human Wiskott–Aldrich syndrome protein-interacting protein. Vrp1p is required for viability at 37°C, but not 24°C. Here, we show that loss of Vrp1p ( vrp1Δ ) leads to a 3–4-fold delay in cytokinesis, wide bud necks, abnormal actomyosin rings, and aberrant septa even at 24°C. Like other mutations affecting the actomyosin ring, vrp1Δ is synthetic lethal with deletion of HOF1 (or CYK2 ), which encodes a protein related to mammalian proline serine threonine phosphatase-interacting protein and Schizosaccharomyces pombe Cdc15p required for an actomyosin ring-independent pathway of cytokinesis in S. cerevisiae . At 37°C, vrp1Δ cells rapidly cease dividing and exhibit a novel terminal phenotype: a single large bud, two well-separated nuclei, and an interphase microtubule array. The arrested cells have a persistent ring containing both actin and myosin at the bud neck. Many also exhibit some polarisation of cortical actin patches to the bud neck. Vrp1p binds an SH3-domain-containing fragment of Hof1p in vitro . Vrp1p is required in vivo for Hof1p relocalisation to a single ring at the bud neck prior to cytokinesis at 37°C, but not at 24°C. Vrp1p thus acts in both actomyosin ring formation and function, as well as in Hof1p localisation during cytokinesis.     

Verprolin cytokinesis function mediated by the Hof one trap domain

In budding yeast, partitioning of the cytoplasm during cytokinesis can proceed via a pathway dependent on the contractile actomyosin ring, as in other eukaryotes, or alternatively via a septum deposition pathway dependent on an SH3 domain protein, Hof1/Cyk2 (the yeast PSTPIP1 ortholog). In dividing yeast cells, Hof1 forms a ring at the bud neck distinct from the actomyosin ring, and this zone is active in septum deposition. We previously showed the yeast Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP) ortholog, verprolin/Vrp1/End5, interacts with Hof1 and facilitates Hof1 recruitment to the bud neck. A Vrp1 fragment unable to interact with yeast WASP (Las17/Bee1), localize to the actin cytoskeleton or function in polarization of the cortical actin cytoskeleton nevertheless retains function in Hof1 recruitment and cytokinesis. Here, we show the ability of this Vrp1 fragment to bind the Hof1 SH3 domain via its Hof one trap (HOT) domain is critical for cytokinesis. The Vrp1 HOT domain consists of three tandem proline-rich motifs flanked by serines. Unexpectedly, the Hof1 SH3 domain itself is not required for cytokinesis and indeed appears to negatively regulate cytokinesis. The Vrp1 HOT domain promotes cytokinesis by binding to the Hof1 SH3 domain and counteracting its inhibitory effect.     

Verprolin function in endocytosis and actin organization. Roles of the Las17p (yeast WASP)-binding domain and a novel C-terminal actin-binding domain

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 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 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 cytoskeleton for Vrp1p function in endocytosis and cell growth, and suggest that Vrp1p binding to individual effectors may alter their physiological activity.     

Saccharomyces cerevisiae Bzz1p is implicated with type I myosins in actin patch polarization and is able to recruit actin-polymerizing machinery in vitro

In Saccharomyces cerevisiae, the WASP (Wiskott-Aldrich syndrome protein) homologue Las17p (also called Bee1p) is an important component of cortical actin patches. Las17p is part of a high-molecular-weight protein complex that regulates Arp2/3 complex-dependent actin polymerization at the cell cortex and that includes the type I myosins Myo3p and Myo5p and verprolin (Vrp1p). To identify other factors implicated with this complex in actin regulation, we isolated proteins that bind to Las17p by two-hybrid screening and affinity chromatography. Here, we report the characterization of Lsb7/Bzz1p (for Las seventeen binding protein 7), an Src homology 3 (SH3) domain protein that interacts directly with Las17p via a polyproline-SH3 interaction. Bzz1p coimmunoprecipitates in a complex with Las17p, Vrp1p, Myo3/5p, Bbc1p, Hsp70p, and actin. It colocalizes with cortical actin patches and with Las17p. This localization is dependent on Las17p, but not on F-actin. Bzz1p interacts physically and genetically with type I myosins. While deletion of BZZ1 shows no obvious phenotype, simultaneous deletion of the BZZ1, MYO3, and MYO5 genes is lethal. Overexpression of Bzz1p inhibits cell growth, and a bzz1Delta myo5Delta double mutant is unable to restore actin polarity after NaCl stress. Finally, Bzz1p in vitro is able to recruit a functional actin polymerization machinery through its SH3 domains. Its interactions with Las17p, Vrp1p, and the type I myosins are essential for this process. This suggests that Bzz1p could be implicated in the regulation of actin polymerization.     

Enhanced membrane fusion in sterol-enriched vacuoles bypasses the Vrp1p requirement

Organization of lipids into membrane microdomains is a vital mechanism of protein processing. Here we show that overexpression of ERG6, a gene involved in ergosterol synthesis, elevates sterol levels 1.5-fold on the vacuole membrane and enhances their homotypic fusion. The mechanism of sterol-enhanced fusion is not via more efficient sorting, but instead promotes increased kinetics of fusion subreactions. We initially isolated ERG6 as a suppressor of a vrp1Delta growth defect selective for vacuole function. VRP1 encodes verprolin, an actin-binding protein that colocalizes to vacuoles. The vrp1Delta mutant has fragmented vacuoles in vivo and isolated vacuoles do not fuse in vitro, indicative of a Vrp1p requirement for membrane fusion. ERG6 overexpression rescues vrp1Delta vacuole fusion in a cytosol-dependent manner. Cytosol prepared from the vrp1Delta strain remains active; therefore, cytosol is not resupplying Vrp1p. Las17p (Vrp1p functional partner) antibodies, which inhibit wild-type vacuole fusion, do not inhibit the fusion of vacuoles from the vrp1Delta-ERG6 overexpression strain. Vacuole-associated actin turnover is decreased in the vrp1Delta strain, but recovered by ERG6 overexpression linking sterol enrichment to actin remodeling. Therefore, the Vrp1p/Las17p requirement for membrane fusion is bypassed by increased sterols, which promotes actin remodeling as part the membrane fusion mechanism.     

The novel adaptor protein, Mti1p, and Vrp1p, a homolog of Wiskott-Aldrich syndrome protein-interacting protein (WIP), may antagonistically regulate type I myosins in Saccharomyces cerevisiae

Type I myosins in yeast, Myo3p and Myo5p (Myo3/5p), are involved in the reorganization of the actin cytoskeleton. The SH3 domain of Myo5p regulates the polymerization of actin through interactions with both Las17p, a homolog of mammalian Wiskott-Aldrich syndrome protein (WASP), and Vrp1p, a homolog of WASP-interacting protein (WIP). Vrp1p is required for both the localization of Myo5p to cortical patch-like structures and the ATP-independent interaction between the Myo5p tail region and actin filaments. We have identified and characterized a new adaptor protein, Mti1p (Myosin tail region-interacting protein), which interacts with the SH3 domains of Myo3/5p. Mti1p co-immunoprecipitated with Myo5p and Mti1p-GFP co-localized with cortical actin patches. A null mutation of MTI1 exhibited synthetic lethal phenotypes with mutations in SAC6 and SLA2, which encode actin-bundling and cortical actin-binding proteins, respectively. Although the mti1 null mutation alone did not display any obvious phenotype, it suppressed vrp1 mutation phenotypes, including temperature-sensitive growth, abnormally large cell morphology, defects in endocytosis and salt-sensitive growth. These results suggest that Mti1p and Vrp1p antagonistically regulate type I myosin functions.     

Actin binding and proline rich motifs of CR16 play redundant role in growth of vrp1Delta cells

CR16, (Glucocorticoid-regulated) belongs to the verprolin family of proteins which are characterized by the presence of a V domain (verprolin) at the N-terminal. Expression of CR16 suppressed the growth and endocytosis defect of vrp1Delta strain without correcting the actin patch polarization defect. The V domain of CR16 is critical for suppression of the growth defect of vrp1Delta strain but not for localisation to cortical actin patches. Mutations in the actin binding motif alone did not abolish the activity of CR16 but the mutations in combination with deletion of N-terminal proline rich motif abolished the ability of CR16 to suppress the growth defect. This suggests that the V domain of CR16 has two functionally redundant motifs and either one of these motifs is sufficient for suppressing the growth defect of vrp1Delta strain. This is in contrast to the observation that both WIP and WIRE require the actin binding motif for their activity.     

Actin-binding Verprolin Is a Polarity Development Protein Required for the Morphogenesis and Function of the Yeast Actin Cytoskeleton

Yeast verprolin, encoded by VRP1, is implicated in cell growth, cytoskeletal organization, endocytosis and mitochondrial protein distribution and function. We show that verprolin is also required for bipolar bud-site selection. Previously we reported that additional actin suppresses the temperature-dependent growth defect caused by a mutation in VRP1. Here we show that additional actin suppresses all known defects caused by vrp1-1 and conclude that the defects relate to an abnormal cytoskeleton. Using the two-hybrid system, we show that verprolin binds actin. An actin-binding domain maps to the LKKAET hexapeptide located in the first 70 amino acids. A similar hexapeptide in other acting-binding proteins was previously shown to be necessary for actin-binding activity. The entire 70– amino acid motif is conserved in novel higher eukaryotic proteins that we predict to be actin-binding, and also in the actin-binding proteins, WASP and N-WASP. Verprolin-GFP in live cells has a cell cycle-dependent distribution similar to the actin cortical cytoskeleton. In fixed cells hemagglutinin-tagged Vrp1p often co-localizes with actin in cortical patches. However, disassembly of the actin cytoskeleton using Latrunculin-A does not alter verprolin's location, indicating that verprolin establishes and maintains its location independent of the actin cytoskeleton. Verprolin is a new member of the actin-binding protein family that serves as a polarity development protein, perhaps by anchoring actin. We speculate that the effects of verprolin upon the actin cytoskeleton might influence mitochondrial protein sorting/function via mRNA distribution.     

The Saccharomyces cerevisiae Arf3 protein is involved in actin cable and cortical patch formation

We show that Arf3p, a member of the ADP ribosylation family, is involved in the organization of actin cables and cortical patches in Saccharomyces cerevisiae. Profilin-deficient cells (pfy1Delta) have severe growth defects and lack actin cables. Overexpression of ARF3 restores actin cables and corrects growth defects in these cells. Cells deficient for the cortical patch proteins Las17p and Vrp1p have growth defects and a random cortical patch distribution. Overexpression of ARF3 in las17Delta and in vrp1Delta cells partially corrects growth defects and restores the polarized distribution of cortical patches. The N-terminal glycine, a myristoylation site in Arf3p, is necessary for its suppressor activity. arf3Delta cells show a random budding pattern. Overexpression of BNI1, GEA2 or SYP1, three genes involved in actin cytoskeleton formation, restores the normal axial budding pattern of arf3Delta cells. BUD6 is a polarity gene and GEA2 is involved in retrograde transport and the organization of the actin cytoskeleton. We have identified genetic interactions between ARF3 and BUD6, and between ARF3 and GEA2. Both double mutant strains have actin cytoskeleton defects. Our results support a role for ARF3 in cell polarity and the organization of the actin cytoskeleton.     

The Src Homology Domain 3 (SH3) of a Yeast Type I Myosin, Myo5p, Binds to Verprolin and Is Required for Targeting to Sites of Actin Polarization

The budding yeast contains two type I myosins, Myo3p and Myo5p, with redundant functions. Deletion of both myosins results in growth defects, loss of actin polarity and polarized cell surface growth, and accumulation of intracellular membranes. Expression of myc-tagged Myo5p in myo3Δ myo5Δ cells fully restores wild-type characteristics. Myo5p is localized as punctate, cortical structures enriched at sites of polarized cell growth. We find that latrunculin-A–induced depolymerization of F-actin results in loss of Myo5p patches. Moreover, incubation of yeast cells at 37°C results in transient depolarization of both Myo5p patches and the actin cytoskeleton. Mutant Myo5 proteins with deletions in nonmotor domains were expressed in myo3Δ myo5Δ cells and the resulting strains were analyzed for Myo5p function. Deletion of the tail homology 2 (TH2) domain, previously implicated in ATP-insensitive actin binding, has no detectable effect on Myo5p function. In contrast, myo3Δ myo5Δ cells expressing mutant Myo5 proteins with deletions of the src homology domain 3 (SH3) or both TH2 and SH3 domains display defects including Myo5p patch depolarization, actin disorganization, and phenotypes associated with actin dysfunction. These findings support a role for the SH3 domain in Myo5p localization and function in budding yeast. The proline-rich protein verprolin (Vrp1p) binds to the SH3 domain of Myo3p or Myo5p in two-hybrid tests, coimmunoprecipitates with Myo5p, and colocalizes with Myo5p. Immunolocalization of the myc-tagged SH3 domain of Myo5p reveals diffuse cytoplasmic staining. Thus, the SH3 domain of Myo5p contributes to but is not sufficient for localization of Myo5p either to patches or to sites of polarized cell growth. Consistent with this, Myo5p patches assemble but do not localize to sites of polarized cell surface growth in a VRP1 deletion mutant. Our studies support a multistep model for Myo5p targeting in yeast. The first step, assembly of Myo5p patches, is dependent upon F-actin, and the second step, polarization of actin patches, requiresVrp1p and the SH3 domain of Myo5p.     

The WASP/Las17p-interacting protein Bzz1p functions with Myo5p in an early stage of endocytosis

Summary. The formation of actin filaments is crucial for endocytosis and other interrelated cellular phenomena such as motility, polarized morphogenesis, and cytokinesis. In this paper we have investigated the role of the WASP/Las17-interacting protein Bzz1p in endocytosis and trafficking to the vacuole. We and others have recently shown that Bzz1p is an actin patch protein that interacts directly with Las17p via a SH3-polyproline interaction. Bzz1p functions with type I myosins to restore polarity of the actin cytoskeleton after NaCl stress. In an in vitro bead assay, GST-Bzz1p fusion protein triggers a functional actin polymerization machinery through its two C-terminal SH3 domains. In this paper we implicate Bzz1p with the type I myosins both in fluid-phase and in the internalization step of receptor-mediated endocytosis. As deduced from their localization as GFP fusions, the vacuolar delivery of endocytic and biosynthetic cargoes as well as the multivesicular body pathway appear unaffected. We further elucidate Bzz1p direct participation in actin polymerization by demonstrating that each of the SH3 domains of Bzz1p individually is able to trigger actin polymerization in a cell-free system dependent on Arp2/3, Las17p, Vrp1p, and the type I myosins. Taken together, our results show that Bzz1p participates, essentially via its SH3 domains, in early steps of endocytosis together with known actin nucleation activators. Correspondence and reprints: Equipe Cytosquelette et Trafic Intracellulaire, UMR7156 du CNRS, Institut de Biologie Moléculaire et Cellulaire du CNRS, 15 rue Descartes, 67084 Strasbourg, France. Present address: Division of Biochemistry, Biozentrum, University of Basel, Basel, Switzerland.     

The Saccharomyces cerevisiae Homologue of Human Wiskott–Aldrich Syndrome Protein Las17p Interacts with the Arp2/3 Complex

Yeast Las17 protein is homologous to the Wiskott-Aldrich Syndrome protein, which is implicated in severe immunodeficiency. Las17p/Bee1p has been shown to be important for actin patch assembly and actin polymerization. Here we show that Las17p interacts with the Arp2/3 complex. LAS17 is an allele-specific multicopy suppressor of ARP2 and ARP3 mutations; overexpression restores both actin patch organization and endocytosis defects in ARP2 temperature-sensitive (ts) cells. Six of seven ARP2 ts mutants and at least one ARP3 ts mutant are synthetically lethal with las17Delta ts confirming functional interaction with the Arp2/3 complex. Further characterization of las17Delta cells showed that receptor-mediated internalization of alpha factor by the Ste2 receptor is severely defective. The polarity of normal bipolar bud site selection is lost. Las17-gfp remains localized in cortical patches in vivo independently of polymerized actin and is required for the polarized localization of Arp2/3 as well as actin. Coimmunoprecipitation of Arp2p with Las17p indicates that Las17p interacts directly with the complex. Two hybrid results also suggest that Las17p interacts with actin, verprolin, Rvs167p and several other proteins including Src homology 3 (SH3) domain proteins, suggesting that Las17p may integrate signals from different regulatory cascades destined for the Arp2/3p complex and the actin cytoskeleton.     

Roles of Hof1p, Bni1p, Bnr1p, and myo1p in cytokinesis in Saccharomyces cerevisiae

Cytokinesis in Saccharomyces cerevisiae occurs by the concerted action of the actomyosin system and septum formation. Here we report on the roles of HOF1, BNI1, and BNR1 in cytokinesis, focusing on Hof1p. Deletion of HOF1 causes a temperature-sensitive defect in septum formation. A Hof1p ring forms on the mother side of the bud neck in G2/M, followed by the formation of a daughter-side ring. Around telophase, Hof1p is phosphorylated and the double rings merge into a single ring that contracts slightly and may colocalize with the actomyosin structure. Upon septum formation, Hof1p splits into two rings, disappearing upon cell separation. Hof1p localization is dependent on septins but not Myo1p. Synthetic lethality suggests that Bni1p and Myo1p belong to one functional pathway, whereas Hof1p and Bnr1p belong to another. These results suggest that Hof1p may function as an adapter linking the primary septum synthesis machinery to the actomyosin system. The formation of the actomyosin ring is not affected by bni1Delta, hof1Delta, or bnr1Delta. However, Myo1p contraction is affected by bni1Delta but not by hof1Delta or bnr1Delta. In bni1Delta cells that lack the actomyosin contraction, septum formation is often slow and asymmetric, suggesting that actomyosin contraction may provide directionality for efficient septum formation.     

A role for myosin-I in actin assembly through interactions with Vrp1p, Bee1p, and the Arp2/3 complex

Type I myosins are highly conserved actin-based molecular motors that localize to the actin-rich cortex and participate in motility functions such as endocytosis, polarized morphogenesis, and cell migration. The COOH-terminal tail of yeast myosin-I proteins, Myo3p and Myo5p, contains an Src homology domain 3 (SH3) followed by an acidic domain. The myosin-I SH3 domain interacted with both Bee1p and Vrp1p, yeast homologues of human WASP and WIP, adapter proteins that link actin assembly and signaling molecules. The myosin-I acidic domain interacted with Arp2/3 complex subunits, Arc40p and Arc19p, and showed both sequence similarity and genetic redundancy with the COOH-terminal acidic domain of Bee1p (Las17p), which controls Arp2/3-mediated actin nucleation. These findings suggest that myosin-I proteins may participate in a diverse set of motility functions through a role in actin assembly.     

Bee1, a Yeast Protein with Homology to Wiscott-Aldrich Syndrome Protein, Is Critical for the Assembly of Cortical Actin Cytoskeleton

Yeast protein, Bee1, exhibits sequence homology to Wiskott-Aldrich syndrome protein (WASP), a human protein that may link signaling pathways to the actin cytoskeleton. Mutations in WASP are the primary cause of Wiskott-Aldrich syndrome, characterized by immuno-deficiencies and defects in blood cell morphogenesis. This report describes the characterization of Bee1 protein function in budding yeast. Disruption of BEE1 causes a striking change in the organization of actin filaments, resulting in defects in budding and cytokinesis. Rather than assemble into cortically associated patches, actin filaments in the buds of Δbee1 cells form aberrant bundles that do not contain most of the cortical cytoskeletal components. It is significant that Δbee1 is the only mutation reported so far that abolishes cortical actin patches in the bud. Bee1 protein is localized to actin patches and interacts with Sla1p, a Src homology 3 domain–containing protein previously implicated in actin assembly and function. Thus, Bee1 protein may be a crucial component of a cytoskeletal complex that controls the assembly and organization of actin filaments at the cell cortex.     

Of Bars and Rings: Hof1-Dependent Cytokinesis in Multiseptated Hyphae of Ashbya gossypii

We analyzed the development of multiple septa in elongated multinucleated cells (hyphae) of the filamentous ascomycete Ashbya gossypii in which septation is apparently uncoupled from nuclear cycles. A key player for this compartmentalization is the PCH protein Hof1. Hyphae that are lacking this protein form neither actin rings nor septa but still elongate at wild-type speed. Using in vivo fluorescence microscopy, we present for the first time the coordination of cytokinesis and septation in multiseptated and multinucleated cells. Hof1, the type II myosin Myo1, the landmark protein Bud3, and the IQGAP Cyk1 form collars of cortical bars already adjacent to hyphal tips, thereby marking the sites of septation. While hyphae continue to elongate, these proteins gradually form cortical rings. This bar-to-ring transition depends on Hof1 and Cyk1 but not Myo1 and is required for actin ring assembly. The Fes/CIP4 homology (FCH) domain of Hof1 ensures efficient localization of Hof1, whereas ring integrity is conferred by the Src homology 3 (SH3) domain. Up to several hours after site selection, actin ring contraction leads to membrane invagination and subsequent cytokinesis. Simultaneously, a septum forms between the adjacent hyphal compartments, which do not separate. During evolution, A. gossypii lost the homologs of two enzymes essential for cell separation in Saccharomyces cerevisiae.     

The human WASP-interacting protein, WIP, activates the cell polarity pathway in yeast.

WIP, the Wiskott-Aldrich syndrome protein-interacting protein, is a human protein involved in actin polymerization and redistribution in lymphoid cells. The mechanism by which WIP reorganizes actin cytoskeleton is unknown. WIP is similar to yeast verprolin, an actin- and myosin-interacting protein required for polarized morphogenesis. To determine whether WIP and verprolin are functional homologues, we analyzed the function of WIP in yeast. WIP suppresses the growth defects of VRP1 missense and null mutations as well as the defects in cytoskeletal organization and endocytosis observed in vrp1-1 cells. The ability of WIP to replace verprolin is dependent on its WH2 actin binding domain and a putative profilin binding domain. Immunofluorescence localization of WIP in yeast cells reveals a pattern consistent with its function at the cortical sites of growth. Thus, like verprolin, WIP functions in yeast to link the polarity development pathway and the actin cytoskeleton to generate cytoskeletal asymmetry. A role for WIP in cell polarity provides a framework for unifying, under a common paradigm, distinct molecular defects associated with immunodeficiencies like Wiskott-Aldrich syndrome.     

Evidence for the genetic interaction between the actin-binding protein Vrp1 and the RhoGAP Rgd1 mediated through Rho3p and Rho4p in Saccharomyces cerevisiae

The non-essential RGD1 gene from Saccharomyces cerevisiae encodes a protein that has been characterized in vitro as a Rho GTPase activating protein (RhoGAP) for the Rho3 and Rho4 proteins. Rgd1p, which displays a conserved FCH-coiled coil-Rho-GAP domain organization, showed a patch-like distribution in the cell, including a localization in growing buds. Using a genetic screen, we found that rgd1delta and vrp1alpha mutations exhibited a synthetic lethality, thus revealing an interaction between these genes. The VRP1 product is an actin and myosin interacting protein involved in polarized growth. Using mutant forms of both Rho3 and Rho4 proteins, we provide evidence for the involvement of these two GTPases in RGD1-VRP1 co-lethality. In addition, these results strongly argue in favour of Rho3p and Rho4p being the targets of Rgd1p RhoGAP activity in vivo. Genetic relationships between either VRP1 or RGD1 and actin cytoskeleton-linked genes were also studied. These and other well-established data support the idea that Vrp1, Las17, Rvs167 proteins belong to the same complex. This protein structure might act with myosins in various actin cytoskeleton-based activities, in co-operation with a Rho3p/Rho4p signalling pathway that is negatively regulated by Rgd1p GAP activity.     

Direct involvement of yeast type I myosins in Cdc42-dependent actin polymerization

The generation of cortical actin filaments is necessary for processes such as cell motility and cell polarization. Several recent studies have demonstrated that Wiskott-Aldrich syndrome protein (WASP) family proteins and the actin-related protein (Arp) 2/3 complex are key factors in the nucleation of actin filaments in diverse eukaryotic organisms. To identify other factors involved in this process, we have isolated proteins that bind to Bee1p/Las17p, the yeast WASP-like protein, by affinity chromatography and mass spectroscopic analysis. The yeast type I myosins, Myo3p and Myo5p, have both been identified as Bee1p-interacting proteins. Like Bee1p, these myosins are essential for cortical actin assembly as assayed by in vitro reconstitution of actin nucleation sites in permeabilized yeast cells. Analysis using this assay further demonstrated that the motor activity of these myosins is required for the polymerization step, and that actin polymerization depends on phosphorylation of myosin motor domain by p21-activated kinases (PAKs), downstream effectors of the small guanosine triphosphatase, Cdc42p. The type I myosins also interact with the Arp2/3 complex through a sequence at the end of the tail domain homologous to the Arp2/3-activating region of WASP-like proteins. Combined deletions of the Arp2/3-interacting domains of Bee1p and the type I myosins abolish actin nucleation sites at the cortex, suggesting that these proteins function redundantly in the activation of the Arp2/3 complex.     

An intact SH3 domain is required for myosin I-induced actin polymerization

The yeast type I myosins (MYO3 and MYO5) are involved in endocytosis and in the polarization of the actin cytoskeleton. The tail of these proteins contains a Tail Homology 2 (TH2) domain that constitutes a putative actin-binding site. Because of the important mechanistic implications of a second ATP-independent actin-binding site, we analyzed its functional relevance in vivo. Even though the myosin tail interacts with actin, and this interaction seems functionally important, deletion of a major portion of the TH2 domain did not abolish interaction. In contrast, we found that the SH3 domain of Myo5p significantly contributes to this interaction, implicating other proteins. We found that Vrp1p, the yeast homolog of WIP [Wiskott-Aldrich syndrome protein (WASP)-interacting protein], seems necessary to sustain the Myo5p tail-F-actin interaction. Consistent with recent results implicating the yeast type I myosins in regulating actin polymerization in vivo, we demonstrate that the C-terminal domain of Myo5p is able to induce cytosol-dependent actin polymerization in vitro, and that this activity requires both an intact Myo5p SH3 domain and Vrp1p.