A myosin light chain mediates the localization of the budding yeast IQGAP-like protein during contractile ring formation

Cytokinesis in animal cells is accomplished through constriction of an actomyosin ring [1] [2] [3], which must assemble at the correct time and place in order to ensure proper division of genetic material and organelles. Budding yeast is a useful model system for determining the biochemical pathway of contractile ring assembly. The budding yeast IQGAP-like protein, Cyk1/Iqg1p, has multiple roles in the assembly and contraction of the actomyosin ring [4] [5] [6]. Previously, the IQ motifs of Cyk1/Iqg1p were shown to be required for the localization of this protein at the bud neck [6]. We have investigated the binding partner of the IQ motifs, which are predicted to interact with calmodulin-like proteins. Mlc1p was originally identified as a light chain for a type V myosin, Myo2p; however, a cytokinesis defect associated with disruption of the MLC1 gene suggested that the essential function of Mlc1p may involve interactions with other proteins [7]. We show that Mlc1p binds the IQ motifs of Cyk1/Iqg1p and present evidence that this interaction recruits Cyk1/Iqg1p to the bud neck. Immunofluorescence staining shows that Mlc1p is localized to sites of polarized cell growth as well as the bud neck before and independently of Cyk1p. These results demonstrate that Mlc1p is important for the assembly of the actomyosin ring in budding yeast and that this function is mediated through interaction with Cyk1/Iqg1p.     

Sequential Assembly of Myosin II, an IQGAP-like Protein, and Filamentous Actin to a Ring Structure Involved in Budding Yeast Cytokinesis

We have identified a Saccharomyces cerevisiae protein, Cyk1p, that exhibits sequence similarity to the mammalian IQGAPs. Gene disruption of Cyk1p results in a failure in cytokinesis without affecting other events in the cell cycle. Cyk1p is diffused throughout most of the cell cycle but localizes to a ring structure at the mother–bud junction after the initiation of anaphase. This ring contains filamentous actin and Myo1p, a myosin II homologue. In vivo observation with green fluorescent protein–tagged Myo1p showed that the ring decreases drastically in size during cell division and therefore may be contractile. These results indicate that cytokinesis in budding yeast is likely to involve an actomyosin-based contractile ring. The assembly of this ring occurs in temporally distinct steps: Myo1p localizes to a ring that overlaps the septins at the G1-S transition slightly before bud emergence; Cyk1p and actin then accumulate in this ring after the activation of the Cdc15 pathway late in mitosis. The localization of myosin is abolished by a mutation in Cdc12p, implicating a role for the septin filaments in the assembly of the actomyosin ring. The accumulation of actin in the cytokinetic ring was not observed in cells depleted of Cyk1p, suggesting that Cyk1p plays a role in the recruitment of actin filaments, perhaps through a filament-binding activity similar to that demonstrated for mammalian IQGAPs.     

Rho1 directs formin-mediated actin ring assembly during budding yeast cytokinesis

In eukaryotic cells, dynamic rearrangement of the actin cytoskeleton is critical for cell division. In the yeast Saccharomyces cerevisiae, three main structures constitute the actin cytoskeleton: cortical actin patches, cytoplasmic actin cables, and the actin-based cytokinetic ring. The conserved Arp2/3 complex and a WASP-family protein mediate actin patch formation, whereas the yeast formins (Bni1 and Bnr1) promote assembly of actin cables. However, the mechanism of actin ring formation is currently unclear. Here, we show that actin filaments are required for cytokinesis in S. cerevisiae, and that the actin ring is a highly dynamic structure that undergoes constant turnover. Assembly of the actin ring requires the formin-like proteins and profilin, but is not Arp2/3-mediated. Furthermore, the formin-dependent actin ring assembly pathway is regulated by the Rho-type GTPase Rho1 but not Cdc42. Finally, we show that the formins are not required for localization of Cyk1/Iqg1, an IQGAP-like protein previously shown to be required for actin ring formation, suggesting that formin-like proteins and Cyk1 act synergistically but independently in assembly of the actin ring.     

Dephosphorylation of Iqg1 by Cdc14 regulates cytokinesis in budding yeast

Cytokinesis separates cells by contraction of a ring composed of filamentous actin (F-actin) and type II myosin. Iqg1, an IQGAP family member, is an essential protein in Saccharomyces cerevisiae required for assembly and contraction of the actomyosin ring. Localization of F-actin to the ring occurs only after anaphase and is mediated by the calponin homology domain (CHD) of Iqg1, but the regulatory mechanisms that temporally restrict actin ring assembly are not well defined. We tested the hypothesis that dephosphorylation of four perfect cyclin-dependent kinase (Cdk) sites flanking the CHD promotes actin ring formation, using site-specific alanine mutants. Cells expressing the nonphosphorylatable iqg1-4A allele formed actin rings before anaphase and exhibited defects in myosin contraction and cytokinesis. The Cdc14 phosphatase is required for normal cytokinesis and acts on specific Cdk phosphorylation sites. Overexpression of Cdc14 resulted in premature actin ring assembly, whereas inhibition of Cdc14 function prevented actin ring formation. Cdc14 associated with Iqg1, dependent on several CHD-flanking Cdk sites, and efficiently dephosphorylated these sites in vitro. Of importance, the iqg1-4A mutant rescued the inability of cdc14-1 cells to form actin rings. Our data support a model in which dephosphorylation of Cdk sites around the Iqg1 CHD by Cdc14 is both necessary and sufficient to promote actin ring formation. Temporal control of actin ring assembly by Cdk and Cdc14 may help to ensure that cytokinesis onset occurs after nuclear division is complete.     

The Saccharomyces cerevisiae actin cytoskeletal component Bsp1p has an auxiliary role in actomyosin ring function and in the maintenance of bud-neck structure

Iqg1p is a component of the actomyosin contractile ring that is required for actin recruitment and septum deposition. Cells lacking Iqg1p function have an altered bud-neck structure and fail to form a functional actomyosin contractile ring resulting in a block to cytokinesis and septation. Here it is demonstrated that increased expression of the actin cytoskeleton associated protein Bsp1p bypasses the requirement for contractile ring function. This also correlates with reduced bud-neck width and remedial septum formation. Increased expression of this protein in a temperature-sensitive iqg1-1 background causes remedial septum formation at the bud neck that is reliant upon chitin synthase III activity and restores cell separation. The observed suppression correlates with a restoration of normal bud-neck structure. While Bsp1p is a component of the contractile ring, its recruitment to the bud neck is not required for the observed suppression. Loss of Bsp1p causes a brief delay in the redistribution of the actin cytoskeleton normally observed at the end of actin ring contraction. Compromise of Iqg1p function, in the absence of Bsp1p function, leads to a profound change in the distribution of actin and the pattern of cell growth accompanied by a failure to complete cytokinesis and cell separation.     

Cyk3, a novel SH3-domain protein, affects cytokinesis in yeast

Cytokinesis requires the wholesale reorganization of the cytoskeleton and secretion to complete the division of one cell into two. In the budding yeast Saccharomyces cerevisiae, the IQGAP-related protein Iqg1 (Cyk1) promotes cytokinetic actin ring formation and is required for cytokinesis and viability [1-3]. As the actin ring is not essential for cytokinesis or viability, Iqg1 must act by another mechanism [4]. To uncover this mechanism, a screen for high-copy suppressors of the iqg1 lethal phenotype was performed. CYK3 suppressed the requirement for IQG1 in viability and cytokinesis without restoration of the actin ring, demonstrating that CYK3 promotes cytokinesis through an actomyosin-ring-independent pathway. CYK3 encodes a novel SH3-domain protein that was found in association with the actin ring and the mother-bud neck. cyk3 null cells had misshapen mother-bud necks and were deficient in cytokinesis. In the cyk3 null strain, actin rearrangements associated with cytokinesis appeared normal, suggesting that the phenotype reflects a defect in secretory targeting or septal synthesis. Deletion of either cyk3 or hof1 alone results in a mild cytokinetic phenotype [5-7], but deletion of both genes resulted in lethality and a complete cytokinetic block, suggesting overlapping function. Thus, Cyk3 appears to be important for cytokinesis and acts potentially downstream of Iqg1.     

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.     

Dual Function of Cyk2, a cdc15/PSTPIP Family Protein,in Regulating Actomyosin Ring Dynamics and Septin Distribution

We previously showed that the budding yeast Saccharomyces cerevisiae assembles an actomyosin-based ring that undergoes a contraction-like size change during cytokinesis. To learn more about the biochemical composition and activity of this ring, we have characterized the in vivo distribution and function of Cyk2p, a budding yeast protein that exhibits significant sequence similarity to the cdc15/PSTPIP family of cleavage furrow proteins. Video microscopy of cells expressing green fluorescent protein (GFP)-tagged Cyk2p revealed that Cyk2p forms a double ring that coincides with the septins through most of the cell cycle. During cytokinesis, however, the Cyk2 double ring merges with the actomyosin ring and exhibits a contraction-like size change that is dependent on Myo1p. The septin double ring, in contrast, does not undergo the contraction-like size change but the separation between the two rings increases during cytokinesis. These observations suggest that the septin-containing ring is dynamically distinct from the actomyosin ring and that Cyk2p transits between the two types of structures. Gene disruption of CYK2 does not affect the assembly of the actomyosin ring but results in rapid disassembly of the ring during the contraction phase, leading to incomplete cytokinesis, suggesting that Cyk2p has an important function in modulating the stability of the actomyosin ring during contraction. Overexpression of Cyk2p also blocks cytokinesis, most likely due to a loss of the septins from the bud neck, indicating that Cyk2p may also play a role in regulating the localization of the septins.     

Saccharomyces cerevisiae Mob1p is required for cytokinesis and mitotic exit

The Saccharomyces cerevisiae mitotic exit network (MEN) is a conserved set of genes that mediate the transition from mitosis to G(1) by regulating mitotic cyclin degradation and the inactivation of cyclin-dependent kinase (CDK). Here, we demonstrate that, in addition to mitotic exit, S. cerevisiae MEN gene MOB1 is required for cytokinesis and cell separation. The cytokinesis defect was evident in mob1 mutants under conditions in which there was no mitotic-exit defect. Observation of live cells showed that yeast myosin II, Myo1p, was present in the contractile ring at the bud neck but that the ring failed to contract and disassemble. The cytokinesis defect persisted for several mitotic cycles, resulting in chains of cells with correctly segregated nuclei but with uncontracted actomyosin rings. The cytokinesis proteins Cdc3p (a septin), actin, and Iqg1p/ Cyk1p (an IQGAP-like protein) appeared to correctly localize in mob1 mutants, suggesting that MOB1 functions subsequent to actomyosin ring assembly. We also examined the subcellular distribution of Mob1p during the cell cycle and found that Mob1p first localized to the spindle pole bodies during mid-anaphase and then localized to a ring at the bud neck just before and during cytokinesis. Localization of Mob1p to the bud neck required CDC3, MEN genes CDC5, CDC14, CDC15, and DBF2, and spindle pole body gene NUD1 but was independent of MYO1. The localization of Mob1p to both spindle poles was abolished in cdc15 and nud1 mutants and was perturbed in cdc5 and cdc14 mutants. These results suggest that the MEN functions during the mitosis-to-G(1) transition to control cyclin-CDK inactivation and cytokinesis.     

Hof1 and Rvs167 Have Redundant Roles in Actomyosin Ring Function during Cytokinesis in Budding Yeast

The Hof1 protein (Homologue of Fifteen) regulates formation of the primary septum during cytokinesis in the budding yeast Saccharomyces cerevisiae, whereas the orthologous Cdc15 protein in fission yeast regulates the actomyosin ring by using its F-BAR domain to recruit actin nucleators to the cleavage site. Here we show that budding yeast Hof1 also contributes to actin ring assembly in parallel with the Rvs167 protein. Simultaneous deletion of the HOF1 and RVS167 genes is lethal, and cells fail to assemble the actomyosin ring as they progress through mitosis. Although Hof1 and Rvs167 are not orthologues, they both share an analogous structure, with an F-BAR or BAR domain at the amino terminus, capable of inducing membrane curvature, and SH3 domains at the carboxyl terminus that bind to specific proline-rich targets. The SH3 domain of Rvs167 becomes essential for assembly of the actomyosin ring in cells lacking Hof1, suggesting that it helps to recruit a regulator of the actin cytoskeleton. This new function of Rvs167 appears to be independent of its known role as a regulator of the Arp2/3 actin nucleator, as actin ring assembly is not abolished by the simultaneous inactivation of Hof1 and Arp2/3. Instead we find that recruitment to the bud-neck of the Iqg1 actin regulator is defective in cells lacking Hof1 and Rvs167, though future studies will be needed to determine if this reflects a direct interaction between these factors. The redundant role of Hof1 in actin ring assembly suggests that the mechanism of actin ring assembly has been conserved to a greater extent across evolution than anticipated previously.     

Identification of yeast IQGAP (Iqg1p) as an anaphase-promoting-complex substrate and its role in actomyosin-ring-independent cytokinesis

In the yeast Saccharomyces cerevisiae, a ring of myosin II forms in a septin-dependent manner at the budding site in late G1. This ring remains at the bud neck until the onset of cytokinesis, when actin is recruited to it. The actomyosin ring then contracts, septum formation occurs concurrently, and cytokinesis is soon completed. Deletion of MYO1 (the only myosin II gene) is lethal on rich medium in the W303 strain background and causes slow-growth and delayed-cell-separation phenotypes in the S288C strain background. These phenotypes can be suppressed by deletions of genes encoding nonessential components of the anaphase-promoting complex (APC/C). This suppression does not seem to result simply from a delay in mitotic exit, because overexpression of a nondegradable mitotic cyclin does not suppress the same phenotypes. Overexpression of either IQG1 or CYK3 also suppresses the myo1Delta phenotypes, and Iqg1p (an IQGAP protein) is increased in abundance and abnormally persistent after cytokinesis in APC/C mutants. In vitro assays showed that Iqg1p is ubiquitinated directly by APC/C(Cdh1) via a novel recognition sequence. A nondegradable Iqg1p (lacking this recognition sequence) can suppress the myo1Delta phenotypes even when expressed at relatively low levels. Together, the data suggest that compromise of APC/C function allows the accumulation of Iqg1p, which then promotes actomyosin-ring-independent cytokinesis at least in part by activation of Cyk3p.     

Cell cycle-dependent roles for the FCH-domain protein Cdc15p in formation of the actomyosin ring in Schizosaccharomyces pombe

Cell division in the fission yeast Schizosaccharomyces pombe requires the formation and constriction of an actomyosin ring at the division site. The actomyosin ring is assembled in metaphase and anaphase A, is maintained throughout mitosis, and constricts after completion of anaphase. Maintenance of the actomyosin ring during late stages of mitosis depends on the septation initiation network (SIN), a signaling cascade that also regulates the deposition of the division septum. However, SIN is not active in metaphase and is not required for the initial assembly of the actomyosin ring early in mitosis. The FER/CIP4-homology (FCH) domain protein Cdc15p is a component of the actomyosin ring. Mutations in cdc15 lead to failure in cytokinesis and result in the formation of elongated, multinucleate cells without a division septum. Here we present evidence that the requirement of Cdc15p for actomyosin ring formation is dependent on the stage of mitosis. Although cdc15 mutants are competent to assemble actomyosin rings in metaphase, they are unable to maintain actomyosin rings late in mitosis when SIN is active. In the absence of functional Cdc15p, ring formation upon metaphase arrest depends on the anillin-like Mid1p. Interestingly, when cytokinesis is delayed due to perturbations to the division machinery, Cdc15p is maintained in a hypophosphorylated form. The dephosphorylation of Cdc15p, which occurs transiently in unperturbed cytokinesis, is partially dependent on the phosphatase Clp1p/Flp1p. This suggests a mechanism where both SIN and Clp1p/Flp1p contribute to maintenance of the actomyosin ring in late mitosis through Cdc15p, possibly by regulating its phosphorylation status.     

Iqg1p links spatial and secretion landmarks to polarity and cytokinesis

Cytokinesis requires the polarization of the actin cytoskeleton, the secretion machinery, and the correct positioning of the division axis. Budding yeast cells commit to their cytokinesis plane by choosing a bud site and polarizing their growth. Iqg1p (Cyk1p) was previously implicated in cytokinesis (Epp and Chant, 1997; Lippincott and Li, 1998; Osman and Cerione, 1998), as well as in the establishment of polarity and protein trafficking (Osman and Cerione, 1998). To better understand how Iqg1p influences these processes, we performed a two-hybrid screen and identified the spatial landmark Bud4p as a binding partner. Iqg1p can be coimmunoprecipitated with Bud4p, and Bud4p requires Iqg1p for its proper localization. Iqg1p also appears to specify axial bud-site selection and mediates the proper localization of the septin, Cdc12p, as well as binds and helps localize the secretion landmark, Sec3p. The double mutants iqg1Deltasec3Delta and bud4Deltasec3Delta display defects in polarity, budding pattern and cytokinesis, and electron microscopic studies reveal that these cells have aberrant septal deposition. Taken together, these findings suggest that Iqg1p recruits landmark proteins to form a targeting patch that coordinates axial budding with cytokinesis.     

Ingression Progression Complexes Control Extracellular Matrix Remodelling during Cytokinesis in Budding Yeast

Eukaryotic cells must coordinate contraction of the actomyosin ring at the division site together with ingression of the plasma membrane and remodelling of the extracellular matrix (ECM) to support cytokinesis, but the underlying mechanisms are still poorly understood. In eukaryotes, glycosyltransferases that synthesise ECM polysaccharides are emerging as key factors during cytokinesis. The budding yeast chitin synthase Chs2 makes the primary septum, a special layer of the ECM, which is an essential process during cell division. Here we isolated a group of actomyosin ring components that form complexes together with Chs2 at the cleavage site at the end of the cell cycle, which we named ‘ingression progression complexes’ (IPCs). In addition to type II myosin, the IQGAP protein Iqg1 and Chs2, IPCs contain the F-BAR protein Hof1, and the cytokinesis regulators Inn1 and Cyk3. We describe the molecular mechanism by which chitin synthase is activated by direct association of the C2 domain of Inn1, and the transglutaminase-like domain of Cyk3, with the catalytic domain of Chs2. We used an experimental system to find a previously unanticipated role for the C-terminus of Inn1 in preventing the untimely activation of Chs2 at the cleavage site until Cyk3 releases the block on Chs2 activity during late mitosis. These findings support a model for the co-ordinated regulation of cell division in budding yeast, in which IPCs play a central role.     

Saccharomyces cerevisiae Dma proteins participate in cytokinesis by controlling two different pathways

Cytokinesis completion in the budding yeast S. cerevisiae is driven by tightly regulated pathways, leading to actomyosin ring contraction coupled to plasma membrane constriction and to centripetal growth of the primary septum, respectively. These pathways can partially substitute for each other, but their concomitant inactivation leads to cytokinesis block and cell death. Here we show that both the lack of the functionally redundant FHA-RING ubiquitin ligases Dma1 and Dma2 and moderate Dma2 overproduction affect actomyosin ring contraction as well as primary septum deposition, although they do not apparently alter cell cycle progression of otherwise wild-type cells. In addition, overproduction of Dma2 impairs the interaction between Tem1 and Iqg1, which is thought to be required for AMR contraction, and causes asymmetric primary septum deposition as well as mislocalization of the Cyk3-positive regulator of this process. In agreement with these multiple inhibitory effects, a Dma2 excess that does not cause any apparent defect in wild-type cells leads to lethal cytokinesis block in cells lacking the Hof1 protein, which is essential for primary septum formation in the absence of Cyk3. Altogether, these findings suggest that the Dma proteins act as negative regulators of cytokinesis.     

Cyk3 acts in actomyosin ring independent cytokinesis by recruiting Inn1 to the yeast bud neck

Cytokinesis in yeast can be achieved by plasma membrane ingression, which is dependent on actomyosin ring constriction. Inn1 presumably couples these processes by interaction with both the plasma membrane and the temporary actomyosin ring component Hof1. In addition, an actomyosin ring independent cytokinesis pathway exists in yeast. We here identified Cyk3, a key component of the alternative pathway, as a novel interaction partner of Inn1. The carboxy-terminal proline rich part of Inn1 binds the SH3 domains of either Cyk3 or Hof1. Strains with truncated proteins lacking either of these SH3 domains do not display any severe phenotypes, but are synthetically lethal, demonstrating their crucial role in cytokinesis. Overexpression of CYK3 leads to an actomyosin ring independent recruitment of Inn1 to the bud neck, further supporting the significance of this interaction in vivo. Moreover, overexpression of CYK3 in a myo1 or an iqg1 deletion not only restores viability, but also the recruitment of Inn1 to the bud neck. We propose that Cyk3 is part of an actomyosin ring independent cytokinesis pathway, which acts as a rescue mechanism to recruit Inn1 to the bud neck.     

IQGAP and mitotic exit network (MEN) proteins are required for cytokinesis and re-polarization of the actin cytoskeleton in the budding yeast, Saccharomyces cerevisiae

In budding yeast the final stages of the cell division cycle, cytokinesis and cell separation, are distinct events that require to be coupled, both together and with mitotic exit. Here we demonstrate that mutations in genes of the mitotic exit network (MEN) prevent cell separation and are synthetically lethal in combination with both cytokinesis and septation defective mutations. Analysis of the synthetic lethal phenotypes reveals that Iqg1p functions in combination with the MEN components, Tem1p, Cdc15p Dbf20p and Dbf2p to govern the re-polarization of the actin cytoskeleton to either side of the bud neck. In addition phosphorylation of the conserved PCH protein, Hof1p, is dependent upon these activities and requires actin ring assembly. Recruitment of Dbf2p to the bud neck is dependent upon actin ring assembly and correlates with Hof1p phosphorylation. Failure to phosphorylate Hof1p results in the increased stability of the protein and its persistence at the bud neck. These data establish a mechanistic dependency of cell separation upon an intermediate step requiring actomyosin ring assembly.     

The IQGAP Iqg1 is a regulatory target of CDK for cytokinesis in Candida albicans

Cyclin-dependent kinases (CDKs) drive and coordinate multiple cell-cycle events, including construction and contraction of the actomyosin ring during cytokinesis. However, it remains unclear whether CDKs regulate cytokinesis by directly targeting components of the ring. In a search for proteins containing consensus CDK phosphorylation sites in Candida albicans, we found that the IQGAP Iqg1 contains two dense clusters of 19 such sites flanking the actin-interacting CH domain. Here, we show that Iqg1 is indeed a phosphoprotein that undergoes cell-cycle-dependent phosphorylation and can be phosphorylated by purified Clb-Cdc28 kinases in vitro. Mass spectrometry identified several phosphoserine and phosphothreonine residues among these CDK sites. Mutating 15 of the CDK phosphorylation sites with alanine markedly reduced Iqg1 phosphorylation in vivo. The 15A mutation greatly stabilized Iqg1, caused both premature assembly and delayed disassembly of the actomyosin ring, blocked Iqg1 interaction with the actin-nucleating proteins Bni1 and Bnr1, and resulted in defects in cytokinesis. Our data therefore strongly support the idea that the Cdc28 CDK regulates cytokinesis partly by directly phosphorylating the actomyosin ring component Iqg1.     

Functions of Vrp1p in cytokinesis and actin patches are distinct and neither requires a WH2/V domain.

Vrp1 (verprolin, End5) is a Saccharomyces cerevisiae actin-associated protein and is related to mammalian Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP). Vrp1-deficient (vrp1 Delta) cells are inviable at high temperature, have partially depolarized cortical actin patches and have defects in both actomyosin ring-dependent and Hof1 (Cyk2)-dependent pathways of cytokinesis. We demonstrate here that N-Vrp1(1-364) and C-Vrp1(364-817) are each sufficient to restore viability, actomyosin ring constriction and Hof1 localization at 37 degrees C to vrp1 Delta. C-Vrp1, like Vrp1, partially co-localizes with cortical actin patches and restores actin patch polarization to vrp1 Delta. Cortical localization of C-Vrp1, but not Vrp1, requires Las17. N-Vrp1 exhibits diffuse cytoplasmic localization and functions in cytokinesis without efficiently restoring polarization of cortical actin patches. N-Vrp1 function is not abolished by mutations affecting the WASP homology 2 (WH2) [verprolin homology (V)] actin-binding domain. N-Vrp1 may function through the type I myosins and actin, while C-Vrp1 may function through both Las17 (Bee1) and type I myosins. The functions of Vrp1 in viability at 37 degrees C and cytokinesis do not require efficient localization to, and function in, the cortical actin cytoskeleton.     

Distinct roles of Rho1, Cdc42, and Cyk3 in septum formation and abscission during yeast cytokinesis

In yeast and animal cytokinesis, the small guanosine triphosphatase (GTPase) Rho1/RhoA has an established role in formation of the contractile actomyosin ring, but its role, if any, during cleavage-furrow ingression and abscission is poorly understood. Through genetic screens in yeast, we found that either activation of Rho1 or inactivation of another small GTPase, Cdc42, promoted secondary septum (SS) formation, which appeared to be responsible for abscission. Consistent with this hypothesis, a dominant-negative Rho1 inhibited SS formation but not cleavage-furrow ingression or the concomitant actomyosin ring constriction. Moreover, Rho1 is temporarily inactivated during cleavage-furrow ingression; this inactivation requires the protein Cyk3, which binds Rho1-guanosine diphosphate via its catalytically inactive transglutaminase-like domain. Thus, unlike the active transglutaminases that activate RhoA, the multidomain protein Cyk3 appears to inhibit activation of Rho1 (and thus SS formation), while simultaneously promoting cleavage-furrow ingression through primary septum formation. This work suggests a general role for the catalytically inactive transglutaminases of fungi and animals, some of which have previously been implicated in cytokinesis.