流感病毒在脂筏上组装与出芽

流感病毒造成的季节性流行性疾病给全世界带来沉重的健康负担.近年来,甲型流感病毒的变种H5N1、H7N9给各国带来了很大危害.流感病毒属于正黏附病毒科,它的遗传物质由多个节段的负链RNA组成,其组装和出芽剪切生殖是一个涉及到多种病毒因子,多步骤、复杂的生化过程.流感病毒会使用宿主的细胞膜上的"脂筏"区域作为病毒出芽位点.首先病毒的两种糖蛋白NA蛋白、HA蛋白会在脂筏区域聚集,造成脂筏区膜变形弯曲,并且发动出芽的过程.接着,流感病毒基质蛋白M1的C端与HA、NA结合,其自身在脂筏区域开始多聚化并使膜向外弯曲形成原始病毒体的内部结构,接着招募病毒的核糖核蛋白复合物(VRNP)与M2蛋白,使组装的过程进一步完成.最后,M2蛋白会富集在原始病毒体的底部,完成膜的剪切和病毒体的释放.     

Analysis of Assembly and Budding of Lujo Virus

The recently identified arenavirus Lujo virus (LUJV) causes fatal hemorrhagic fever in humans. We analyzed its mechanism of viral release driven by matrix protein Z and the cell surface glycoprotein precursor GPC. The L domains in Z are required for efficient virus-like particle release, but Tsg101, ALIX/AIP1, and Vps4A/B are unnecessary for budding. LUJV GPC is cleaved by site 1 protease (S1P) at the RKLM motif, and treatment with the S1P inhibitor PF-429242 reduced LUJV production.     

Insertion of Capsid Proteins from Nonenveloped Viruses into the Retroviral Budding Pathway

Retroviral Gag proteins direct the assembly and release of virus particles from the plasma membrane. The budding machinery consists of three small domains, the M (membrane-binding), I (interaction), and L (late or "pinching-off") domains. In addition, Gag proteins contain sequences that control particle size. For Rous sarcoma virus (RSV), the size determinant maps to the capsid (CA)-spacer peptide (SP) sequence, but it functions only when I domains are present to enable particles of normal density to be produced. Small deletions throughout the CA-SP sequence result in the release of particles that are very large and heterogeneous, even when I domains are present. In this report, we show that particles of relatively uniform size and normal density are released by budding when the size determinant and I domains in RSV Gag are replaced with capsid proteins from two unrelated, nonenveloped viruses: simian virus 40 and satellite tobacco mosaic virus. These results indicate that capsid proteins of nonenveloped viruses can interact among themselves within the context of Gag and be inserted into the retroviral budding pathway merely by attaching the M and L domains to their amino termini. Thus, the differences in the assembly pathways of enveloped and nonenveloped viruses may be far simpler than previously thought.     

Virus Entry, Assembly, Budding, and Membrane Rafts

As intracellular parasites, viruses rely heavily on the use of numerous cellular machineries for completion of their replication cycle. The recent discovery of the heterogeneous distribution of the various lipids within cell membranes has led to the proposal that sphingolipids and cholesterol tend to segregate in microdomains called membrane rafts. The involvement of membrane rafts in biosynthetic traffic, signal transduction, and endocytosis has suggested that viruses may also take advantage of rafts for completion of some steps of their replication cycle, such as entry into their cell host, assembly, and budding. In this review, we have attempted to delineate all the reliable data sustaining this hypothesis and to build some models of how rafts are used as platforms for assembly of some viruses. Indeed, if in many cases a formal proof of raft involvement in a virus replication cycle is still lacking, one can reasonably suggest that, owing to their ability to specifically attract some proteins, lipid microdomains provide a particular milieu suitable for increasing the efficiency of many protein-protein interactions which are crucial for virus infection and growth.     

Conditions for Copackaging Rous Sarcoma Virus and Murine Leukemia Virus Gag Proteins during Retroviral Budding

Rous sarcoma virus (RSV) and murine leukemia virus (MLV) are examples of distantly related retroviruses that normally do not encounter one another in nature. Their Gag proteins direct particle assembly at the plasma membrane but possess very little sequence similarity. As expected, coexpression of these two Gag proteins did not result in particles that contain both. However, when the N-terminal membrane-binding domain of each molecule was replaced with that of the Src oncoprotein, which is also targeted to the cytoplasmic face of the plasma membrane, efficient copackaging was observed in genetic complementation and coimmunoprecipitation assays. We hypothesize that the RSV and MLV Gag proteins normally use distinct locations on the plasma membrane for particle assembly but otherwise have assembly domains that are sufficiently similar in function (but not sequence) to allow heterologous interactions when these proteins are redirected to a common membrane location.     

The Ecm11-Gmc2 Complex Promotes Synaptonemal Complex Formation through Assembly of Transverse Filaments in Budding Yeast

During meiosis, homologous chromosomes pair at close proximity to form the synaptonemal complex (SC). This association is mediated by transverse filament proteins that hold the axes of homologous chromosomes together along their entire length. Transverse filament proteins are highly aggregative and can form an aberrant aggregate called the polycomplex that is unassociated with chromosomes. Here, we show that the Ecm11-Gmc2 complex is a novel SC component, functioning to facilitate assembly of the yeast transverse filament protein, Zip1. Ecm11 and Gmc2 initially localize to the synapsis initiation sites, then throughout the synapsed regions of paired homologous chromosomes. The absence of either Ecm11 or Gmc2 substantially compromises the chromosomal assembly of Zip1 as well as polycomplex formation, indicating that the complex is required for extensive Zip1 polymerization. We also show that Ecm11 is SUMOylated in a Gmc2-dependent manner. Remarkably, in the unSUMOylatable ecm11 mutant, assembly of chromosomal Zip1 remained compromised while polycomplex formation became frequent. We propose that the Ecm11-Gmc2 complex facilitates the assembly of Zip1 and that SUMOylation of Ecm11 is critical for ensuring chromosomal assembly of Zip1, thus suppressing polycomplex formation.     

Simulations Show that Virus Assembly and Budding Are Facilitated by Membrane Microdomains

For many viruses, assembly and budding occur simultaneously during virion formation. Understanding the mechanisms underlying this process could promote biomedical efforts to block viral propagation and enable use of capsids in nanomaterials applications. To this end, we have performed molecular dynamics simulations on a coarse-grained model that describes virus assembly on a fluctuating lipid membrane. Our simulations show that the membrane can promote association of adsorbed subunits through dimensional reduction, but it also introduces thermodynamic and kinetic effects that can inhibit complete assembly. We find several mechanisms by which membrane microdomains, such as lipid rafts, reduce these effects, and thus, enhance assembly. We show how these predicted mechanisms can be experimentally tested. Furthermore, the simulations demonstrate that assembly and budding depend crucially on the system dynamics via multiple timescales related to membrane deformation, protein diffusion, association, and adsorption onto the membrane.     

Initial Steps in RNA Processing and Ribosome Assembly Occur at Mitochondrial DNA Nucleoids

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In Vitro Reconstitution of Cortical Actin Assembly Sites in Budding Yeast

We have developed a biochemical approach for identifying the components of cortical actin assembly sites in polarized yeast cells, based on a permeabilized cell assay that we established for actin assembly in vitro. Previous analysis indicated that an activity associated with the cell cortex promotes actin polymerization in the bud. After inactivation by a chemical treatment, this activity can be reconstituted back to the permeabilized cells from a cytoplasmic extract. Fractionation of the extract revealed that the reconstitution depends on two sequentially acting protein factors. Bee1, a cortical actin cytoskeletal protein with sequence homology to Wiskott-Aldrich syndrome protein, is required for the first step of the reconstitution. This finding, together with the severe defects in actin organization associated with the bee1 null mutation, indicates that Bee1 protein plays a direct role in controlling actin polymerization at the cell cortex. The factor that acts in the second step of the reconstitution has been identified by conventional chromatography. It is composed of a novel protein, Pca1. Sequence analysis suggests that Pca1 has the potential to interact with SH3 domain-containing proteins and phospholipids.     

TSG101在病毒出芽过程中的作用及其机制

TSG101基因是新发现的抑癌基因候选者,定位于人类 11号染色体 p1511-p1512,其编码产物TSG101蛋白N端区域与泛素结合酶(UBC)同源。近年来研究发现,TSG101基因具有多种重要的功能,与多种病毒出芽密切相关,所以TSG101可作为一个新的抗病毒靶点。本文主要从TSG101在多种病毒（HIV、IAV、MARV、ASV等）出芽过程中扮演的角色,TSG101与多种蛋白(泛素、Nedd4、ARMMs、Tom1、Gag、VP40、NP等)的相互作用进而辅助病毒出芽的机制,以及TSG101抑制剂的研究等方面进行阐述。     

Cis‐Golgi Cisternal Assembly and Biosynthetic Activation Occur Sequentially in Plants and Algae

The cisternal progression/maturation model of Golgi trafficking predicts that cis‐Golgi cisternae are formed de novo on the cis‐side of the Golgi. Here we describe structural and functional intermediates of the cis cisterna assembly process in high‐pressure frozen algae (Scherffelia dubia, Chlamydomonas reinhardtii) and plants (Arabidopsis thaliana, Dionaea muscipula; Venus flytrap) as determined by electron microscopy, electron tomography and immuno‐electron microscopy techniques. Our findings are as follows: (i) The cis‐most (C1) Golgi cisternae are generated de novo from cisterna initiators produced by the fusion of 3–5 COPII vesicles in contact with a C2 cis cisterna. (ii) COPII vesicles fuel the growth of the initiators, which then merge into a coherent C1 cisterna. (iii) When a C1 cisterna nucleates its first cisterna initiator it becomes a C2 cisterna. (iv) C2‐Cn cis cisternae grow through COPII vesicle fusion. (v) ER‐resident proteins are recycled from cis cisternae to the ER via COPIa‐type vesicles. (vi) In S. dubia the C2 cisternae are capable of mediating the self‐assembly of scale protein complexes. (vii) In plants, ～90% of native α‐mannosidase I localizes to medial Golgi cisternae. (viii) Biochemical activation of cis cisternae appears to coincide with their conversion to medial cisternae via recycling of medial cisterna enzymes. We propose how the different cis cisterna assembly intermediates of plants and algae may actually be related to those present in the ERGIC and in the pre‐cis Golgi cisterna layer in mammalian cells.     

Retroviral Capsid Assembly: A Role for the CA Dimer in Initiation

In maturing retroviral virions, CA protein assembles to form a capsid shell that is essential for infectivity. The structure of the two folded domains [N-terminal domain (NTD) and C-terminal domain (CTD)] of CA is highly conserved among various retroviruses, and the capsid assembly pathway, although poorly understood, is thought to be conserved as well. In vitro assembly reactions with purified CA proteins of the Rous sarcoma virus (RSV) were used to define factors that influence the kinetics of capsid assembly and provide insights into underlying mechanisms. CA multimerization was triggered by multivalent anions providing evidence that in vitro assembly is an electrostatically controlled process. In the case of RSV, in vitro assembly was a well-behaved nucleation-driven process that led to the formation of structures with morphologies similar to those found in virions. Isolated RSV dimers, when mixed with monomeric protein, acted as efficient seeds for assembly, eliminating the lag phase characteristic of a monomer-only reaction. This demonstrates for the first time the purification of an intermediate on the assembly pathway. Differences in the intrinsic tryptophan fluorescence of monomeric protein and the assembly-competent dimer fraction suggest the involvement of the NTD in the formation of the functional dimer. Furthermore, in vitro analysis of well-characterized CTD mutants provides evidence for assembly dependence on the second domain and suggests that the establishment of an NTD-CTD interface is a critical step in capsid assembly initiation. Overall, the data provide clear support for a model whereby capsid assembly within the maturing virion is dependent on the formation of a specific nucleating complex that involves a CA dimer and is directed by additional virion constituents.     

Wave Transmission through an Assembly of Randomly Branching Elastic Tubes

Calculations are presented of the transmission of oscillations through an assembly of randomly branching elastic tubes, as a model of not only the major arteries, but also a peripheral vascular bed. It appears that the viscosity of the arterial wall must be the major source of attenuation in the larger arteries, while the viscosity of the blood plays a significant role only in the smaller vessels. In all situations, variations of cross-sectional area have a considerable effect on wave transmission, causing a general decrease in amplitude and an accentuation of reflection from the terminations. The effects of variation in cross-sectional area are sufficiently great to indicate that they should be included in future models of the arterial system. Finally, it is argued that because of the presence of random branching and elastic nonuniformity, the determination of the reflection coefficient for a system such as the arterial tree may be quite misleading.     

Processing Body and Stress Granule Assembly Occur by Independent and Differentially Regulated Pathways in Saccharomyces cerevisiae

A variety of ribonucleoprotein (RNP) granules form in eukaryotic cells to regulate the translation, decay, and localization of the encapsulated messenger RNA (mRNAs). The work here examined the assembly and function of two highly conserved RNP structures, the processing body (P body) and the stress granule, in the yeast Saccharomyces cerevisiae. These granules are induced by similar stress conditions and contain translationally repressed mRNAs and a partially overlapping set of protein constituents. However, despite these similarities, the data indicate that these RNP complexes are independently assembled and that this assembly is controlled by different signaling pathways. In particular, the cAMP-dependent protein kinase (PKA) was found to control P body formation under all conditions examined. In contrast, the assembly of stress granules was not affected by changes in either PKA or TORC1 signalling activity. Both of these RNP granules were also detected in stationary-phase cells, but each appears at a distinct time. P bodies were formed prior to stationary-phase arrest, and the data suggest that these foci are important for the long-term survival of these quiescent cells. Stress granules, on the other hand, were not assembled until after the cells had entered into the stationary phase of growth and their appearance could therefore serve as a specific marker for the entry into this quiescent state. In all, the results here provide a framework for understanding the assembly of these RNP complexes and suggest that these structures have distinct but important activities in quiescent cells.EUKARYOTIC cells contain a number of membrane-bound compartments that partition the cytoplasm into distinct functional units. Proteins that act in similar pathways are often localized to the same compartment whereas those with competing activities are sequestered within different environments. Interestingly, recent data suggest that particular proteins and RNAs are also concentrated in what can be thought of as nontraditional compartments that lack a boundary membrane. These ribonucleoprotein (RNP) complexes, or granules, are more dynamic in nature and are found in both the nucleus and the cytoplasm of the cell (Anderson and Kedersha 2006; Mao et al. 2011; Weber and Brangwynne 2012). The formation of these granules can be induced by a variety of cues, including an exposure to stress or specific developmental transitions. In some cases, the underlying reasons for this reorganization of protein and RNA are known. For example, the polar granules present in germ cells store maternal mRNAs that are translated following fertilization (Schisa et al. 2001; Leatherman and Jongens 2003). However, for most RNP granules, the physiological role of the larger aggregate-like structures remains unclear. Nonetheless, the prevalence and evolutionary conservation of these complexes suggests that they serve important functions in the cell.Two of the better-characterized cytoplasmic RNPs are the processing bodies (P bodies) and stress granules that form in response to a variety of stress conditions. These particles contain translationally repressed messenger RNA (mRNAs) and a partially overlapping set of protein constituents (Kedersha and Anderson 2002; Anderson and Kedersha 2009; Balagopal and Parker 2009). Since a number of factors important for protein translation are also found in stress granules, these structures have been suggested to be sites of mRNA storage (Yamasaki and Anderson 2008). In contrast, P bodies were originally identified as cytoplasmic foci containing proteins important for mRNA decay (Sheth and Parker 2003; Eulalio et al. 2007a). Although this initially led to speculation that these foci were sites of mRNA turnover, more recent studies have found that this decay proceeds normally in cells lacking the larger P body complexes (Stoecklin et al. 2006; Decker et al. 2007; Eulalio et al. 2007b). As a result, the biological functions associated with P body foci remain unclear. However, an intriguing possibility has been suggested by studies demonstrating that P bodies contain proteins that do not appear to have a direct role in mRNA decay. These proteins include the phosphatase, calcineurin, and the catalytic subunits of the cAMP-dependent protein kinase (PKA) (Tudisca et al. 2010; Kozubowski et al. 2011). P bodies may therefore carry out specific functions that are dictated by the particular proteins present within these cytoplasmic structures. These functions may vary depending upon the particular cell type and stress condition used to induce the foci.A significant body of work has linked the induction of both P bodies and stress granules to the inhibition of protein synthesis, but less is known about the mechanisms regulating the subsequent formation of the larger aggregate-like assemblies (Franks and Lykke-Andersen 2008). These latter structures appear to form by a self-assembly process that involves the prion-like domains present in a number of granule proteins (Gilks et al. 2004; Decker et al. 2007; Reijns et al. 2008). Some insight into the regulation of this latter process was provided by a recent study of the P bodies that form in response to glucose deprivation in Saccharomyces cerevisiae (Ramachandran et al. 2011). This work showed that the inactivation of the PKA signaling pathway was both a necessary and a sufficient condition for P body foci formation. PKA directly phosphorylates Pat1, a conserved core constituent of these RNP structures, and thereby disrupts Pat1 interactions with a number of P body components, including the RNA helicase Dhh1 (Ramachandran et al. 2011). In contrast, defects in other nutrient-sensing pathways, including those involving the TORC1 or Snf1 protein kinases, did not have a significant effect upon P body formation. This work also suggested that P body foci were important for the long-term survival of cells that had entered into the stationary phase of growth. In particular, mutants that were defective for foci formation lost viability more rapidly during this period of quiescence (Ramachandran et al. 2011). This latter result is of interest in light of other work indicating that as much as 20% of the yeast proteome might relocalize to cytoplasmic foci when cells enter into this G₀-like resting state (Narayanaswamy et al. 2009; Noree et al. 2010). Since this phenomenon might not be restricted to yeast (An et al. 2008; Noree et al. 2010), the concentration of material at discrete sites in the cytoplasm may be generally important for the biology of the quiescent cell. Determining the underlying reasons for this relocalization of protein will therefore be critical for a complete understanding of the physiology of the eukaryotic cell.In S. cerevisiae, PKA is an essential component of one of the key signaling pathways responsible for coordinating cell growth with nutrient availability (Bahn et al. 2007; Zaman et al. 2008). This pathway also involves the GTP-binding Ras proteins Ras1 and Ras2 and is thought to respond, either directly or indirectly, to the levels of glucose present within cells (Santangelo 2006; Slattery et al. 2008; Zaman et al. 2009). The active, GTP-bound forms of the Ras proteins interact with the adenylyl cyclase Cyr1 and stimulate the production of cAMP (Field et al. 1990; Suzuki et al. 1990). This cyclic nucleotide is then bound by Bcy1, the regulatory subunit of the PKA enzyme, leading to the subsequent release of the active catalytic subunits; the basal state of PKA is an inactive heterotetramer made up of two catalytic and two regulatory subunits (Uno et al. 1982; Toda et al. 1987a; Taylor et al. 2008). These catalytic subunits are then free to phosphorylate their respective targets and thereby influence cell growth (Budovskaya et al. 2005). The existing genetic data suggest that this Ras/PKA pathway might play an important role in regulating the entry into stationary phase. For example, mutants that inactivate this pathway result in a growth arrest that resembles stationary phase (Iida and Yahara 1984; Schneper et al. 2004). Conversely, cells with constitutively elevated levels of PKA activity fail to arrest normally in stationary phase when nutrients are limiting (Broek et al. 1985; Broach 1991). The above results with Pat1 suggest that the PKA-mediated control of P body formation is one important component of this regulation of stationary-phase biology.In this study, we examined the regulation of P body and stress granule assembly in response to a variety of environmental cues, including several that can induce both of these RNP foci. This work demonstrated that the PKA pathway has a general role in the regulation of P body foci formation as mutants with constitutive PKA signaling were defective for P body assembly in all conditions tested. In contrast, stress granule formation was not influenced by changes in either PKA or TORC1 signalling activity. The results here also demonstrate that both types of RNP foci are present in stationary-phase cells and provide further support for a role for P bodies in the long-term survival of these quiescent cells. Finally, we show that P bodies and stress granules form at different times during batch culture growth and that stress granules in particular appear only after cells enter into stationary phase. Therefore, stress granule formation could serve as a useful marker for cell entry into this quiescent state. In all, this work indicates that P bodies and stress granules form independently of one another and that each assembly pathway is regulated by distinct signaling mechanisms.     

ATP-dependent Pre-replicative Complex Assembly Is Facilitated by Adk1p in Budding Yeast

Pre-replicative complex (pre-RC) assembly is a critical part of the mechanism that controls the initiation of DNA replication, and ATP binding and hydrolysis by multiple pre-RC proteins are essential for pre-RC assembly and activation. Here, we demonstrate that Adk1p (adenylate kinase 1 protein) plays an important role in pre-RC assembly in Saccharomyces cerevisiae. Isolated from a genetic screen, adk1^G20S cells with a mutation within the nucleotide-binding site were defective in replication initiation. adk1Δ cells were viable at 25 °C but not at 37°C. Flow cytometry indicated that both the adk1-td (temperature-inducible degron) and adk1^G20S mutants were defective in S phase entry. Furthermore, Adk1p bound to chromatin throughout the cell cycle and physically interacted with Orc3p, whereas the Adk1^G20S protein had a reduced ability to bind chromatin and Orc3p without affecting the cellular ATP level. In addition, Adk1p associated with replication origins by ChIP assay. Finally, Adk1-td protein depletion prevented pre-RC assembly during the M-to-G₁ transition. We suggest that Adk1p regulates ATP metabolism on pre-RC proteins to promote pre-RC assembly and activation.     

Structural Evidence of Glycoprotein Assembly in Cellular Membrane Compartments prior to Alphavirus Budding

Membrane glycoproteins of alphavirus play a critical role in the assembly and budding of progeny virions. However, knowledge regarding transport of viral glycoproteins to the plasma membrane is obscure. In this study, we investigated the role of cytopathic vacuole type II (CPV-II) through in situ electron tomography of alphavirus-infected cells. The results revealed that CPV-II contains viral glycoproteins arranged in helical tubular arrays resembling the basic organization of glycoprotein trimers on the envelope of the mature virions. The location of CPV-II adjacent to the site of viral budding suggests a model for the transport of structural components to the site of budding. Thus, the structural characteristics of CPV-II can be used in evaluating the design of a packaging cell line for replicon production.Semliki Forest virus (SFV) is an enveloped alphavirus belonging to the family Togaviridae. This T=4 icosahedral virus particle is approximately 70 nm in diameter (30) and consists of 240 copies of E1/E2 glycoprotein dimers (3, 8, 24). The glycoproteins are anchored in a host-derived lipid envelope that encloses a nucleocapsid, made of a matching number of capsid proteins and a positive single-stranded RNA molecule. After entry of the virus via receptor-mediated endocytosis, a low-pH-induced fusion of the viral envelope with the endosomal membrane delivers the nucleocapsid into the cytoplasm, where the replication events of SFV occur (8, 19, 30). Replication of the viral genome and subsequent translation into structural and nonstructural proteins followed by assembly of the structural proteins and genome (7) lead to budding of progeny virions at the plasma membrane (18, 20). The synthesis of viral proteins shuts off host cell macromolecule synthesis, which allows for efficient intracellular replication of progeny virus (7). The expression of viral proteins leads to the formation of cytopathic vacuolar compartments as the result of the reorganization of cellular membrane in the cytoplasm of an infected cell (1, 7, 14).Early studies using electron microscopy (EM) have characterized the cytopathic vacuoles (CPVs) in SFV-infected cells (6, 13, 14) and identified two types of CPV, namely, CPV type I (CPV-I) and CPV-II. It was found that CPV-I is derived from modified endosomes and lysosomes (18), while CPV-II is derived from the trans-Golgi network (TGN) (10, 11). Significantly, the TGN and CPV-II vesicles are the major membrane compartments marked with E1/E2 glycoproteins (9, 11, 12). Inhibition by monensin results in the accumulation of E1/E2 glycoproteins in the TGN (12, 26), thereby indicating the origin of CPV-II. While CPV-II is identified as the predominant vacuolar structure at the late stage of SFV infection, the exact function of this particular cytopathic vacuole is less well characterized than that of CPV-I (2, 18), although previous observations have pointed to the involvement of CPV-II in budding, because an associated loss of viral budding was observed when CPV-II was absent (9, 36).In this study, we characterized the structure and composition of CPV-II in SFV-infected cells in situ with the aid of electron tomography and immuno-electron microscopy after physical fixation of SFV-infected cells by high-pressure freezing and freeze substitution (21, 22, 33). The results revealed a helical array of E1/E2 glycoproteins within CPV-II and indicate that CPV-II plays an important role in intracellular transport of glycoproteins prior to SFV budding.     

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.