Presumptive Bud Site Research Articles

Septin Assembly and Remodeling at the Cell Division Site During the Cell Cycle.

The septin family of proteins can assemble into filaments that further organize into different higher order structures to perform a variety of different functions in different cell types and organisms. In the budding yeast Saccharomyces cerevisiae, the septins localize to the presumptive bud site as a cortical ring prior to bud emergence, expand into an hourglass at the bud neck (cell division site) during bud growth, and finally “split” into a double ring sandwiching the cell division machinery during cytokinesis. While much work has been done to understand the functions and molecular makeups of these structures, the mechanisms underlying the transitions from one structure to another have largely remained elusive. Recent studies involving advanced imaging and in vitro reconstitution have begun to reveal the vast complexity involved in the regulation of these structural transitions, which defines the focus of discussion in this mini-review.

Temporal regulation of morphogenetic events in Saccharomyces cerevisiae.

Tip growth in fungi involves highly polarized secretion and modification of the cell wall at the growing tip. The genetic requirements for initiating polarized growth are perhaps best understood for the model budding yeast Saccharomyces cerevisiae. Once the cell is committed to enter the cell cycle by activation of G1 cyclin/cyclin-dependent kinase (CDK) complexes, the polarity regulator Cdc42 becomes concentrated at the presumptive bud site, actin cables are oriented toward that site, and septin filaments assemble into a ring around the polarity site. Several minutes later, the bud emerges. Here, we investigated the mechanisms that regulate the timing of these events at the single-cell level. Septin recruitment was delayed relative to polarity establishment, and our findings suggest that a CDK-dependent septin “priming” facilitates septin recruitment by Cdc42. Bud emergence was delayed relative to the initiation of polarized secretion, and our findings suggest that the delay reflects the time needed to weaken the cell wall sufficiently for the cell to bud. Rho1 activation by Rom2 occurred at around the time of bud emergence, perhaps in response to local cell-wall weakening. This report reveals regulatory mechanisms underlying the morphogenetic events in the budding yeast.

Sensing a bud in the yeast morphogenesis checkpoint: a role for Elm1.

Bud formation by Saccharomyces cerevisiae must be coordinated with the nuclear cycle to enable successful proliferation. Many environmental stresses temporarily disrupt bud formation, and in such circumstances, the morphogenesis checkpoint halts nuclear division until bud formation can resume. Bud emergence is essential for degradation of the mitotic inhibitor, Swe1. Swe1 is localized to the septin cytoskeleton at the bud neck by the Swe1-binding protein Hsl7. Neck localization of Swe1 is required for Swe1 degradation. Although septins form a ring at the presumptive bud site before bud emergence, Hsl7 is not recruited to the septins until after bud emergence, suggesting that septins and/or Hsl7 respond to a "bud sensor." Here we show that recruitment of Hsl7 to the septin ring depends on a combination of two septin-binding kinases: Hsl1 and Elm1. We elucidate which domains of these kinases are needed and show that artificial targeting of those domains suffices to recruit Hsl7 to septin rings even in unbudded cells. Moreover, recruitment of Elm1 is responsive to bud emergence. Our findings suggest that Elm1 plays a key role in sensing bud emergence.

A novel role for the alcohol sensitive ring/PHD finger protein Asr1p in regulating cell cycle mediated by septin-dependent assembly in yeast

Septin is a conserved eukaryotic family of GTP-binding filament-forming proteins with functions in cytokinesis and other processes. It has been suggested that the dynamic assembly of septin, including the processes from septin initially localizing to the presumptive bud site to the septin collar finally splitting into two cells, coordinates closely with the checkpoint response of cell cycle. Here, we discovered that over-expression of Alcohol sensitive Ring/PHD finger 1 protein (Asr1p) in Saccharomyces cerevisiae triggered the Swe1p-dependent cell cycle checkpoint for a G2/M transition delay, and this G2/M transition delay was caused by the septin defect. Since it was shown that Asr1p affected actin dynamics through the interaction with Crn1p and crn1 should be epistatic to asr1 in the regulation of actin, the gene knockout of crn1 in the Asr1p over-expression strain restored the defects in septin and cell cycle along with the disordered actin dynamics. Our investigation further showed that the disturbed septin assembly caused by abnormal Asr1p lead to the abnormal localization of the checkpoint proteins such as Gla4/PAK and Cdc5/Polo, and finally triggered the Swe1p-dependent G2/M transition arrest. Additionally, the Ring finger/PHD domains of Asr1p were illustrated to be required but not sufficient for its role in septin. Taken together, our current data suggested a close relationship in the assembly between septin and actin cytoskeleton, which also partially explained how actin cytoskeleton participated in the regulation of the checkpoint of G2/M.

Does the External Environment of Cells, in Addition to their Genetic Programs, Play a Role in the Localization of their Division Sites ?

Oriented cell division and establishment of cell polarity are central to development of many organisms [Drubin & Nelson 96]. Yeast cells exhibit defined patterns of oriented cell divisions by choosing a specific bud site on their cell cortex. The current understanding is that the site for bud formation is determined genetically, depending on cell type [Ni & Snyder 01, Aronov et al 07]. In haploid cells, bud sites are adjacent to the previous bud neck site: This is the axial pattern. In diploid cells, mother cells select bud sites either adjacent to their previous daughter cells or on their opposite end, whereas daughter cells always choose a bud site directed away from their mother: This is the bipolar pattern. It is believed that cells respond to cortical cues that mark positions on the cell cortex to establish these cell type-specific budding patterns. Recruitment of proteins to the presumptive bud site is thought to direct the cytoskeleton and secretory apparatus toward the bud site, thereby restricting new growth to the bud.Might the external environment play an additional role in the localization of bud sites, such as confinement via some mechanical and/or chemical sensing? To address this question, we perform image analysis of budding yeast cells in a two-dimensional micro-fluidic chamber, allowing for a controlled growth environment over multiple cell cycles [Charvin et al 08].

Symmetry-Breaking Polarization Driven by a Cdc42p GEF-PAK Complex

In 1952, Alan Turing suggested that spatial patterns could arise from homogeneous starting conditions by feedback amplification of stochastic fluctuations. One example of such self-organization, called symmetry breaking, involves spontaneous cell polarization in the absence of spatial cues. The conserved GTPase Cdc42p is essential for both guided and spontaneous polarization, and in budding yeast cells Cdc42p concentrates at a single site (the presumptive bud site) at the cortex. Cdc42p concentrates at a random cortical site during symmetry breaking in a manner that requires the scaffold protein Bem1p. The mechanism whereby Bem1p promotes this polarization was unknown. Here we show that Bem1p promotes symmetry breaking by assembling a complex in which both a Cdc42p-directed guanine nucleotide exchange factor (GEF) and a Cdc42p effector p21-activated kinase (PAK) associate with Bem1p. Analysis of Bem1p mutants indicates that both GEF and PAK must bind to the same molecule of Bem1p, and a protein fusion linking the yeast GEF and PAK bypasses the need for Bem1p. Although mammalian cells lack a Bem1p ortholog, they contain more complex multidomain GEFs that in some cases can directly interact with PAKs, and we show that yeast containing an artificial GEF with similar architecture can break symmetry even without Bem1p. Yeast symmetry-breaking polarization involves a GEF-PAK complex that binds GTP-Cdc42p via the PAK and promotes local Cdc42p GTP-loading via the GEF. By generating fresh GTP-Cdc42p near pre-existing GTP-Cdc42p, the complex amplifies clusters of GTP-Cdc42p at the cortex. Our findings provide mechanistic insight into an evolutionarily conserved pattern-forming positive-feedback pathway.

Dynamics of Cdc42 network embodies a Turing-type mechanism of yeast cell polarity

Complex biochemical networks can be understood by identifying their principal regulatory motifs and mode of action. We model the early phase of budding yeast cellular polarization and show that the biochemical processes in the presumptive bud site comprise a Turing-type mechanism. The roles of the prototypical activator and substrate are played by GTPase Cdc42 in its active and inactive states, respectively. We demonstrate that the nucleotide cycling of Cdc42 converts cellular energy into a stable cluster of activated Cdc42. This energy drives a continuous membrane-cytoplasmic exchange of the cluster components to counteract diffusive spread of the cluster. This exchange explains why only one bud forms per cell cycle, because the winner-takes-all competition of candidate sites inevitably selects a single site.

Role of a Cdc42p effector pathway in recruitment of the yeast septins to the presumptive bud site.

The septins are GTP-binding, filament-forming proteins that are involved in cytokinesis and other processes. In the yeast Saccharomyces cerevisiae, the septins are recruited to the presumptive bud site at the cell cortex, where they form a ring through which the bud emerges. We report here that in wild-type cells, the septins typically become detectable in the vicinity of the bud site several minutes before ring formation, but the ring itself is the first distinct structure that forms. Septin recruitment depends on activated Cdc42p but not on the normal pathway for bud-site selection. Recruitment occurs in the absence of F-actin, but ring formation is delayed. Mutant phenotypes and suppression data suggest that the Cdc42p effectors Gic1p and Gic2p, previously implicated in polarization of the actin cytoskeleton, also function in septin recruitment. Two-hybrid, in vitro protein binding, and coimmunoprecipitation data indicate that this role involves a direct interaction of the Gic proteins with the septin Cdc12p.

Cell Polarity Protein Spa2P Associates With Proteins Involved In Actin Function InSaccharomyces Cerevisiae

Spa2p is a nonessential protein that regulates yeast cell polarity. It localizes early to the presumptive bud site and remains at sites of growth throughout the cell cycle. To understand how Spa2p localization is regulated and to gain insight into its molecular function in cell polarity, we used a coimmunoprecipitation strategy followed by tandem mass spectrometry analysis to identify proteins that associate with Spa2p in vivo. We identified Myo1p, Myo2p, Pan1p, and the protein encoded by YFR016c as proteins that interact with Spa2p. Strikingly, all of these proteins are involved in cell polarity and/or actin function. Here we focus on the functional significance of the interactions of Spa2p with Myo2p and Myo1p. We find that localization of Spa2GFP to sites of polarized growth depends on functional Myo2p but not on Myo1p. We also find that Spa2p, like Myo2p, cosediments with F-actin in an ATP-sensitive manner. We hypothesize that Spa2p associates with actin via a direct or indirect interaction with Myo2p and that Spa2p may be involved in mediating polarized localization of polarity proteins via Myo2p. In addition, we observe an enhanced cell-separation defect in a myo1spa2 strain at 37 degrees C. This provides further evidence that Spa2p is involved in cytokinesis and cell wall morphogenesis.

Role of a Cdc42p Effector Pathway in Recruitment of the Yeast Septins to the Presumptive Bud Site

Role of a Cdc42p Effector Pathway in Recruitment of the Yeast Septins to the Presumptive Bud Site

Local exposure of phosphatidylethanolamine on the yeast plasma membrane is implicated in cell polarity.

Cell surface phosphatidylethanolamine (PE) of the yeast cell was probed by biotinylated Ro09-0198 (Bio-Ro), which specifically binds to PE and was visualized with fluorescein-labelled streptavidin. In Saccharomyces cerevisiae, the signals were observed at the presumptive bud site, the emerging small bud cortex, the bud neck of the late mitotic large-budded cells and the tip of the mating projection. In Schizosaccharomyces pombe, the signals were observed at one end or both ends of mono-nucleated cells and the division plane of the late mitotic cells. These sites were polarized ends in the yeast cells, implying that PE is exposed on the cell surface at cellular polarized ends. Treatment of S. cerevisiae cells with Ro09-0198 resulted in aberrant F-actin accumulation at the above sites, implying that limited surface exposure of PE is involved in the polarized organization of the actin cytoskeleton. Furthermore, S. cerevisiae ros3, dnf1 and dnf2 null mutants, which were known to be defective in the internalization of fluorescence-labelled PE, as well as the combinatorial mutants, were stained with Bio-Ro at the enlarging bud cortex, in addition to the Bio-Ro-staining sites of wild-type cells, suggesting that Ros3p, Dnf1p and Dnf2p are involved in the retrieval of exposed PE at the bud cortex.

The role of Cdc42p GTPase-activating proteins in assembly of the septin ring in yeast.

The septins are a conserved family of GTP-binding, filament-forming proteins. In the yeast Saccharomyces cerevisiae, the septins form a ring at the mother-bud neck that appears to function primarily by serving as a scaffold for the recruitment of other proteins to the neck, where they participate in cytokinesis and a variety of other processes. Formation of the septin ring depends on the Rho-type GTPase Cdc42p but appears to be independent of the actin cytoskeleton. In this study, we investigated further the mechanisms of septin-ring formation. Fluorescence-recovery-after-photobleaching (FRAP) experiments indicated that the initial septin structure at the presumptive bud site is labile (exchanges subunits freely) but that it is converted into a stable ring as the bud emerges. Mutants carrying the cdc42V36G allele or lacking two or all three of the known Cdc42p GTPase-activating proteins (GAPs: Bem3p, Rga1p, and Rga2p) could recruit the septins to the cell cortex but were blocked or delayed in forming a normal septin ring and had accompanying morphogenetic defects. These phenotypes were dramatically enhanced in mutants that were also defective in Cla4p or Gin4p, two protein kinases previously shown to be important for normal septin-ring formation. The Cdc42p GAPs colocalized with the septins both early and late in the cell cycle, and overexpression of the GAPs could suppress the septin-organization and morphogenetic defects of temperature-sensitive septin mutants. Taken together, the data suggest that formation of the mature septin ring is a process that consists of at least two distinguishable steps, recruitment of the septin proteins to the presumptive bud site and their assembly into the stable septin ring. Both steps appear to depend on Cdc42p, whereas the Cdc42p GAPs and the other proteins known to promote normal septin-ring formation appear to function in a partially redundant manner in the assembly step. In addition, because the eventual formation of a normal septin ring in a cdc42V36G or GAP mutant was invariably accompanied by a switch from an abnormally elongated to a more normal bud morphology distal to the ring, it appears that the septin ring plays a direct role in determining the pattern of bud growth.

Bud morphogenesis and the actin and microtubule cytoskeletons during budding in the corn smut fungus, Ustilago maydis

Ustilago maydis is a dimorphic Basidiomycete fungus with a yeast-like form and a hyphal form. Here we present a comprehensive analysis of bud formation and the actin and microtubule cytoskeletons of the yeast-like form during the cell cycle. We show that bud morphogenesis entails a series of shape changes, initially a tubular or conical structure, culminating in a cigar-shaped cell connected to the mother cell by a narrow neck. Labelling of cells with concanavalin A demonstrated that growth occurs at bud tip. Indirect immunofluorescence studies revealed that the actin cytoskeleton consists of patches and cables that polarize to the presumptive bud site and the bud tip and an actin ring that forms at the neck region. Because the bud tip corresponds to the site of active cell wall growth, we hypothesize that actin is involved in secretion of cell wall components. The microtubule cytoskeleton has recently been shown to consist of a cytoplasmic network during interphase that disassembles at mitosis when a spindle and astral microtubules are formed. We have carried out studies of U. maydis cells synchronized by the microtubule-depolymerizing drug thiabendazole which allow us to construct a temporal sequence of steps in spindle formation and spindle elongation during the cell cycle. These studies suggest that astral microtubules may be involved in early stages of spindle orientation and migration of the nucleus into the bud and that the spindle pole bodies may be involved in reestablishment of the cytoplasmic microtubule network.

The role of cell cycle-regulated expression in the localization of spatial landmark proteins in yeast.

In Saccharomyces cerevisiae, Bud8p and Bud9p are homologous plasma membrane glycoproteins that appear to mark the distal and proximal cell poles, respectively, as potential sites for budding in the bipolar pattern. Here we provide evidence that Bud8p is delivered to the presumptive bud site (and thence to the distal pole of the bud) just before bud emergence, and that Bud9p is delivered to the bud side of the mother-bud neck (and thence to the proximal pole of the daughter cell) after activation of the mitotic exit network, just before cytokinesis. Like the delivery of Bud8p, that of Bud9p is actin dependent; unlike the delivery of Bud8p, that of Bud9p is also septin dependent. Interestingly, although the transcription of BUD8 and BUD9 appears to be cell cycle regulated, the abundance of BUD8 mRNA peaks in G2/M and that of BUD9 mRNA peaks in late G1, suggesting that the translation and/or delivery to the cell surface of each protein is delayed and presumably also cell cycle regulated. The importance of time of transcription in localization is supported by promoter-swap experiments: expression of Bud8p from the BUD9 promoter leads to its localization predominantly to the sites typical for Bud9p, and vice versa. Moreover, expression of Bud8p from the BUD9 promoter fails to rescue the budding-pattern defect of a bud8 mutant but fully rescues that of a bud9 mutant. However, although expression of Bud9p from the BUD8 promoter fails to rescue a bud9 mutant, it also rescues only partially the budding-pattern defect of a bud8 mutant, suggesting that some feature(s) of the Bud8p protein is also important for Bud8p function. Experiments with chimeric proteins suggest that the critical element(s) is somewhere in the extracytoplasmic domain of Bud8p.

Bud8p and Bud9p, proteins that may mark the sites for bipolar budding in yeast.

The bipolar budding pattern of a/alpha Saccharomyces cerevisiae cells appears to depend on persistent spatial markers in the cell cortex at the two poles of the cell. Previous analysis of mutants with specific defects in bipolar budding identified BUD8 and BUD9 as potentially encoding components of the markers at the poles distal and proximal to the birth scar, respectively. Further genetic analysis reported here supports this hypothesis. Mutants deleted for BUD8 or BUD9 grow normally but bud exclusively from the proximal and distal poles, respectively, and the double-mutant phenotype suggests that the bipolar budding pathway has been totally disabled. Moreover, overexpression of these genes can cause either an increased bias for budding at the distal (BUD8) or proximal (BUD9) pole or a randomization of bud position, depending on the level of expression. The structures and localizations of Bud8p and Bud9p are also consistent with their postulated roles as cortical markers. Both proteins appear to be integral membrane proteins of the plasma membrane, and they have very similar overall structures, with long N-terminal domains that are both N- and O-glycosylated followed by a pair of putative transmembrane domains surrounding a short hydrophilic domain that is presumably cytoplasmic. The putative transmembrane and cytoplasmic domains of the two proteins are very similar in sequence. When Bud8p and Bud9p were localized by immunofluorescence and tagging with GFP, each protein was found predominantly in the expected location, with Bud8p at presumptive bud sites, bud tips, and the distal poles of daughter cells and Bud9p at the necks of large-budded cells and the proximal poles of daughter cells. Bud8p localized approximately normally in several mutants in which daughter cells are competent to form their first buds at the distal pole, but it was not detected in a bni1 mutant, in which such distal-pole budding is lost. Surprisingly, Bud8p localization to the presumptive bud site and bud tip also depends on actin but is independent of the septins.

A GDP/GTP exchange factor involved in linking a spatial landmark to cell polarity.

In both animal and yeast cells, signaling pathways involving small guanosine triphosphatases (GTPases) regulate polarized organization of the actin cytoskeleton. In the budding yeast Saccharomyces cerevisiae, the Ras-like GTPase Bud1/Rsr1 and its guanosine 5'-diphosphate (GDP)/guanosine 5'-triphosphate (GTP) exchange factor Bud5 are involved in the selection of a specific site for growth, thus determining cell polarity. We found that Bud5 is localized at the cell division site and the presumptive bud site. Its localization is dependent on potential cellular landmarks, such as Bud3 and Axl2/Bud10 in haploid cells and Bud8 and Bud9 in diploid cells. Bud5 also physically interacts with Axl2/Bud10, a transmembrane glycoprotein, suggesting that a receptor-like transmembrane protein recruits a GDP/GTP exchange factor to connect an intrinsic spatial signal to oriented cell growth.

Identification of novel, evolutionarily conserved Cdc42p-interacting proteins and of redundant pathways linking Cdc24p and Cdc42p to actin polarization in yeast.

In the yeast Saccharomyces cerevisiae, Cdc24p functions at least in part as a guanine-nucleotide-exchange factor for the Rho-family GTPase Cdc42p. A genetic screen designed to identify possible additional targets of Cdc24p instead identified two previously known genes, MSB1 and CLA4, and one novel gene, designated MSB3, all of which appear to function in the Cdc24p-Cdc42p pathway. Nonetheless, genetic evidence suggests that Cdc24p may have a function that is distinct from its Cdc42p guanine-nucleotide-exchange factor activity; in particular, overexpression of CDC42 in combination with MSB1 or a truncated CLA4 in cells depleted for Cdc24p allowed polarization of the actin cytoskeleton and polarized cell growth, but not successful cell proliferation. MSB3 has a close homologue (designated MSB4) and two more distant homologues (MDR1 and YPL249C) in S. cerevisiae and also has homologues in Schizosaccharomyces pombe, Drosophila (pollux), and humans (the oncogene tre17). Deletion of either MSB3 or MSB4 alone did not produce any obvious phenotype, and the msb3 msb4 double mutant was viable. However, the double mutant grew slowly and had a partial disorganization of the actin cytoskeleton, but not of the septins, in a fraction of cells that were larger and rounder than normal. Like Cdc42p, both Msb3p and Msb4p localized to the presumptive bud site, the bud tip, and the mother-bud neck, and this localization was Cdc42p dependent. Taken together, the data suggest that Msb3p and Msb4p may function redundantly downstream of Cdc42p, specifically in a pathway leading to actin organization. From previous work, the BNI1, GIC1, and GIC2 gene products also appear to be involved in linking Cdc42p to the actin cytoskeleton. Synthetic lethality and multicopy suppression analyses among these genes, MSB, and MSB4, suggest that the linkage is accomplished by two parallel pathways, one involving Msb3p, Msb4p, and Bni1p, and the other involving Gic1p and Gic2p. The former pathway appears to be more important in diploids and at low temperatures, whereas the latter pathway appears to be more important in haploids and at high temperatures.

Localization of Bud2p, a GTPase-activating protein necessary for programming cell polarity in yeast to the presumptive bud site.

Yeast cells of different cell type exhibit distinct budding patterns that reflect the organization of the actin cytoskeleton. Bud1p (Rsr1p), a Ras-like GTPase, and Bud2p, a GTPase-activating protein for Bud1p, are essential for proper budding pattern. We show that Bud2p is localized at the presumptive bud site in G(1) cells in all cell types and that this localization is independent of Bud1p. Bud2p subsequently localizes to the mother-bud neck after bud emergence; this localization depends on the integrity of the septins. These observations indicate that Bud2p becomes positioned in G(1) cells by recognizing cell type-specific landmarks at the presumptive bud site.

Involvement of an actomyosin contractile ring in Saccharomyces cerevisiae cytokinesis.

In Saccharomyces cerevisiae, the mother cell and bud are connected by a narrow neck. The mechanism by which this neck is closed during cytokinesis has been unclear. Here we report on the role of a contractile actomyosin ring in this process. Myo1p (the only type II myosin in S. cerevisiae) forms a ring at the presumptive bud site shortly before bud emergence. Myo1p ring formation depends on the septins but not on F-actin, and preexisting Myo1p rings are stable when F-actin is depolymerized. The Myo1p ring remains in the mother-bud neck until the end of anaphase, when a ring of F-actin forms in association with it. The actomyosin ring then contracts to a point and disappears. In the absence of F-actin, the Myo1p ring does not contract. After ring contraction, cortical actin patches congregate at the mother-bud neck, and septum formation and cell separation rapidly ensue. Strains deleted for MYO1 are viable; they fail to form the actin ring but show apparently normal congregation of actin patches at the neck. Some myo1Delta strains divide nearly as efficiently as wild type; other myo1Delta strains divide less efficiently, but it is unclear whether the primary defect is in cytokinesis, septum formation, or cell separation. Even cells lacking F-actin can divide, although in this case division is considerably delayed. Thus, the contractile actomyosin ring is not essential for cytokinesis in S. cerevisiae. In its absence, cytokinesis can still be completed by a process (possibly localized cell-wall synthesis leading to septum formation) that appears to require septin function and to be facilitated by F-actin.

The spa2-related protein, sph1p, is important for polarized growth in yeast

The Saccharomyces cerevisiae protein Sph1p is both structurally and functionally related to the polarity protein, Spa2p. Sph1p and Spa2p are predicted to share three 100-amino acid domains each exceeding 30% sequence identity, and the amino-terminal domain of each protein contains a direct repeat common to Homo sapiens and Caenorhabditis elegans protein sequences. sph1- and spa2-deleted cells possess defects in mating projection morphology and pseudohyphal growth. sph1(Delta) spa2(Delta) double mutants also exhibit a strong haploid invasive growth defect and an exacerbated mating projection defect relative to either sph1(Delta) or spa2(Delta) single mutants. Consistent with a role in polarized growth, Sph1p localizes to growth sites in a cell cycle-dependent manner: Sph1p concentrates as a cortical patch at the presumptive bud site in unbudded cells, at the tip of small, medium and large buds, and at the bud neck prior to cytokinesis. In pheromone-treated cells, Sph1p localizes to the tip of the mating projection. Proper localization of Sph1p to sites of active growth during budding and mating requires Spa2p. Sph1p interacts in the two-hybrid system with three mitogen-activated protein (MAP) kinase kinases (MAPKKs): Mkk1p and Mkk2p, which function in the cell wall integrity/cell polarization MAP kinase pathway, and Ste7p, which operates in the pheromone and pseudohyphal signaling response pathways. Sph1p also interacts weakly with STE11, the MAPKKK known to activate STE7. Moreover, two-hybrid interactions between SPH1 and STE7 and STE11 occur independently of STE5, a proposed scaffolding protein which interacts with several members of this MAP kinase module. We speculate that Spa2p and Sph1p may function during pseudohyphal and haploid invasive growth to help tether this MAP kinase module to sites of polarized growth. Our results indicate that Spa2p and Sph1p comprise two related proteins important for the control of cell morphogenesis in yeast.