Purification, characterization, and immunofluorescence localization of Saccharomyces cerevisiae capping protein

Capping protein binds the barbed ends of actin filaments and nucleates actin filament assembly in vitro. We purified capping protein from Saccharomyces cervisiae. One of the two subunits is the product of the CAP2 gene, which we previously identified as the gene encoding the beta subunit of capping protein based on its sequence similarity to capping protein beta subunits in chicken and Dictyostelium (Amatruda, J. F., J. F. Cannon, K. Tatchell, C. Hug, and J. A. Cooper. 1990. Nature (Lond.) 344:352-354). Yeast capping protein has activity in critical concentration and low-shear viscometry assays consistent with barbed- end capping activity. Like chicken capping protein, yeast capping protein is inhibited by PIP2. By immunofluorescence microscopy yeast capping protein colocalizes with cortical actin spots at the site of bud emergence and at the tips of growing buds and shmoos. In contrast, capping protein does not colocalize with actin cables or with actin rings at the site of cytokinesis.     

Mutations That Enhance the Cap2 Null Mutant Phenotype in Saccharomyces Cerevisiae Affect the Actin Cytoskeleton, Morphogenesis and Pattern of Growth

Mutations conferring synthetic lethality in combination with null mutations in CAP2, the gene encoding the β subunit of capping protein of Saccharomyces cerevisiae, were obtained in a colony color assay. Monogenic inheritance was found for four mutations, which were attributed to three genetic loci. One mutation, sac6-69, is in the gene encoding fimbrin, another actin-binding protein, which was expected because null mutations in SAC6 and CAP2 are known to be synthetic-lethal. The other two loci were designated slc for synthetic lethality with cap2. These loci include the mutations slc1-66, slc1-87 and slc2-107. The slc mutations are semi-dominant, as shown by incomplete complementation in slc/SLC cap2/cap2 heterozygotes. The slc mutations and sac6-69 interact with each other, as shown by enhanced phenotypes in diheterozygotes. Moreover, the haploid slc2-107 sac6-69 double mutant is inviable. In a CAP2 background, the slc mutations lead to temperature and osmotic sensitivity. They alter the distribution of the actin cytoskeleton, including deficits in the presence of actin cables and the polarization of cortical actin patches. The slc mutations also lead to a pseudomycelial growth pattern. Together these results suggest that slc1 and slc2 encode components of the actin cytoskeleton in yeast and that the actin cytoskeleton can regulate the patterns of growth.     

Unexpected combinations of null mutations in genes encoding the actin cytoskeleton are lethal in yeast.

To understand the role of the actin cytoskeleton in cell physiology, and how actin-binding proteins regulate the actin cytoskeleton in vivo, we and others previously identified actin-binding proteins in Saccharomyces cerevisiae and studied the effect of null mutations in the genes for these proteins. A null mutation of the actin gene (ACT1) is lethal, but null mutations in the tropomyosin (TPM1), fimbrin (SAC6), Abp1p (ABP1), and capping protein (CAP1 and CAP2) genes have relatively mild or no effects. We have now constructed double and triple mutants lacking 2 or 3 of these actin-binding proteins, and studied the effect of the combined mutations on cell growth, morphology, and organization of the actin cytoskeleton. Double mutants lacking fimbrin and either Abp1p or capping protein show negative synthetic effects on growth, in the most extreme case resulting in lethality. All other combinations of double mutations and the triple mutant lacking tropomyosin, Abp1p, and capping protein, are viable and their phenotypes are similar to or only slightly more severe than those of the single mutants. Therefore, the synthetic phenotypes are highly specific. We confirmed this specificity by overexpression of capping protein and Abp1p in strains lacking fimbrin. Thus, while overexpression of these proteins has deleterious effects on actin organization in wild-type strains, no synthetic phenotype was observed in the absence of fimbrin. We draw two important conclusions from these results. First, since mutations in pairs of actin-binding protein genes cause inviability, the actin cytoskeleton of yeast does not contain a high degree of redundancy. Second, the lack of structural and functional homology among these genetically redundant proteins (fimbrin and capping protein or Abp1p) indicates that they regulate the actin cytoskeleton by different mechanisms. Determination of the molecular basis for this surprising conclusion will provide unique insights into the essential mechanisms that regulate the actin cytoskeleton.     

Mutational analysis of capping protein function in Saccharomyces cerevisiae.

To investigate physiologic functions and structural correlates for actin capping protein (CP), we analyzed site-directed mutations in CAP1 and CAP2, which encode the alpha and beta subunits of CP in Saccharomyces cerevisiae. Mutations in four different regions caused a loss of CP function in vivo despite the presence of mutant protein in the cells. Mutations in three regions caused a complete loss of all aspects of function, including the actin distribution, viability with sac6, and localization of CP to actin cortical patches. Mutation of the fourth region led to partial loss of only one function-formation of actin cables. Some mutations retained function and exhibited the complete wild-type phenotype, and some mutations led to a complete loss of protein and therefore loss of function. The simplest hypothesis that can explain these results is that a single biochemical property is necessary for all in vivo functions. This biochemical property is most likely binding to actin filaments, because the nonfunctional mutant CPs no longer co-localize with actin filaments in vivo and because direct binding of CP to actin filaments has been well established by studies with purified proteins in vitro. More complex hypotheses, involving the existence of additional biochemical properties important for function, cannot be excluded by this analysis.     

Cdc50p,a conserved endosomal membrane protein,controls polarized growth in Saccharomyces cerevisiae

During the cell cycle of the yeast Saccharomyces cerevisiae, the actin cytoskeleton and the growth of cell surface are polarized, mediating bud emergence, bud growth, and cytokinesis. We identified CDC50 as a multicopy suppressor of the myo3 myo5-360 temperature-sensitive mutant, which is defective in organization of cortical actin patches. The cdc50 null mutant showed cold-sensitive cell cycle arrest with a small bud as reported previously. Cortical actin patches and Myo5p, which are normally localized to polarization sites, were depolarized in the cdc50 mutant. Furthermore, actin cables disappeared, and Bni1p and Gic1p, effectors of the Cdc42p small GTPase, were mislocalized in the cdc50 mutant. As predicted by its amino acid sequence, Cdc50p appears to be a transmembrane protein because it was solubilized from the membranes by detergent treatment. Cdc50p colocalized with Vps21p in endosomal compartments and was also localized to the class E compartment in the vps27 mutant. The cdc50 mutant showed defects in a late stage of endocytosis but not in the internalization step. It showed, however, only modest defects in vacuolar protein sorting. Our results indicate that Cdc50p is a novel endosomal protein that regulates polarized cell growth.     

Spindle orientation in Saccharomyces cerevisiae depends on the transport of microtubule ends along polarized actin cables

Microtubules and actin filaments interact and cooperate in many processes in eukaryotic cells, but the functional implications of such interactions are not well understood. In the yeast Saccharomyces cerevisiae, both cytoplasmic microtubules and actin filaments are needed for spindle orientation. In addition, this process requires the type V myosin protein Myo2, the microtubule end-binding protein Bim1, and Kar9. Here, we show that fusing Bim1 to the tail of the Myo2 is sufficient to orient spindles in the absence of Kar9, suggesting that the role of Kar9 is to link Myo2 to Bim1. In addition, we show that Myo2 localizes to the plus ends of cytoplasmic microtubules, and that the rate of movement of these cytoplasmic microtubules to the bud neck depends on the intrinsic velocity of Myo2 along actin filaments. These results support a model for spindle orientation in which a Myo2-Kar9-Bim1 complex transports microtubule ends along polarized actin cables. We also present data suggesting that a similar process plays a role in orienting cytoplasmic microtubules in mating yeast cells.     

Identification of Genes Controlling Growth Polarity in the Budding Yeast Saccharomyces Cerevisiae: A Possible Role of N-Glycosylation and Involvement of the Exocyst Complex

The regulation of secretion polarity and cell surface growth during the cell cycle is critical for proper morphogenesis and viability of Saccharomyces cerevisiae. A shift from isotropic cell surface growth to polarized growth is necessary for bud emergence and a repolarization of secretion to the bud neck is necessary for cell separation. Although alterations in the actin cytoskeleton have been implicated in these changes in secretion polarity, clearly other cellular systems involved in secretion are likely to be targets of cell cycle regulation. To investigate mechanisms coupling cell cycle progression to changes in secretion polarity in parallel with and downstream of regulation of actin polarization, we implemented a screen for mutants defective specifically in polarized growth but with normal actin cytoskeleton structure. These mutants fell into three classes: those partially defective in N-glycosylation, those linked to specific defects in the exocyst, and a third class neither defective in glycosylation nor linked to the exocyst. These results raise the possibility that changes in N-linked glycosylation may be involved in a signal linking cell cycle progression and secretion polarity and that the exocyst may have regulatory functions in coupling the secretory machinery to the polarized actin cytoskeleton.     

How capping protein binds the barbed end of the actin filament

Cytoskeletal filaments are often capped at one end, regulating assembly and cellular location. The actin filament is a right-handed, two-strand long-pitch helix. The ends of the two protofilaments are staggered in relation to each other, suggesting that capping could result from one protein binding simultaneously to the ends of both protofilaments. Capping protein (CP), a ubiquitous alpha/beta heterodimer in eukaryotes, tightly caps (K(d) approximately 0.1-1 nM) the barbed end of the actin filament (the end favored for polymerization), preventing actin subunit addition and loss. CP is critical for actin assembly and actin-based motility in vivo and is an essential component of the dendritic nucleation model for actin polymerization at the leading edge of cells. However, the mechanism by which CP caps actin filaments is not well understood. The X-ray crystal structure of CP has inspired a model where the C termini ( approximately 30 amino acids) of the alpha and beta subunits of CP are mobile extensions ("tentacles"), and these regions are responsible for high-affinity binding to, and functional capping of, the barbed end. We tested the tentacle model in vitro with recombinant mutant CPs. Loss of both tentacles causes a complete loss of capping activity. The alpha tentacle contributes more to capping affinity and kinetics; its removal reduces capping affinity by 5000-fold and the on-rate of capping by 20-fold. In contrast, removal of the beta tentacle reduced the affinity by only 300-fold and did not affect the on-rate. These two regions are not close to each other in the three-dimensional structure, suggesting CP uses two independent actin binding tentacles to cap the barbed end. CP with either tentacle alone can cap, as can the isolated beta tentacle alone, suggesting that the individual tentacles interact with more than one actin subunit at a subunit interface at the barbed end.     

High Rates of Actin Filament Turnover in Budding Yeast and Roles for Actin in Establishment and Maintenance of Cell Polarity Revealed Using the Actin Inhibitor Latrunculin-A

We report that the actin assembly inhibitor latrunculin-A (LAT-A) causes complete disruption of the yeast actin cytoskeleton within 2–5 min, suggesting that although yeast are nonmotile, their actin filaments undergo rapid cycles of assembly and disassembly in vivo. Differences in the LAT-A sensitivities of strains carrying mutations in components of the actin cytoskeleton suggest that tropomyosin, fimbrin, capping protein, Sla2p, and Srv2p act to increase actin cytoskeleton stability, while End3p and Sla1p act to decrease stability. Identification of three LAT-A resistant actin mutants demonstrated that in vivo effects of LAT-A are due specifically to impairment of actin function and implicated a region on the three-dimensional actin structure as the LAT-A binding site.

LAT-A was used to determine which of 19 different proteins implicated in cell polarity development require actin to achieve polarized localization. Results show that at least two molecular pathways, one actindependent and the other actin-independent, underlie polarity development. The actin-dependent pathway localizes secretory vesicles and a putative vesicle docking complex to sites of cell surface growth, providing an explanation for the dependence of polarized cell surface growth on actin function. Unexpectedly, several proteins that function with actin during cell polarity development, including an unconventional myosin (Myo2p), calmodulin, and an actin-interacting protein (Bud6/Aip3p), achieved polarized localization by an actin-independent pathway, revealing interdependence among cell polarity pathways. Finally, transient actin depolymerization caused many cells to abandon one bud site or mating projection and to initiate growth at a second site. Thus, actin filaments are also required for maintenance of an axis of cell polarity.

     

The rho-GAP encoded by BEM2 regulates cytoskeletal structure in budding yeast.

Microfilaments are required for polarized growth and morphogenesis in Saccharomyces cerevisiae. To accomplish this, actin cables and patches are redistributed during the cell cycle to direct secretory components to appropriate sites for cell growth. A major component of actin cables is tropomyosin I, encoded by TPM1, that determines or stabilizes these structures. Disruption of TPM1 is not lethal but results in the loss of actin cables and confers a partial defect in polarized secretion. Using a synthetic lethal screen, we have identified seven mutations residing in six genes whose products are required in the absence of Tpm1p. Each mutant exhibited a morphological defect, suggesting a functional link to the actin cytoskeleton. Complementation cloning of one mutation revealed that it lies in BEM2, which encodes a GTPase-activating protein for the RHO1 product. bem2 mutations also show synthetic lethality with rho1 and mutations in certain other cytoskeletal genes (ACT1, MYO1, MYO2, and SAC6) but not with mutations in several noncytoskeletal genes. These data therefore provide a genetic link between the GAP encoded by BEM2 and the functional organization of microfilaments. In addition, we show that bem2 mutations confer benomyl sensitivity and have abnormal microtubule arrays, suggesting that the BEM2 product may also be involved directly or indirectly in regulating microtubule function.     

A role for actin, Cdc1p, and Myo2p in the inheritance of late Golgi elements in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, Golgi elements are present in the bud very early in the cell cycle. We have analyzed this Golgi inheritance process using fluorescence microscopy and genetics. In rapidly growing cells, late Golgi elements show an actin-dependent concentration at sites of polarized growth. Late Golgi elements are apparently transported into the bud along actin cables and are also retained in the bud by a mechanism that may involve actin. A visual screen for mutants defective in the inheritance of late Golgi elements yielded multiple alleles of CDC1. Mutations in CDC1 severely depolarize the actin cytoskeleton, and these mutations prevent late Golgi elements from being retained in the bud. The efficient localization of late Golgi elements to the bud requires the type V myosin Myo2p, further suggesting that actin plays a role in Golgi inheritance. Surprisingly, early and late Golgi elements are inherited by different pathways, with early Golgi elements localizing to the bud in a Cdc1p- and Myo2p-independent manner. We propose that early Golgi elements arise from ER membranes that are present in the bud. These two pathways of Golgi inheritance in S. cerevisiae resemble Golgi inheritance pathways in vertebrate cells.     

PAK-family kinases regulate cell and actin polarization throughout the cell cycle of Saccharomyces cerevisiae

During the cell cycle of the yeast Saccharomyces cerevisiae, the actin cytoskeleton and cell surface growth are polarized, mediating bud emergence, bud growth, and cytokinesis. We have determined whether p21-activated kinase (PAK)-family kinases regulate cell and actin polarization at one or several points during the yeast cell cycle. Inactivation of the PAK homologues Ste20 and Cla4 at various points in the cell cycle resulted in loss of cell and actin cytoskeletal polarity, but not in depolymerization of F-actin. Loss of PAK function in G1 depolarized the cortical actin cytoskeleton and blocked bud emergence, but allowed isotropic growth and led to defects in septin assembly, indicating that PAKs are effectors of the Rho-guanosine triphosphatase Cdc42. PAK inactivation in S/G2 resulted in depolarized growth of the mother and bud and a loss of actin polarity. Loss of PAK function in mitosis caused a defect in cytokinesis and a failure to polarize the cortical actin cytoskeleton to the mother-bud neck. Cla4-green fluorescent protein localized to sites where the cortical actin cytoskeleton and cell surface growth are polarized, independently of an intact actin cytoskeleton. Thus, PAK family kinases are primary regulators of cell and actin cytoskeletal polarity throughout most or all of the yeast cell cycle. PAK-family kinases in higher organisms may have similar functions.     

Stable and dynamic axes of polarity use distinct formin isoforms in budding yeast

Bud growth in yeast is guided by myosin-driven delivery of secretory vesicles from the mother cell to the bud. We find transport occurs along two sets of actin cables assembled by two formin isoforms. The Bnr1p formin assembles cables that radiate from the bud neck into the mother, providing a stable mother-bud axis. These cables also depend on septins at the neck and are required for efficient transport from the mother to the bud. The Bni1p formin assembles cables that line the bud cortex and target vesicles to varying locations in the bud. Loss of these cables results in morphological defects as vesicles accumulate at the neck. Assembly of these cables depends on continued polarized secretion, suggesting vesicular transport provides a positive feedback signal for Bni1p activation, possibly by rho-proteins. By coupling different formin isoforms to unique cortical landmarks, yeast uses common cytoskeletal elements to maintain stable and dynamic axes in the same cell.     

Differential activities and regulation of Saccharomyces cerevisiae formin proteins Bni1 and Bnr1 by Bud6

Formins are conserved proteins that nucleate actin assembly and tightly associate with the fast growing barbed ends of actin filaments to allow insertional growth. Most organisms express multiple formins, but it has been unclear whether they have similar or distinct activities and how they may be regulated differentially. We isolated and compared the activities of carboxyl-terminal fragments of the only two formins expressed in Saccharomyces cerevisiae, Bni1 and Bnr1. Bnr1 was an order of magnitude more potent than Bni1 in actin nucleation and processive capping, and unlike Bni1, Bnr1 bundled actin filaments. Profilin bound directly to Bni1 and Bnr1 and regulated their activities similarly. However, the cell polarity factor Bud6/Aip3 specifically bound to and stimulated Bni1, but not Bnr1. This was unexpected, since previous two-hybrid studies suggested Bud6 interacts with both formins. We mapped Bud6 binding activity to specific residues in the carboxyl terminus of Bni1 that are adjacent to its diaphanous autoregulatory domain (DAD). Fusion of the carboxyl terminus of Bni1 to Bnr1 conferred Bud6 stimulation to a Bnr1-Bni1 chimera. Thus, Bud6 differentially stimulates Bni1 and not Bnr1. We found that Bud6 is up-regulated during bud growth, when it is delivered to the bud tip on Bni1-nucleated actin cables. We propose that Bud6 stimulation of Bni1 promotes robust cable formation, which in turn delivers more Bud6 to the bud tip, reinforcing polarized cell growth through a positive feedback loop.     

Interactions with PIP2, ADP-actin monomers, and capping protein regulate the activity and localization of yeast twinfilin

Twinfilin is a ubiquitous actin monomer-binding protein that regulates actin filament turnover in yeast and mammalian cells. To elucidate the mechanism by which twinfilin contributes to actin filament dynamics, we carried out an analysis of yeast twinfilin, and we show here that twinfilin is an abundant protein that localizes to cortical actin patches in wild-type yeast cells. Native gel assays demonstrate that twinfilin binds ADP-actin monomers with higher affinity than ATP-actin monomers. A mutant twinfilin that does not interact with actin monomers in vitro no longer localizes to cortical actin patches when expressed in yeast, suggesting that the ability to interact with actin monomers may be essential for the localization of twinfilin. The localization of twinfilin to the cortical actin cytoskeleton is also disrupted in yeast strains where either the CAP1 or CAP2 gene, encoding for the alpha and beta subunits of capping protein, is deleted. Purified twinfilin and capping protein form a complex on native gels. Twinfilin also interacts with phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2), and its actin monomer-sequestering activity is inhibited by PI(4,5)P2. Based on these results, we propose a model for the biological role of twinfilin as a protein that localizes actin monomers to the sites of rapid filament assembly in cells.     

A high-affinity interaction with ADP-actin monomers underlies the mechanism and in vivo function of Srv2/cyclase-associated protein

Cyclase-associated protein (CAP), also called Srv2 in Saccharomyces cerevisiae, is a conserved actin monomer-binding protein that promotes cofilin-dependent actin turnover in vitro and in vivo. However, little is known about the mechanism underlying this function. Here, we show that S. cerevisiae CAP binds with strong preference to ADP-G-actin (Kd 0.02 microM) compared with ATP-G-actin (Kd 1.9 microM) and competes directly with cofilin for binding ADP-G-actin. Further, CAP blocks actin monomer addition specifically to barbed ends of filaments, in contrast to profilin, which blocks monomer addition to pointed ends of filaments. The actin-binding domain of CAP is more extensive than previously suggested and includes a recently solved beta-sheet structure in the C-terminus of CAP and adjacent sequences. Using site-directed mutagenesis, we define evolutionarily conserved residues that mediate binding to ADP-G-actin and demonstrate that these activities are required for CAP function in vivo in directing actin organization and polarized cell growth. Together, our data suggest that in vivo CAP competes with cofilin for binding ADP-actin monomers, allows rapid nucleotide exchange to occur on actin, and then because of its 100-fold weaker binding affinity for ATP-actin compared with ADP-actin, allows other cellular factors such as profilin to take the handoff of ATP-actin and facilitate barbed end assembly.     

Phosphatidylserine is polarized and required for proper Cdc42 localization and for development of cell polarity

Polarity is key to the function of eukaryotic cells. On the establishment of a polarity axis, cells can vectorially target secretion, generating an asymmetric distribution of plasma membrane proteins. From Saccharomyces cerevisiae to mammals, the small GTPase Cdc42 is a pivotal regulator of polarity. We used a fluorescent probe to visualize the distribution of phosphatidylserine in live S. cerevisiae. Remarkably, phosphatidylserine was polarized in the plasma membrane, accumulating in bud necks, the bud cortex and the tips of mating projections. Polarization required vectorial delivery of phosphatidylserine-containing secretory vesicles, and phosphatidylserine was largely excluded from endocytic vesicles, contributing to its polarized retention. Mutants lacking phosphatidylserine synthase had impaired polarization of the Cdc42 complex, leading to a delay in bud emergence, and defective mating. The addition of lysophosphatidylserine resulted in resynthesis and polarization of phosphatidylserine, as well as repolarization of Cdc42. The results indicate that phosphatidylserine--and presumably its polarization--are required for optimal Cdc42 targeting and activation during cell division and mating.     

Role of actin and Myo2p in polarized secretion and growth of Saccharomyces cerevisiae

We examined the role of the actin cytoskeleton in secretion in Saccharomyces cerevisiae with the use of several quantitative assays, including time-lapse video microscopy of cell surface growth in individual living cells. In latrunculin, which depolymerizes filamentous actin, cell surface growth was completely depolarized but still occurred, albeit at a reduced level. Thus, filamentous actin is necessary for polarized secretion but not for secretion per se. Consistent with this conclusion, latrunculin caused vesicles to accumulate at random positions throughout the cell. Cortical actin patches cluster at locations that correlate with sites of polarized secretion. However, we found that actin patch polarization is not necessary for polarized secretion because a mutant, bee1Delta(las17Delta), which completely lacks actin patch polarization, displayed polarized growth. In contrast, a mutant lacking actin cables, tpm1-2 tpm2Delta, had a severe defect in polarized growth. The yeast class V myosin Myo2p is hypothesized to mediate polarized secretion. A mutation in the motor domain of Myo2p, myo2-66, caused growth to be depolarized but with only a partial decrease in the level of overall growth. This effect is similar to that of latrunculin, suggesting that Myo2p interacts with filamentous actin. However, inhibition of Myo2p function by expression of its tail domain completely abolished growth.     

Evidence for a functional link between profilin and CAP in the yeast S. cerevisiae

CAP is a component of the S. cerevisiae adenylyl cyclase complex. The N-terminal domain is required for cellular RAS responsiveness. Loss of the C-terminal domain is associated with morphological and nutritional defects. Here we report that cap- cells bud randomly and are defective in actin distribution. The morphological and nutritional defects associated with loss of the CAP C-terminal domain are suppressed by over-expression of PFY, the gene encoding profilin, an actin- and polyphosphoinositide-binding protein. The phenotype of cells lacking PFY resembles that of cells lacking the CAP C-terminal domain. Study of mutated yeast profilins and profilins from Acanthamoeba suggests that the ability of profilin to suppress cap- cells is dependent upon a property other than, or in addition to, its ability to bind actin. This property may be its ability to bind polyphosphoinositides. We propose that CAP and profilin provide a link between growth signals and remodeling of the cellular cytoskeleton.     

Genetic and biochemical analysis of the adenylyl cyclase-associated protein, cap, in Schizosaccharomyces pombe.

We have identified, cloned, and studied a gene, cap, encoding a protein that is associated with adenylyl cyclase in the fission yeast Schizosaccharomyces pombe. This protein shares significant sequence homology with the adenylyl cyclase-associated CAP protein in the yeast Saccharomyces cerevisiae. CAP is a bifunctional protein; the N-terminal domain appears to be involved in cellular responsiveness to RAS, whereas loss of the C-terminal portion is associated with morphological and nutritional defects. S. pombe cap can suppress phenotypes associated with deletion of the C-terminal CAP domain in S. cerevisiae but does not suppress phenotypes associated with deletion of the N-terminal domain. Analysis of cap disruptants also mapped the function of cap to two domains. The functional loss of the C-terminal region of S. pombe cap results in abnormal cellular morphology, slow growth, and failure to grow at 37 degrees C. Increases in mating and sporulation were observed when the entire gene was disrupted. Overproduction of both cap and adenylyl cyclase results in highly elongated large cells that are sterile and have measurably higher levels of adenylyl cyclase activity. Our results indicate that cap is required for the proper function of S. pombe adenylyl cyclase but that the C-terminal domain of cap has other functions that are shared with the C-terminal domain of S. cerevisiae CAP.