The Schizosaccharomyces pombe cdc3+ gene encodes a profilin essential for cytokinesis

The fission yeast Schizosaccharomyces pombe divides by medial fission and, like many higher eukaryotic cells, requires the function of an F- actin contractile ring for cytokinesis. In S. pombe, a class of cdc- mutants defective for cytokinesis, but not for DNA replication, mitosis, or septum synthesis, have been identified. In this paper, we present the characterization of one of these mutants, cdc3-124. Temperature shift experiments reveal that mutants in cdc3 are incapable of forming an F-actin contractile ring. We have molecularly cloned cdc3 and used the cdc3+ genomic DNA to create a strain carrying a cdc3 null mutation by homologous recombination in vivo. Cells bearing a cdc3-null allele are inviable. They arrest the cell cycle at cytokinesis without forming a contractile ring. DNA sequence analysis of the cdc3+ gene reveals that it encodes profilin, an actin-monomer-binding protein. In light of recent studies with profilins, we propose that Cdc3-profilin plays an essential role in cytokinesis by catalyzing the formation of the F-actin contractile ring. Consistent with this proposal are our observations that Cdc3-profilin localizes to the medial region of the cell where the F-actin contractile ring forms, and that it is essential for F-actin ring formation. Cells overproducing Cdc3-profilin become elongated, dumbbell shaped, and arrest at cytokinesis without any detectable F-actin staining. This effect of Cdc3-profilin overproduction is relieved by introduction of a multicopy plasmid carrying the actin encoding gene, act1+. We attribute these effects to potential sequestration of actin monomers by profilin, when present in excess.     

Cdc4p, a contractile ring protein essential for cytokinesis in Schizosaccharomyces pombe, interacts with a phosphatidylinositol 4-kinase

The proposed function of Cdc4p, an essential contractile ring protein in Schizosaccharomyces pombe, is that of a myosin essential light chain. However, five conditionally lethal cdc4 alleles exhibit complementation in diploids. Such interallelic complementation is not readily explained if the sole function of Cdc4p is that of a myosin essential light chain. Complementation of cdc4 alleles could occur only if different mutant forms can assemble into an active oligomeric complex or if Cdc4p has more than one essential function. To search for other proteins that may interact with Cdc4p, we performed a two-hybrid screen and identified two such candidates: one similar to Saccharomyces cerevisiae Vps27p and the other a putative phosphatidylinositol (PI) 4-kinase. Binding of Cdc4p to the latter and to myosin heavy chain (Myo2p) was confirmed by immunosorbent assays. Deletion studies demonstrated interaction between the Cdc4p C-terminal domain and the PI 4-kinase C-terminal domain. Furthermore, interaction was abolished by the Cdc4p C-terminal domain point mutation, Gly107 to Ser. This allele also causes failure of cytokinesis. Ectopic expression of the PI 4-kinase C-terminal domain caused cytokinesis defects that were most extreme in cells carrying the G107S allele. We suggest that Cdc4p plays multiple roles in cytokinesis and that interaction with a PI 4-kinase may be important for contractile ring assembly and/or function.     

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.     

Isolation and characterization of new fission yeast cytokinesis mutants.

Schizosaccharomyces pombe is an excellent organism in which to study cytokinesis as it divides by medial fission using an F-actin contractile ring. To enhance our understanding of the cell division process, a large genetic screen was carried out in which 17 genetic loci essential for cytokinesis were identified, 5 of which are novel. Mutants identifying three genes, rng3(+), rng4(+), and rng5(+), were defective in organizing an actin contractile ring. Four mutants defective in septum deposition, septum initiation defective (sid)1, sid2, sid3, and sid4, were also identified and characterized. Genetic analyses revealed that the sid mutants display strong negative interactions with the previously described septation mutants cdc7-24, cdc11-123, and cdc14-118. The rng5(+), sid2(+), and sid3(+) genes were cloned and shown to encode Myo2p (a myosin heavy chain), a protein kinase related to budding yeast Dbf2p, and Spg1p, a GTP binding protein that is a member of the ras superfamily of GTPases, respectively. The ability of Spg1p to promote septum formation from any point in the cell cycle depends on the activity of Sid4p. In addition, we have characterized a phenotype that has not been described previously in cytokinesis mutants, namely the failure to reorganize actin patches to the medial region of the cell in preparation for septum formation.     

A novel suppressor of ras1 in fission yeast, byr4, is a dosage- dependent inhibitor of cytokinesis

A novel gene, designated byr4, was identified in Schizosaccharomyces pombe that affects the mitotic cell cycle and shows genetic interactions with the ras1 signaling pathways. Null alleles of byr4 cause cell cycle arrest in late mitosis and permit multiple rounds of septation. The multiple septa typically divide two nuclei, but the nuclei frequently do not stain equally with 4',6-diamidino-2- phenylindole (DAPI), suggesting that byr4 is required for proper karyokinesis. Overexpression of byr4 inhibits cytokinesis, but cell cycle progression continues leading to multinucleate cells. When byr4 is overexpressed, the early steps in the cytokinesis pathway, including formation of the medial F-actin ring, occur normally; however, the later steps in the pathway, including contraction of the F-actin ring, septation, and rearrangement of the medial F-actin following mitosis, rarely occur, byr4 shows two genetic interactions with ras1. The inhibition of cytokinesis by byr4 overexpression was exacerbated by null alleles of ras1 and scd1, suggesting a link between pathways needed for cell polarity and cytokinesis. Overexpression of byr4 also partially bypasses the need for ras1 for sporulation. The electrophoretic mobility of the byr4 protein varied in response to mutants that perturb cytokinesis and karyokinesis, suggesting interactions between byr4 and these gene products. A more rapidly migrating byr4 protein was found in cells with mutations in cdc16, which undergo repeated septation, and in cdc15, which fail to form a medial F-actin ring in mitosis. A slower migrating byr4 protein was found in cells with a mutation in the beta-tubulin gene, which arrests cells at the metaphase-anaphase transition.     

Interactions of Cdc4p, a myosin light chain, with IQ-domain containing proteins in Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe undergoes cell division through a medially placed actomyosin-based contractile ring. One of the key components of this ring is the F-actin based motor protein myosin II. The myosin II heavy chain Myo2p has two light-chain-binding domains, IQl and IQ2, which bind the essential light chain, Cdc4p, and the regulatory light chain, Rlc1p. Previously, we have reported the characterization of cells expressing Myo2p lacking the IQ2 domain that facilitates Myo2p interaction with Rlc1p. In this study, we have created and characterized S. pombe strains carrying precise deletions of IQ1 and the entire neck region encompassing the IQ1 and IQ2 domains. Surprisingly, we found that the entire neck region of Myo2p is dispensable for Myo2p function. Cells deleted for IQ1, IQ2 and the entire neck region of Myo2p do not display any obvious cytoskeletal abnormalities. Immunofluorescence studies indicated that Cdc4p localizes at the ring in early and late mitotic cells in a strain in which interactions of Cdc4p with both the myosin II heavy chains (Myo2p and Myp2p) are abolished. Unlike mutations in Rlc1p that are suppressed by a simultaneous deletion of its binding site on Myo2p, mutations in the essential light chain Cdc4p are not suppressed by deletion of its binding sites on Myo2p, suggesting that Cdc4p may have additional partners essential for cytokinesis. Consistent with this, we provide evidence that two other IQ-domain containing actomyosin ring proteins, Rng2p (an IQGAP-related protein) and Myo51p (a type V myosin heavy chain), physically interact with Cdc4p. We concluded that Cdc4p, a novel myosin light chain, interacts with multiple actomyosin ring components to effect cytokinesis.     

Dissecting the domain structure of Cdc4p, a myosin essential light chain involved in Schizosaccharomyces pombe cytokinesis

Cytokinesis is the process by which one cell divides into two. Key in the cytokinetic mechanism of Schizosaccharomyces pombe is the contractile ring myosin, which consists of two heavy chains (Myo2p), two essential light chains (Cdc4p), and two regulatory light chains (Rlc1p). Cdc4p is a dumbbell-shaped EF-hand protein composed of N- and C-terminal domains separated by a flexible linker. The properties of these two domains are of particular interest because each is hypothesized to have independent functions in binding different components of the cytokinesis machinery. To help define these properties, we used NMR spectroscopy to compare the structure, stability, and dynamics of the isolated N- and C-terminal domains with one another and with native Cdc4p. On the basis of invariant chemical shifts, the N-domain retains the same structure in isolation as in the context of the full-length Cdc4p, whereas the C-domain appears markedly perturbed. This perturbation results from intramolecular binding of the residual linker sequence at the N-terminus of the C-domain in a mode similar to that used by native Cdc4p to associate with target polypeptide sequences. NMR relaxation, thermal denaturation, and amide hydrogen exchange experiments also indicate that the C-domain is less stable and more dynamic than the N-domain, both in isolation and in the full-length protein. We hypothesize that these properties reflect a conformational plasticity of the C-domain, which may allow Cdc4p to interact with several regulatory or contractile ring proteins necessary for cytokinesis.     

Loss of CDC5 function in Saccharomyces cerevisiae leads to defects in Swe1p regulation and Bfa1p/Bub2p-independent cytokinesis

In many organisms, polo kinases appear to play multiple roles during M-phase progression. To provide new insights into the function of budding yeast polo kinase Cdc5p, we generated novel temperature-sensitive cdc5 mutants by mutagenizing the C-terminal domain. Here we show that, at a semipermissive temperature, the cdc5-3 mutant exhibited a synergistic bud elongation and growth defect with loss of HSL1, a component important for normal G(2)/M transition. Loss of SWE1, which phosphorylates and inactivates the budding yeast Cdk1 homolog Cdc28p, suppressed the cdc5-3 hsl1Delta defect, suggesting that Cdc5p functions at a point upstream of Swe1p. In addition, the cdc5-4 and cdc5-7 mutants exhibited chained cell morphologies with shared cytoplasms between the connected cell bodies, indicating a cytokinetic defect. Close examination of these mutants revealed delayed septin assembly at the incipient bud site and loosely organized septin rings at the mother-bud neck. Components in the mitotic exit network (MEN) play important roles in normal cytokinesis. However, loss of BFA1 or BUB2, negative regulators of the MEN, failed to remedy the cytokinetic defect of these mutants, indicating that Cdc5p promotes cytokinesis independently of Bfa1p and Bub2p. Thus, Cdc5p contributes to the activation of the Swe1p-dependent Cdc28p/Clb pathway, normal septin function, and cytokinesis.     

Structure of Cdc4p, a contractile ring protein essential for cytokinesis in Schizosaccharomyces pombe

The Schizosaccharomyces pombe Cdc4 protein is required for the formation and function of the contractile ring, presumably acting as a myosin light chain. By using NMR spectroscopy, we demonstrate that purified Cdc4p is a monomeric protein with two structurally independent domains, each exhibiting a fold reminiscent of the EF-hand class of calcium-binding proteins. Although Cdc4p has one potentially functional calcium-binding site, it does not bind calcium in vitro. Three variants of Cdc4p containing single point mutations responsible for temperature-sensitive arrest of the cell cycle at cytokinesis (Gly-19 to Glu, Gly-82 to Asp, and Gly-107 to Ser) were also characterized by NMR and circular dichroism spectroscopy. In each case, the amino acid substitution only leads to small perturbations in the conformation of the protein. Furthermore, thermal unfolding studies indicate that, like wild-type Cdc4p, the three mutant forms are all extremely stable, remaining completely folded at temperatures significantly above those causing failure of cytokinesis in intact cells. Therefore, the altered phenotype must arise directly from a disruption of the function of Cdc4p rather than indirectly through a disruption of its overall structure. Several mutant alleles of Cdc4p also show interallelic complementation in diploid cells. This phenomenon can be explained if Cdcp4 has more than one essential function or, alternatively, if two mutant proteins assemble to form a functional complex. Based on the structure of Cdc4p, possible models for interallelic complementation including interactions with partner proteins and the formation of a myosin complex with Cdc4p fulfilling the role of both an essential and regulatory light chain are proposed.     

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.     

Bni5p,a septin-interacting protein,is required for normal septin function and cytokinesis in Saccharomyces cerevisiae

In the budding yeast Saccharomyces cerevisiae, the Cdc3p, Cdc10p, Cdc11p, Cdc12p, and Sep7p/Shs1p septins assemble early in the cell cycle in a ring that marks the future cytokinetic site. The septins appear to be major structural components of a set of filaments at the mother-bud neck and function as a scaffold for recruiting proteins involved in cytokinesis and other processes. We isolated a novel gene, BNI5, as a dosage suppressor of the cdc12-6 growth defect. Overexpression of BNI5 also suppressed the growth defects of cdc10-1, cdc11-6, and sep7Delta strains. Loss of BNI5 resulted in a cytokinesis defect, as evidenced by the formation of connected cells with shared cytoplasms, and deletion of BNI5 in a cdc3-6, cdc10-1, cdc11-6, cdc12-6, or sep7Delta mutant strain resulted in enhanced defects in septin localization and cytokinesis. Bni5p localizes to the mother-bud neck in a septin-dependent manner shortly after bud emergence and disappears from the neck approximately 2 to 3 min before spindle disassembly. Two-hybrid, in vitro binding, and protein-localization studies suggest that Bni5p interacts with the N-terminal domain of Cdc11p, which also appears to be sufficient for the localization of Cdc11p, its interaction with other septins, and other critical aspects of its function. Our data suggest that the Bni5p-septin interaction is important for septin ring stability and function, which is in turn critical for normal cytokinesis.     

A novel role for Cdc5p in DNA replication.

DNA replication initiates from specific chromosomal sites called origins, and in the budding yeast Saccharomyces cerevisiae these sites are occupied by the origin recognition complex (ORC). Dbf4p is proposed to play a role in targeting the G1/S kinase Cdc7p to initiation complexes late in G1. We report that Dbf4p may also recruit Cdc5p to origin complexes. Cdc5p is a member of the Polo family of kinases that is required for the completion of mitosis. Cdc5p and Cdc7p each interact with a distinct domain of Dbf4p. cdc5-1 mutants have a plasmid maintenance defect that can be suppressed by the addition of multiple origins. cdc5-1 orc2-1 double mutants are synthetically lethal. Levels of Cdc5p were found to be cell cycle regulated and peaked in G2/M. These results suggest a role for Cdc5p and possibly Polo-like kinases at origin complexes.     

Parameters that specify the timing of cytokinesis.

One model for the timing of cytokinesis is based on findings that p34(cdc2) can phosphorylate myosin regulatory light chain (LC20) on inhibitory sites (serines 1 and 2) in vitro (Satterwhite, L.L., M.H. Lohka, K.L. Wilson, T.Y. Scherson, L.J. Cisek, J.L. Corden, and T.D. Pollard. 1992. J. Cell Biol. 118:595-605), and this inhibition is proposed to delay cytokinesis until p34(cdc2) activity falls at anaphase. We have characterized previously several kinase activities associated with the isolated cortical cytoskeleton of dividing sea urchin embryos (Walker, G.R., C.B. Shuster, and D.R. Burgess. 1997. J. Cell Sci. 110:1373-1386). Among these kinases and substrates is p34(cdc2) and LC20. In comparison with whole cell activity, cortical H1 kinase activity is delayed, with maximum levels in cortices prepared from late anaphase/telophase embryos. To determine whether cortical-associated p34(cdc2) influences cortical myosin II activity during cytokinesis, we labeled eggs in vivo with [(32)P]orthophosphate, prepared cortices, and mapped LC20 phosphorylation through the first cell division. We found no evidence of serine 1,2 phosphorylation at any time during mitosis on LC20 from cortically associated myosin. Instead, we observed a sharp rise in serine 19 phosphorylation during anaphase and telophase, consistent with an activating phosphorylation by myosin light chain kinase. However, serine 1,2 phosphorylation was detected on light chains from detergent-soluble myosin II. Furthermore, cells arrested in mitosis by microinjection of nondegradable cyclin B could be induced to form cleavage furrows if the spindle poles were physically placed in close proximity to the cortex. These results suggest that factors independent of myosin II inactivation, such as the delivery of the cleavage stimulus to the cortex, determine the timing of cytokinesis.     

Evidence for F-actin-dependent and -independent mechanisms involved in assembly and stability of the medial actomyosin ring in fission yeast

Cell division in a number of eukaryotes, including the fission yeast Schizosaccharomyces pombe, is achieved through a medially placed actomyosin-based contractile ring. Although several components of the actomyosin ring have been identified, the mechanisms regulating ring assembly are still not understood. Here, we show by biochemical and mutational studies that the S.pombe actomyosin ring component Cdc4p is a light chain associated with Myo2p, a myosin II heavy chain. Localization of Myo2p to the medial ring depended on Cdc4p function, whereas localization of Cdc4p at the division site was independent of Myo2p. Interestingly, the actin-binding and motor domains of Myo2p are not required for its accumulation at the division site although the motor activity of Myo2p is essential for assembly of a normal actomyosin ring. The initial assembly of Myo2p and Cdc4p at the division site requires a functional F-actin cytoskeleton. Once established, however, F-actin is not required for the maintenance of Cdc4p and Myo2p medial rings, suggesting that the attachment of Cdc4p and Myo2p to the division site involves proteins other than actin itself.     

Phosphorylation of the myosin-II light chain does not regulate the timing of cytokinesis in fission yeast.

Proper coordination of cytokinesis with chromosome separation during mitosis is crucial to ensure that each daughter cell inherits an equivalent set of chromosomes. It has been proposed that one mechanism by which this is achieved is through temporally regulated myosin regulatory light chain (RLC) phosphorylation (Satterwhite, L. L., and Pollard, T. D. (1992) Curr. Opin. Cell Biol. 4, 43-52). A variety of evidence is consistent with this model. A direct test of the importance of RLC phosphorylation in vivo has been done only in Dictyostelium and Drosophila; phosphorylation of the RLC is essential in Drosophila (Jordan, P., and Karess, R. (1997) J. Cell Biol. 139, 1805-1819) but not essential in Dictyostelium (Ostrow, B. D., Chen, P., and Chisholm, R. L. (1994) J. Cell Biol. 127, 1945-1955). The Schizosaccharomyces pombe myosin light chain Cdc4p is essential for cytokinesis, but it was unknown whether phosphorylation played a role in its regulation. Here we show that the S. pombe myosin light chain Cdc4p is phosphorylated in vivo on either serine 2 or 6 but not both. Mutation of either or both of these sites to alanine did not effect the ability of Cdc4p to bind the type II myosin Myo2p, and cells expressing only these mutated versions of Cdc4p grew and divided normally. Similarly, mutation of Ser-2, Ser-6, or both residues to aspartic acid did not affect growth or division of cells. Thus we conclude that phosphorylation of Cdc4p is not essential in vivo for the function of the protein.     

The Cdc42p GTPase is targeted to the site of cell division in the fission yeast Schizosaccharomyces pombe

The Rho-family GTPase Cdc42p regulates many aspects of cell polarity and growth in eukaryotic cells, including the organization of the actin cytoskeleton. To further examine Cdc42p function in the fission yeast Schizosaccharomyces pombe, a functional green fluorescent protein (GFP)-Cdc42p fusion protein was generated. GFP-Cdc42p was observed at the medial region of the cell at the cell-division site early in cytokinesis and remained there through cell separation, and was also localized to the periphery of the cell and to internal membranes. Unexpectedly, treatment with the actin-depolymerizing drug latrunculin-A disrupted the medial region targeting pattern, and cells deficient in the actin-binding proteins tropomyosin and profilin also did not exhibit medial GFP-Cdc42p staining. In addition, medial GFP-Cdc42p localization was eliminated in a number of cytokinesis mutants, including strains defective in assembling the medial actinomyosin ring, medial ring contraction, and septum assembly. GFP-Cdc42p targeting was less affected in mutants that formed misplaced or multiple septa. These results suggest that the localization of Cdc42p at the cell-division site was dependent upon the actin cytoskeleton and that Cdc42p may function in the interdependent processes of cytokinesis and septation.     

Shs1 plays separable roles in septin organization and cytokinesis in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, five septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1/Sep7) form the septin ring at the bud neck during vegetative growth. We show here that disruption of SHS1 caused cold-sensitive growth in the W303 background, with cells arrested in chains, indicative of a cytokinesis defect. Surprisingly, the other four septins appeared to form an apparently normal septin ring in shs1Delta cells grown under the restrictive condition. We found that Myo1 and Iqg1, two components of the actomyosin contractile ring, and Cyk3, a component of the septum formation, were either delocalized or mislocalized in shs1Delta cells, suggesting that Shs1 plays supportive roles in cytokinesis. We also found that deletion of SHS1 enhanced or suppressed the septin defect in cdc10Delta and cdc11Delta cells, respectively, suggesting that Shs1 is involved in septin organization, exerting different effects on septin-ring assembly, depending on the composition of the septin subunits. Furthermore, we constructed an shs1-100c allele that lacks the coding sequence for the C-terminal 32 amino acids. This allele still displayed the genetic interactions with the septin mutants, but did not show cytokinesis defects as described above, suggesting that the roles of Shs1 in septin organization and cytokinesis are separable.     

The role of the sid1p kinase and cdc14p in regulating the onset of cytokinesis in fission yeast

Coordination of mitosis and cytokinesis is crucial for ensuring proper chromosome segregation and genomic stability. In Schizosaccharomyces pombe, the sid genes (cdc7, cdc11, cdc14, spg1, sid1, sid2 and sid4) define a signaling pathway that regulates septation and cytokinesis. Here we describe the characterization of a novel protein kinase, Sid1p. Sid1p localizes asymmetrically to one spindle pole body (SPB) in anaphase. Sid1p localization is maintained during medial ring constriction and septum synthesis and disappears prior to cell separation. Additionally, we found that Cdc14p is in a complex with Sid1p. Epistasis analysis places Sid1p-Cdc14p downstream of Spg1p-Cdc7p but upstream of Sid2p. Finally, we show that cyclin proteolysis during mitosis is unaffected by inactivating the sid pathway; in fact, loss of Cdc2-cyclin activity promotes Sid1p-Cdc14p association with the SPB, possibly providing a mechanism that couples cytokinesis with mitotic exit.     

Analysis of the S. pombe signalling scaffold protein Cdc11p reveals an essential role for the N-terminal domain in SIN signalling

The initiation of cytokinesis in the fission yeast Schizosaccharomyces pombe is signalled by the septation initiation network (SIN). Signalling originates from the spindle pole body (SPB), where SIN proteins are anchored by a scaffold composed of cdc11p and sid4p. Cdc11p links the other SIN proteins to sid4p and the SPB. Homologues of cdc11p have been identified in Saccharomyes cerevisiae (Nud1p) and human cells (Centriolin). We have defined functional domains of cdc11p by analysis of deletion mutants. We demonstrate that the C-terminal end of cdc11p is necessary for SPB localisation. We also show that the N-terminal domain is necessary and sufficient for signal transduction, since tethering of this domain to the SPB will substitute for cdc11p in SIN function.