The key to S. pombe cytokinesis is the activity of a signaling cascade termed the septation initiation network (SIN; reviewed in Cerutti and Simanis, 2000 ; McCollum and Gould, 2001 ). The SIN is required for the final steps in cell division including contraction of the actomyosin ring and formation of the septum. Mutants in the SIN give rise to the septation initiation defective (sid) phenotype in which cells become highly elongated and multinucleate. The SIN is triggered by the activity of Spg1p (Schmidt et al., 1997 ), a small Ras-superfamily GTPase that resides at the SPB(s) throughout the cell cycle (Sohrmann et al., 1998 ). During interphase, Spg1p is in an inactive GDP-bound form. During metaphase, it becomes activated at both SPBs (GTP-bound form), but during anaphase B, it becomes inactivated at only one pole giving rise to a poorly understood asymmetric state (Sohrmann et al., 1998 ). The GTP-bound form of Spg1p recruits the Cdc7p protein kinase resulting in Cdc7p localization to both SPBs during metaphase and just one SPB during anaphase B (Sohrmann et al., 1998 ). The Sid1p protein kinase, in a complex with Cdc14p, is then recruited to the SPB that contains Cdc7p and activated Spg1p at this time (Guertin et al., 2000 ). The Sid2p protein kinase is also found constitutively at SPBs (Sparks et al., 1999 ; Hou et al., 2000 ; Salimova et al., 2000 ). After recruitment of the Sid1p-Cdc14p complex to a single SPB during anaphase, Sid2p is activated and recruited to the medial ring (Sparks et al., 1999 ). The recruitment of Sid2p to either the SPB or the medial ring depends on the function of its binding partner, Mob1p (Hou et al., 2000 ; Salimova et al., 2000 ).

Sid4p functions upstream of all the aforementioned SIN components. It resides at the SPB throughout the cell cycle and is required for the SPB localization of all other tested SIN components (Sparks et al., 1999 ; Chang and Gould, 2000 ; Hou et al., 2000 ; Salimova et al., 2000 ), including the two-component GAP for Spg1p that is comprised of Cdc16p and Byr4p (Furge et al., 1998 ; Li et al., 2000 ). Although the dependency on Sid4p is consistent with its functioning as a SPB scaffold for the SIN, a direct interaction between Sid4p and other SIN components has not been detected.

Strains containing mutations in the cdc11⁺ gene were first identified as part of a screen for cell cycle mutants (Nurse et al., 1976 ). They were members of a family of mutants that failed to undergo septation but continued to elongate and formed multiple nuclei. This phenotype has since been given the name of sid. Subsequent genetic analyses indicated that cdc11 mutants displayed numerous genetic interactions with other sid mutants, indicating that Cdc11p was likely to participate in the SIN pathway (Marks et al., 1992 ). However, although all other genetically defined members of the pathway have been cloned and studied (for review, see Cerutti and Simanis, 2000 ; McCollum and Gould, 2001 ), throughout this time the identity of cdc11⁺ has eluded discovery. In retrospect, this may be due to its relatively large ORF (>3 kb), making it poorly represented in most gene libraries. The genetic studies mentioned above had previously placed Cdc11p toward the top of the SIN pathway. cdc11 mutants rescue a mutation in cdc16⁺, one of the GAP components of the system (Marks et al., 1992 ). In addition, increased expression of Cdc7p or Spg1p could rescue mutations in cdc11 (Fankhauser and Simanis, 1994 ; Schmidt et al., 1997 ). Our localization data support placing Cdc11p function near the top of the SIN pathway and indicate that Cdc11p most likely functions with Sid4p to provide a SPB platform on which the SIN pathway might organize. This interpretation is consistent with the recent characterization of S. cerevisiae Nud1p, a homolog of Cdc11p (Bardin et al., 2000 ; Gruneberg et al., 2000 ). Nud1p was found to interact with Bfa1p and Bub2p (Gruneberg et al., 2000 ), the S. cerevisiae homologues of Byr4p and Cdc16p, respectively. It was also reported that nud1 mutants mislocalized Tem1p, the S. cerevisiae equivalent of Spg1p, and failed to exit mitosis (Bardin et al., 2000 ; Gruneberg et al., 2000 ).

A search for motifs in the Cdc11p sequence revealed that the C terminus contains a total of 11 leucine-rich repeats arranged into three sets (Figure ​(Figure1A).1A). These structures are known to act as favorable surfaces for protein interactions and to be particularly common in those involved in signal transduction pathways (for review, see Kobe and Deisenhofer, 1995 ). No motif involved in catalytic activity was identified in these searches. Based on this information, it seems reasonable to suggest that Cdc11p may act in the capacity of a scaffolding protein for the SIN pathway. Our finding that the C-terminal region is responsible for binding to Sid4p will probably prove to be but one of a number of interactions in which Cdc11p participates. On this note, we found that Spg1p could not localize to SPBs in cells overproducing the C terminus of Cdc11p. It is likely, therefore, that residues 631-1045 lack an intact binding site for Spg1p or another protein that tethers Spg1p to Cdc11p.

The findings of ourselves and others (Sparks et al., 1999 ; Guertin et al., 2000 ; Hou et al., 2000 ; Li et al., 2000 ) that members of the SIN including its negative regulators depend upon Cdc11p, which in turn depends upon Sid4p, for localization to the SPB allow us to conclude that Sid4p is the SIN pathway component most proximal to the center of the SPB. Its role in the SIN may be purely a structural one, linking the “active” members of the SIN to the SPB. The previously reported ability of Sid4p to dimerize (Chang and Gould, 2000 ) has now been found to be the function of the two central coiled-coil domains within the protein, and this dimerization is essential for proper Sid4p SPB localization. We reported previously that the Sid4p N-terminus was responsible for SPB localization. This conclusion was based on the observations that Sid4ΔNp overexpression prevented assembly of other pathway components at the SPB, that Sid4ΔNp was not detected at the SPB (this was apparently due to a technical problem that we have since corrected), and that the Sid4p N-terminal portion was detected at the SPB when it was highly overproduced (Chang and Gould, 2000 ). However, the more detailed analysis reported here has shown that the C terminus of Sid4p, not the N terminus, is responsible for SPB localization. The Sid4p C terminus readily localizes to SPBs, and it is most likely the last coiled-coil region of the protein that interacts with another SPB component. When the Sid4p C terminus is overproduced, it blocks the ability of full-length Sid4p, Cdc11p, and Spg1p to localize to SPBs and hence produces a sid phenotype. This indicates that the C terminus binds efficiently to the Sid4p docking site on SPBs, and it will be interesting to learn what other SPB protein(s) the C terminus of Sid4p interacts with. In contrast, the Sid4p N-terminus, even when highly overproduced, does not disturb the localization of other SIN components (our unpublished results and Chang and Gould, 2000 ). Our explanation of why the Sid4p N terminus was previously detected at SPBs, when overproduced, is that this fragment, as we have demonstrated here, is able to bind Cdc11p. Presumably, the weak localization we observed was due to its ability to interact with some Cdc11p at SPBs (Chang and Gould, 2000 ).

Sid4p does not have any immediately obvious homolog in S. cerevisiae. However, it is worth considering the possibility that Cnm67p may be the S. cerevisiae counterpart of Sid4p. Although sequence similarity between the two proteins is undetectable, Cnm67p is of similar size to Sid4p and contains central coiled-coil domains (Brachat et al., 1998 ). The N-terminal region of Cnm67p interacts with the C terminus of Nud1p (Elliott et al., 1999 ), these proteins can be found in a cytoplasmic complex together (Elliott et al., 1999 ), and Cnm67p is required for the localization of Nud1p to the outer plaque of the SPB (Adams and Kilmartin, 1999 ). Further, it is the C terminus of Cnm67p, like the C terminus of Sid4p, that tethers it to the SPB, and the loss of Cnm67p leads to the formation of multinucleate cells (Schaerer et al., 2001 ). Importantly, loss of Cnm67p has been shown to result in spindle and/or astral microtubule defects resulting from the loss of the outer plaque of the SPB (Hoepfner et al., 2000 ). Although we have not detected any obvious microtubule or nuclear positioning defects in sid4-SA1 (our unpublished results; Chang and Gould, 2000 ), it will be important to examine sid4 null mutants by electron microscopy to determine whether they exhibit SPB defects.

While this article was under review, Krapp et al. (2001) reached similar conclusions regarding the identity and role of Cdc11p in the SIN. Furthermore, these authors reported that cdc11::ura4⁺ cells displayed defects in astral microtubule attachment to the SPB (Krapp et al., 2001 ) similar to those observed in S. cerevisiae nud1 mutants (Gruneberg et al., 2000 ). Although we detected extra Spg1p “dots” in certain cdc11 mutants at permissive temperature, indicating a potential abnormality in SPB structure, both interphase and mitotic microtubule structures in cdc11 temperature-sensitive mutants appeared normal as determined by indirect immunofluoresence microcopy (our unpublished results). Thus, some SPB functions are likely to be retained in the temperature-sensitive cdc11 mutants relative to the null.