Ppc89 links multiple proteins, including the septation initiation network, to the core of the fission yeast spindle-pole body.

The spindle-pole body (SPB), the yeast analog of the centrosome, serves as the major microtubule (MT) organizing center in the yeast cell. In addition to this central function, the SPB organizes and concentrates proteins required for proper coordination between the nuclear-division cycle and cytokinesis. For example, the Schizosaccharomyces pombe septation-initiation network (SIN), which is responsible for initiating actomyosin ring constriction and septation, is assembled at the SPB through its two scaffolding components, Sid4 and Cdc11. In an effort to identify novel SIN interactors, we purified Cdc11 and identified by mass spectrometry a previously uncharacterized protein associated with it, Ppc89. Ppc89 localizes constitutively to the SPB and interacts directly with Sid4. A fusion between the N-terminal 300 amino acids of Sid4 and a SPB targeting domain of Ppc89 supplies the essential function of Sid4 in anchoring the SIN. ppc89Delta cells are inviable and exhibit defects in SPB integrity, and hence in spindle formation, chromosome segregation, and SIN localization. Ppc89 overproduction is lethal, resulting primarily in a G2 arrest accompanied by massive enlargement of the SPB and increased SPB MT nucleation. These results suggest a fundamental role for Ppc89 in organization of the S. pombe SPB.

Sid4p-Cdc11p assembles the septation initiation network and its regulators at the S. pombe SPB.

The Schizosaccharomyces pombe septation initiation network (SIN) triggers actomyosin ring constriction, septation, and cell division. It is organized at the spindle pole body (SPB) by the scaffold proteins Sid4p and Cdc11p. Here, we dissect the contributions of Sid4p and Cdc11p in anchoring SIN components and SIN regulators to the SPB. We find that Sid4p interacts with the SIN activator, Plo1p, in addition to Cdc11p and Dma1p. While the C terminus of Cdc11p is involved in binding Sid4p, its N-terminal half is involved in a wide variety of direct protein-protein interactions, including those with Spg1p, Sid2p, Cdc16p, and Cdk1p-Cdc13p. Given that the localizations of the remaining SIN components depend on Spg1p or Cdc16p, these data allow us to build a comprehensive model of SIN component organization at the SPB. FRAP experiments indicate that Sid4p and Cdc11p are stable SPB components, whereas signaling components of the SIN are dynamically associated with these structures. Our results suggest that the Sid4p-Cdc11p complex organizes a signaling hub on the SPB and that this hub coordinates cell and nuclear division.

The spindle pole body protein Cdc11p links Sid4p to the fission yeast septation initiation network.

The Schizosaccharomyces pombe septation initiation network (SIN) signals the onset of cell division from the spindle pole body (SPB) and is regulated by the small GTPase Spg1p. The localization of SIN components including Spg1p to the SPB is required for cytokinesis and is dependent on Sid4p, a constitutive resident of SPBs. However, a direct interaction between Sid4p and other members of the SIN has not been detected. To understand how Sid4p is linked to other SIN components, we have begun to characterize an S. pombe homolog of the Saccharomyces cerevisiae SPB protein Nud1p. We have determined that this S. pombe Nud1p homolog corresponds to Cdc11p, a previously uncharacterized SIN element. We report that Cdc11p is present constitutively at SPBs and that its function appears to be required for the localization of all other SIN components to SPBs with the exception of Sid4p. The Cdc11p C terminus localizes the protein to SPBs in a Sid4p-dependent manner, and we demonstrate a direct Cdc11p-Sid4p interaction. The N-terminus of Cdc11p is required for Spg1p binding to SPBs. Our studies indicate that Cdc11p provides a physical link between Sid4p and the Spg1p signaling pathway.

Suppression of sorbitol dependence in a strain bearing a mutation in the SRB1/PSA1/VIG9 gene encoding GDP-mannose pyrophosphorylase by PDE2 overexpression suggests a role for the Ras/cAMP signal-transduction pathway in the control of yeast cell-wall biogenesis.

Complementation studies and allele replacement in Saccharomyces cerevisiae revealed that PSA1/VIG9, an essential gene that encodes GDP-mannose pyrophosphorylase, is the wild-type SRB1 gene. Cloning and sequencing of the srb1-1 allele showed that it determines a single amino acid change from glycine to aspartic acid at residue 276 (srb1(D276)). Genetic evidence is presented showing that at least one further mutation is required for the sorbitol dependence of srb1(D276). A previously reported complementing gene, which this study has now identified as PDE2, is a multi-copy suppressor of sorbitol dependence and is not, as was previously suggested, the SRB1 gene. srb and pde2 mutants share a number of phenotypes, including lysis upon hypotonic shock and enhanced transformability. These data are consistent with the idea that the Ras/cAMP pathway might modulate cell-wall construction.