Spc42p: A Phosphorylated Component of the S. cerevisiae Spindle Pole Body (SPB) With an Essential Function During SPB Duplication

The 42-kD component of the S. cerevisiae spindle pole body (SPB) localizes to the electron-dense central plaque of the SPB (Rout, M.P., and J.V. Kilmartin. 1991. Cold Spring Harbor Symp. Quant. Biol. 56:687--691). We have cloned the corresponding gene SPC42 (spindle pole component) and show that it is essential. Seven temperature-sensitive (ts) mutants in SPC42 were prepared by error-prone PCR. We found that a change to a proline residue in a potential coiledcoil region of Spc42p was responsible for the ts phenotype in at least three alleles, suggesting that formation of the coiled-coil is essential for normal function. The mutant cells showed a phenotype of predominantly single or bilobed SPBs often with an accumulation of unstructured electron-dense material associated with the bridge structure adjacent to the SPB. This phenotype suggests a defect in SPB duplication. This was confirmed by examining synchronized mutant cells that lose viability when SPB duplication is attempted. Spc42p is a phosphoprotein which shows some cell cycle-regulated phosphorylation. Overexpression of Spc42p causes the formation of a discor dome-shaped polymer composed of phosphorylated Spc42p, which is attached to the central plaque and associated with the outer nuclear membrane. Taken together, these data suggest that Spc42p forms a polymeric layer at the periphery of the SPB central plaque which has an essential function during SPB duplication and may facilitate attachment of the SPB to the nuclear membrane. T hE spindle pole body (SPB) 1 of Saccharomyces cerevisiae is the functional equivalent of the centrosome in the yeast cell. It is a complex cylindrical multi-layered structure lying in the nuclear envelope. The central layer or plaque lies in the plane of the nuclear membrane. On the cytoplasmic side is the outer plaque that organizes cytoplasmic microtubules, while nuclear or spindle microtubules are organized from a more illdefined inner plaque (Byers, 1981a; Rose et al., 1993; Winey and Byers, 1993). Like the centrosome, the SPB is duplicated during G1/S. The daughter SPB assembles on the satellite structure, which is cytoplasmic and attached to the mother SPB by a short electron-dense segment of the nuclear membrane called the half-bridge (Byers and Goetsch, 1975; Winey et al., 1991a). During or after duplication the daughter SPB is inserted into the nuclear envelope. Both SPBs remain connected by the bridge until this structure is somehow partitioned between the two SPBs as they migrate to opposite positions on the nuclear envelope and form the mitotic spindle (Byers and Goetsch, 1974). Address correspondence to J.V. Kilmartin, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England. Tel.: 44 1223 402242. Fax: 44 1223 412142. E-mail: [email protected] 1. Abbreviat ions used in this paper: CIP, calf intestinal alkaline phosphatase; MCB, MluI cell cycle box; SPB, spindle pole body; SPC, spindle pole component; ts, temperature-sensitive. At present an explanation of SPB functions in molecular terms is in its very early stages. Genetic and biochemical approaches have identified a number of structural components of the SPB. Cdc31p (Baum et al., 1986; Spang et al., 1993), Karlp (Rose and Fink, 1987; Spang et al., 1995), and calmodulin (Geiser et al., 1993; Stirling et al., 1994) were identified from genetic screens, while 110-, 90-, and 42-kD components were identified using mAbs prepared against an enriched SPB preparation (Rout and Kilmartin, 1990, 1991). Screens for mutants with defects in SPB assembly have identified MPS1, MPS2 (Winey et al., 1991b), and NDC1 (Thomas and Botstein, 1986; Winey et al., 1993), although it has not yet been established what precise role these gene products have in SPB duplication. A combination of genetic and biochemical experiments has provided a partial understanding of the functions of some of these SPB components. Cdc31p and Karlp are both essential proteins required for SPB duplication. They interact with each other (Biggins and Rose, 1994; Vallen et al., 1994; Spang et al., 1995) and temperature-sensitive (ts) mutants in both proteins show the same phenotype: cells arrest with a single enlarged SPB (Byers, 1981b; Rose and Fink, 1987). SPB components in these cells appear to assemble around the preexisting SPB rather than the satellite, perhaps due to a defect in the associated half-bridge, since this is where both Cdc31p and Karlp are localized (Spang et al., 1993; Spang et al., 1995). Cdc31p is a calmod© The Rockefeller University Press, 0021-9525/96/03/887/15 $2.00 The Journal of CeU Biology, Volume 132, Number 5, March 1996 887-901 887 on Jauary 2, 2018 jcb.rress.org D ow nladed fom ulin-like protein (Baum et al., 1986) that has homologues in Chlamydomonas (Huang et al., 1988) and mammals (Lee and Huang, 1993; Errabolu et al., 1994). Karlp is a membrane protein (Rose and Fink, 1987) with no homologues yet known in higher eukaryotes. The gene for the l l0 -kD SPB component, SPCllO (Kilmartin et al., 1993) is identical to NUF1 (Mirzayan et al., 1992). Spcl l0p contains a long central coiled-coil region. Deletions in this coiled-coil show that Spcl l0p acts as a spacer protein between the central plaque and the ends of the nuclear microtubules (Kilmartin et al., 1993). Spcl l0p is a calmodulin-binding protein and the essential function of calmodulin during mitosis occurs via an interaction with Spcl l0p (Geiser et al., 1993; Stirling et al., 1994). While some progress has been made in understanding the molecular architecture of the SPB, very little is known about the regulation of its assembly at G1/S. The CDC28/ cdc2 kinase clearly has a pivotal role in both daughter SPB assembly and the initiation of S phase (Hartwell, 1974; Nurse, 1990). However in budding yeast few potential substrates of the CDC28 kinase have been identified. The kinase Mpslp (Lauze et al., 1995) may also be involved in SPB duplication but its substrates are also unknown. In this paper we show that the 42-kD component, which localizes to the electron-dense central plaque of the SPB (Rout and Kilmartin, 1991), is a phosphoprotein showing some cell cycle-regulated phosphorylation. Its gene, SPC42 (Spindle Pole Component), is essential and ts mutants show alterations in the central plaque structure and defects in SPB duplication. Experiments on synchronized cells suggest that SPC42 exercises an essential function during SPB duplication. Overexpression of Spc42p causes the formation of a polymer associated with the central plaque which is composed of phosphorylated Spc42p. Materials and Methods