Direct Interaction between Yeast Spindle Pole Body Components: Karlp Is Required for Cdc31p Localization to the Spindle Pole Body

The Saccharomyces cerevisiae genes KAR/ and CDC31 are required for the initial stages of spindie pole body (SPB) duplication in yeast. The Cdc31 protein is most related to caltractin/centrin, a calciumbinding protein present in microtubule organizing centers in many organisms. Because of a variety of genetic interactions between CDC31 and KARl (VaUen, E. A., W. Ho, M. Winey, and M. D. Rose. 1994. Genetics. In press), we wanted to determine whether Cdc31p and Karlp physically interact. Cdc31p was expressed and purified from Escherichia coli and active for binding calcium. Using a protein blotting technique, Cdc31p bound to Karlp in vitro via an essential domain in Karlp required for SPB duplication (Vallen, E. A., M. A. Hiller, T. Y. Scherson, and M. D. Rose. 1992a. J. Cell Biol. 117:1277-1287). By immunofluorescence microscopy, we determined that the interaction also occurs in vivo. Cdc31p was localized to the SPB in wild-type cells but was mislocalized in a karl mutant strain. In a karl mutant containing a dominant CDC31 suppressor, Cdc31p was again localized to the SPB. Furthermore, the localization of Cdc31p to the SPB was affected by the overexpression of Karlp-/~-galactosidase hybrids. Based on these data, we propose that the essential function of Karlp is to localize Cdc31p to the SPB, and that this interaction is normally required for SPB duplication. ~ eukaryotic organisms contain microtubule organizing centers that mediate the assembly of the microtubule-based cytoskeleton. Like the centrosome of higher eukaryotes, the microtubule organizing center in Saccharomyces cerevisiae, called the spindle pole body (SPB) m, mediates the assembly of the mitotic and meiotic spindles (for reviews see Rose et al., 1993; W'mey and Byers, 1993). In addition, the SPB also assembles cytoplasmic microtubules that are essential for the process of nuclear fusion during mating (karyogamy). The SPB is a disc-shaped trilaminar structure embedded in the nuclear envelope (Byers and Goetsch, 1974; Byers and Goetsch, 1975). The central plaque is within the plane of the membrane, whereas the inner and outer plaques face the nucleoplasm and cytoplasm, respectively. In addition, the "half-bridge ~ structure appears to be a distinct modification of the nuclear envelope on one side of the SPB. The inner plaque organiT~s the nuclear microtubules that form the mitotic and meiotic spindles, while the outer plaque organizes the cytoplasmic microtubules that are required for nuclear Address all correspondence to Mark D. Rose, Department of Molecular Biology, Princeton University, Washington Road, Princeton, NJ 08544-1014. 1. Abbwviations used in this paper: IPTG, isopr~yl ~-D-thiogalactopyranoside; SPB, spindle pole body. positioning, spindle orientation, and karyogamy (Jacobs et al., 1988; Delgado and Conde, 1984; Huffaker et al., 1988). As is the case for centriole duplication (Kochanski and Borisy, 1990), SPB duplication appears to be a conservative process that occurs early in the cell cycle before S phase (Winey et al, 1991; Vallen et al., 1992b). The earliest known event in SPB duplication is the formation of a precursor "satellite" structure on the cytoplasmic face of the half-bridge. This is followed by the appearance of two morphologically identical SPBs connected by a complete "bridge" structure. The bridge splits when one SPB migrates to the opposite side of the nucleus to form a bipolar spindle, resulting in two halfbridge structures, one of which remains attached to each SPB. Several genes required for SPB duplication have been identified by mutations that affect microtubule-mediated processes. KAR/ was identified by its role in karyogamy (Conde and Fink, 1976), and further genetic analysis demonstrated that it is required for SPB duplication (Rose and Fink, 1987). These two functions of KAR/are mediated by two separate domains, the karyogamy and SPB domains (Vallen et al., 1992a). Because Kaxlp-~-galactosidase hybrid proteins localize to the SPB, it was proposed that Karlp is a component of the SPB (Vallen et al., 1992b). The SPB domain was found to be both necessary and sufficient for localization of Karlp-B-galactosidase hybrids to the SPB. There© The Rockefeller University Press, 0021-9525/94105/843110 $2.00 The Journal of Cell Biology, Volume 125, Number 4, May 1994 843-852 843 on July 8, 2017 jcb.rress.org D ow nladed fom fore, localization to the SPB probably reflects the essential role of KAR/in SPB duplication. Mutations in KARl result in phenotypes very similar to those produced by mutations in the CDC31 gene. Both/car/ and cdc31 mutations block SPB duplication and the mutants arrest as large budded cells with increased ploidy (Rose and Fink, 1987; Schild et al., 1981). Both mutations block very early in SPB duplication, resulting in a single, abnormally enlarged SPB lacking the associated half-bridge and satellite structures (Byers, 1981; Rose and Fink, 1987). Cdc31p is a member of the caimodulin family of calcium-binding proteins (Baum et al., 1986) and shares greatest homology to the protein caltractin]centrin from Chlamydomonas reinhardtii (Huang et al., 1988b; Salisbury et al., 1988). Caltractin was identified as a major caicium-binding protein in the basal body (Huang et al., 1988a) which serves as the microtubule organizing center in Chlamydomonas. Because mutations in centrin/caltractin are defective for basal body localization and/or segregation (Taillon et al.,' 1992), it is likely that this centrosomal component has a conserved function among diverse organisms. A human homologue of caltractin that localizes to the centrosome was identified (Lee and Huang, 1993), providing further support for the fundamental role of this protein. Based on its homology to caltractin and its mutant phenotype, Cdc3 lp was predicted to be a SPB component. This was recently confirmed by immunoelectron microscopy, which determined that Cdc31p is a component of the half-bridge of the SPB (Spang et al.,