The Schizosaccharomyces pombe CLIP170-associated protein (CLASP) Peg1 was identified in a screen for mutants with spindle formation defects and a screen for molecules that antagonized EB1 function. The conditional peg1.1 mutant enabled us to identify key features of Peg1 function. First, Peg1 was required to form a spindle and astral microtubules, yet destabilized interphase microtubules. Second, Peg1 was required to slow the polymerization rate of interphase microtubules that establish end-on contact with the cortex at cell tips. Third, Peg1 antagonized the action of S. pombe CLIP170 (Tip1) and EB1 (Mal3). Fourth, although Peg1 resembled higher eukaryotic CLASPs by physically associating with both Mal3 and Tip1, neither Tip1 nor Mal3 was required for Peg1 to destabilize interphase microtubules or for it to associate with microtubules. Conversely, neither Mal3 nor Tip1 required Peg1 to associate with microtubules or cell tips. Consistently, while mal3.⌬ and tip1.⌬ disrupted linear growth, corrupting peg1 + did not. Fifth, peg1.1 phenotypes resembled those arising from deletion of the single heavy or both light chains of fission yeast dynein. Furthermore, all interphase phenotypes arising from peg1 + manipulation relied on dynein function. Thus, the impact of S. pombe CLASP on interphase microtubule behavior is more closely aligned to dynein than EB1 or CLIP170.[Keywords: CLASP; dynein; S. pombe; fission yeast; CLIP170; EB1] Supplemental material is available at http://www.genesdev.org.

A critical aspect of mitosis is the interaction of the kinetochore with spindle microtubules. Fission yeast Mal3 is a member of the EB1 family of microtubule plus-end binding proteins, which have been implicated in this process. However, the Mal3 interaction partner at the kinetochore had not been identified. Here, we show that the mal3 mutant phenotype can be suppressed by the presence of extra Spc7, an essential kinetochore protein associated with the central centromere region. Mal3 and Spc7 interact physically as both proteins can be coimmunoprecipitated. Overexpression of a Spc7 variant severely compromises kinetochore-microtubule interaction, indicating that the Spc7 protein plays a role in this process. Spc7 function seems to be conserved because, Spc105, a Saccharomyces cerevisiae homolog of Spc7, identified by mass spectrometry as a component of the conserved Ndc80 complex, can rescue mal3 mutant strains. INTRODUCTIONSegregation of chromosomes requires the association of spindle microtubules and chromosomes. Attachment of the mitotic spindle fibers occurs at a multicomponent protein complex, the kinetochore, that is assembled on centromeric DNA. This DNA region differs greatly in structure and size among various organisms (reviewed in Pidoux and Allshire, 2000;Cleveland et al., 2003). The budding yeast centromere DNA is the simplest one described and consists of a very well defined 125-base pair region, whereas in higher eucaryotes, centromeric DNA is made up of highly repetitive sequences encompassing up to millions of base pairs. The centromere DNA of the fission yeast Schizosaccharomyces pombe lies in between these two extremes: it occupies between 40 and 100 kb on each chromosome and is composed of a central region flanked by inner and outer repetitive sequences. To date, proteins found to be associated with these regions either bind to the central core region or to the outer repeats, thus pointing to the existence of two distinct domains in the fission yeast centromere (reviewed in Pidoux and Allshire, 2000). The heterochromatic outer repeats are required for centromere cohesion (reviewed in Bernard and Allshire, 2002), whereas the central region is needed for the assembly of the kinetochore per se (Saitoh et al., 1997;Goshima et al., 1999;Jin et al., 2002;Pidoux et al., 2003). However, in spite of the different cis-acting DNA requirements, a substantial number of kinetochore proteins have been conserved from yeast to humans, among them the four-component Ndc80 complex. This complex is required for kinetochore-microtubule association and spindle checkpoint signaling (He et al., 2001;Janke et al., 2001;Wigge and Kilmartin, 2001;Bharadwaj et al., 2004;McCleland et al., 2004).The spindle microtubules that attach to kinetochores are highly dynamic structures that alternate between phases of growth and shrinkage (Kirschner and Mitchison, 1986). This dynamic behavior is also observed after microtubules are attached to kinetochores and is coregulated by components of the kinetochore complex and microtubule...

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