The Fission Yeast Kinetochore Component Spc7 Associates with the EB1 Family Member Mal3 and Is Required for Kinetochore–Spindle Association

Abstract

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...

KNL1 and the CENP-H/I/K Complex Coordinately Direct Kinetochore Assembly in Vertebrates

“…Recent work in fission yeast demonstrated that the KNL-1 homologue Spc7 is not required for Ndc80 localization, but also reported synthetic lethal genetic interactions between spc7 and mis6 (CENP-I) or sim4 (CENP-K) mutants (Kerres et al, 2004), consistent with the synergistic defects we describe here for hKNL1/ CENP-K double inhibitions. Overall, these results suggest evolutionary plasticity in kinetochore assembly, specifically in the recruitment of the critical Ndc80 complex.…”

“…A key player in kinetochore assembly in C. elegans is KNL-1, which functions downstream of the inner kinetochore components such as CENP-A, but upstream of all known outer kinetochore proteins (Desai et al, 2003;Cheeseman et al, 2004). KNL-1 belongs to a conserved protein family that includes budding yeast Spc105 (Nekrasov et al, 2003), fission yeast Spc7 (Kerres et al, 2004), Drosophila Spc105R (Przewloka et al, 2007), and a novel protein previously identified in human cells known as AF15q14, D40, CASC5, or Blinkin (Cheeseman et al, 2004;Obuse et al, 2004;Kiyomitsu et al, 2007). AF15q14 is present in all vertebrates including chicken (Gallus gallus).…”

Asp1, a Conserved 1/3 Inositol Polyphosphate Kinase, Regulates the Dimorphic Switch inSchizosaccharomyces pombe

“…S. pombe strains were grown in rich Protein analysis. Protein extracts were prepared as described previously (35). Blots were probed with anti-green fluorescent protein (anti-GFP) antibody (polyclonal rabbit; Invitrogen) followed by alkaline phosphatase (AP)-conjugated secondary antibody (affinity-purified goat anti-rabbit IgG [Fc]; Promega, Madison, WI) and detected with 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium (BCIP/NBT) as the substrate.…”

The V260I Mutation in Fission Yeast α-Tubulin Atb2 Affects Microtubule Dynamics and EB1-Mal3 Localization and Activates the Bub1 Branch of the Spindle Checkpoint

“…Recent work in fission yeast indicates that Mal3 interacts with the kinetochore component Spc7 (homologue of budding yeast Spc105 and human KIAA1570) during mitosis and is important for accurate chromosome segregation (Kerres et al, 2004). We have recently shown that in the absence of Mal3, mitotic delay is induced, in which the Bub1 branch of the spindle checkpoint is activated, and Bub1-localizing kinetochores are associated for a prolonged period of time with only one of two separating spindle pole bodies (SPBs) Asakawa and Toda, 2006).…”