Evidence that the MIF2 gene of Saccharomyces cerevisiae encodes a centromere protein with homology to the mammalian centromere protein CENP-C.

Abstract

The MIF2 gene of Saccharomyces cerevisiae has been implicated in mitosis. Here we provide genetic evidence that MIF2 encodes a centromere protein. Specifically, we found that mutations in MIF2 stabilize dicentric minichromosomes and confer high instability (i.e., a synthetic acentric phenotype) to chromosomes that bear a cis-acting mutation in element I of the yeast centromeric DNA (CDEI). Similarly, we observed synthetic phenotypes between mutations in MIF2 and trans-acting mutations in three known yeast centromere protein genes-CEP1/CBF1/CPF1, NDC1/CBF2, and CEP3/CBF3B. In addition, the mif2 temperature-sensitive phenotype can be partially rescued by increased dosage of CEPI. Synthetic lethal interactions between a cepl null mutation and mutations in either NDC1O or CEP3 were also detected. Taken together, these data suggest that the Mif2 protein interacts with Ceplp at the centromere and that the yeast centromere indeed exists as a higher order protein-DNA complex. The Mif2 and Cepl proteins contain motifs of known transcription factors, suggesting that assembly of the yeast centromere is analogous to that of eukaryotic enhancers and origins of replication. We also show that the predicted Mif2 protein shares two short regions of homology with the mammalian centromere Ag CENP-C and that two temperature-sensitive mutations in MIF2 lie within these regions. These results provide evidence for structural conservation between yeast and mammalian centromeres. INTRODUCTION The proper segregation of eukaryotic chromosomes is mediated by a specialized chromosomal structure, termed the centromere or kinetochore. Studies of the centromere in vitro and in vivo suggest it is a multifunctional complex that can capture and stabilize microtubules, promote bidirectional chromosome movement along microtubules, facilitate polymerization and depolymerization of microtubules, and mediate sister chromatid association until the onset of anaphase (reviewed in Mitchison, 1988;Schulman and Bloom, 1991). To understand the molecular basis of these activities, it is necessary to identify the cis-and trans-acting components of the centromere and to elu-* Corresponding author. cidate how they assemble into a higher order structure with the appropriate biochemical properties.Although some progress has been made in the molecular analysis of larger centromeres from mammals and fission yeast, the less complex centromeres of the yeast Saccharomyces cerevisiae have proved more amenable to detailed molecular genetic analysis of structure and function. Functional yeast centromeric DNA sequences (-125 bp) were identified by their ability to confer mitotic and meiotic stability to small recombinant DNA plasmids (minichromosomes) in yeast cells (Clarke and Carbon, 1980;Fitzgerald-Hayes et al., 1982;Hieter et al., 1985). These sequences are comprised of three conserved centromere DNA elements (Figure 1) Hegemann and Fleig, 1993). In addition, in vivo, a unique nuclease-resistant chromatin structure (encompassing -200 bp) is associated with the centro...

The Saccharomyces cerevisiae kinetochore

“…Mif2p, originally assigned to have a role in spindle integrity during anaphase [23], may also be a component of the S. cerevisiae kinetochore [24]. This is supported by two lines of evidence.…”

A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex.

“…It remains to be determined whether these properties result strictly from a failure to reimport NuMA into the nucleus after mitosis. Finally, the yeast homologue of GMP1, Smt3p, was originally identified as a suppressor of the MIF2 gene, whose protein product is a centromere protein required for mitotic spindle integrity (Meluh and Koshland, 1995). It is presently unkown whether Smt3p also localizes to the spindle in yeast and whether it is conjugated to the RanGAP1 homologue.…”

“…Overall identities between GMP1 and the worm, rice, and yeast homologues are 59, 39, and 40%, respectively, whereas identities between the carboxyl-terminal domains are 64, 42, and 46%, respectively. Interestingly, the yeast homologue (Smt3p) was originally identified as a suppressor of MIF2, a gene encoding a centromere-associated protein required for mitotic spindle integrity (Meluh and Koshland, 1995).…”

“…D76147, D73158, and T02042, and the rice homologue (O. sativa) was derived from clones D15376 and D22620. The yeast sequence (S. cerevisiae) is derived from a cDNA clone coding Smt3p (Meluh and Koshland, 1995). precursors (all known ubiquitins are synthesized as precursors with carboxyl-terminal extensions, or as polyubiquitin), the double glycines present in GMP1 and in the GMPl-related proteins are not at the extreme carboxyl terminus.…”

Drosophila Smt3 negatively regulates JNK signaling through sequestering Hipk in the nucleus

“…The gene that encodes SUMO was initially identified in Saccharomyces cerevisiae (Meluh and Koshland, 1995). In mammalian cells, there are three SUMO genes, whereas only a single gene, smt3, exists in Drosophila (Huang et al, 1998;Johnson et al, 1997;Su and Li, 2002), making Drosophila a useful experimental system in which to study the biological functions of sumoylation.…”