Structural and functional characterization of the unique N-terminus of Cse4p, A histone H3-like protein at the Saccharomyces cerevisiae centromere
The budding yeast (S. cerevisiae) centromere component, Cse4p is an evolutionarily conserved histone H3-like protein, with homologues identified in fission yeast, worm, fly and human. All histone H3-like proteins have C-terminal histone fold domains (HFD) that are highly similar to the HFD of H3, but carry very different N-termini with unknown functions. The Cse4p N-terminus contains 135 residues, with a large portion of charged amino acids and a high concentration of serines within the first 22 residues. Based on the current model that suggests that Cse4p replaces H3 in a specialized centromeric nucleosome, the Cse4p N-terminus would extend out from the putative Cse4p-nucleosome and may play a variety of roles in centromere function.
To elucidate the function of the Cse4p N-terminus, we conducted two comprehensive and systematic mutagenesis studies involving alanine scanning and sequence deletions, and we defined a 33-amino acid domain that is essential for cell viability and chromosome segregation. This essential N-terminal domain (END) has functions distinct from that of the HFD as demonstrated by interallelic complementation between cse4 END and HFD mutant alleles and heterodimer formation of END-HFD mutant proteins. Mutating all the potential posttranslational sites in the END indicates that the END function does not require posttranslational phosphorylation or acetylation. Genetic studies involving dosage suppression, synthetic lethality and two-hybrid analysis reveal that the END interacts with the Ctf19p/Mcm21p/Okp1p kinetochore complex. These results are consistent with the current Cse4p-nucleosome model.
Although Cse4p has an HFD resembling that of H3, unlike H3, Cse4p exclusively localizes at the centromere. An important question is whether the N-terminus of Cse4p is responsible for the specific centromere targeting of the protein. Lethal Cse4p proteins lacking regions of the N-terminus can localize to the centromere in the presence or absence of wildtype Cse4p as determined by chromatin immunoprecipitation. In contrast, some lethal Cse4p HFD mutant proteins as well as chimeric proteins consisting of the Cse4p N-terminus fused to the HFD of either H3 or the Cse4p human homologue, CENP-A, fail to localize to the centromere. We conclude that the N-terminus of Cse4p is not required for centromere targeting of the protein and that the Cse4p HFD is necessary and sufficient to confer centromere localization.