Functional Complementation of Human Centromere Protein A (CENP-A) by Cse4p from Saccharomyces cerevisiae

Abstract

We have employed a novel in vivo approach to study the structure and function of the eukaryotic kinetochore multiprotein complex. RNA interference (RNAi) was used to block the synthesis of centromere protein A (CENP-A) and Clip-170 in human cells. By coexpression, homologous kinetochore proteins from Saccharomyces cerevisiae were then tested for the ability to complement the RNAi-induced phenotypes. Cse4p, the budding yeast CENP-A homolog, was specifically incorporated into kinetochore nucleosomes and was able to complement RNAi-induced cell cycle arrest in CENP-A-depleted human cells. Thus, Cse4p can structurally and functionally substitute for CENP-A, strongly suggesting that the basic features of centromeric chromatin are conserved between yeast and mammals. Bik1p, the budding yeast homolog of human CLIP-170, also specifically localized to kinetochores during mitosis, but Bik1p did not rescue CLIP-170 depletion-induced cell cycle arrest. Generally, the newly developed in vivo complementation assay provides a powerful new tool for studying the function and evolutionary conservation of multiprotein complexes from yeast to humans.Centromeres are eukaryotic cellular structures that are essential for faithful chromosomal segregation during mitotic and meiotic cell division. The kinetochore complex is a defined multiprotein structure on the mitotic chromosome that adheres to the centromere (18, 61). The kinetochore serves as the site of attachment for spindle microtubules, which facilitate the alignment and separation of chromosomes during mitosis (12,13). Although the centromere's function is highly conserved among eukaryotes, centromeric morphology varies significantly, ranging from small, simple kinetochores in the budding yeast Saccharomyces cerevisiae to complex centromeres in multicellular eukaryotes (14). In mammalian cells, the centromere forms a visible primary constriction during metaphase and the kinetochore is a distinct structure that can be resolved into subregions (45,47,67). Finally, in holokinetic organisms such as the nematode Caenorhabditis elegans, multiple centromeres are dispersed throughout the chromosomes (1, 2). The primary nucleotide sequences of centromeric DNAs are also not conserved across phylogenies. The absolute size of the centromeric DNA can vary from ca. 125 bp in S. cerevisiae to tens of megabases in higher eukaryotes (11). Beyond the lack of size and sequence conservation between organisms, the centromere's function may be established not only at predefined sequences, but also at noncentromeric DNA elements, as illustrated by neocentromeres in human (11) and plant (93) cells. Finally, while in budding yeast the centromere DNA alone can nucleate centromere formation de novo, centromeres of metazoan cells strongly depend on epigenetic factors rather than DNA sequences for their activity (90). Thus, there is no primary sequence determinant in centromeric DNA that is conserved among eukaryotic species.At the protein level, a series of kinetochore components show homology to proteins...

Cdc2-mediated Phosphorylation of CLIP-170 Is Essential for Its Inhibition of Centrosome Reduplication

“…Recently, it was shown that knockdown of CLIP-170 with RNAi results in an almost complete block of cells in mitosis (13). Considering the essential role of Cdc2 in mitosis, we explored the possibility of a potential direct connection between CLIP-170 and Cdc2.…”

“…The role of CLIP-170 during mitosis was recently examined by loss-offunction approaches. It was shown that CLIP-170 localizes to unattached kinetochores in prometaphase and that such localization is essential for the formation of kinetochore-microtubule attachments (12,13).…”

CLIP-170 facilitates the formation of kinetochore–microtubule attachments

“…In mammalian cells, inhibition of CLIP-170 function using a dominant-negative approach results in a slight increase of mitotic cells and a relative increase in prometaphase cells (Dujardin et al, 1998). A more recent report showed that knockdown of CLIP-170 proteins levels through RNA interference (RNAi) results in a near complete block of cells in mitosis (Wieland et al, 2004), suggesting that CLIP-170 has an important role in mitotic progression. Here, we have addressed the molecular function of CLIP-170 in mitosis and the mechanisms that control its localization.…”

Phylogenetic Analysis of Fungal Centromere H3 Proteins

“…The respective CenH3's from S. cerevisiae (Cse4), C. elegans (HCP-3, DH6H3), and human (CENP-A) localize to pericentric heterochromatin when expressed in Drosophila cells, and Cse4 and HCP-3 localize to centromeric regions in HeLa cells (Henikoff et al 2000). Wieland et al (2004) found that S. cerevisiae Cse4 functionally complements lethal CENP-A defects induced by RNAi in human tissue culture cells. The species specificity observed among Drosophila CenH3's may be exceptional, in that it can be attributed to the rapidly evolving centromere DNAs within the Drosophila lineage .…”