Separase Cleaves the N-Tail of the CENP-A Related Protein CPAR-1 at the Meiosis I Metaphase-Anaphase Transition in C. elegans.
Centromeres are defined epigenetically in the majority of eukaryotes by the presence of chromatin containing the centromeric histone H3 variant CENP-A. Most species have a single gene encoding a centromeric histone variant whereas C. elegans has two: HCP-3 (also known as CeCENP-A) and CPAR-1. Prior RNAi replacement experiments showed that HCP-3 is the functionally dominant isoform, consistent with CPAR-1 not being detectable in embryos. GFP::CPAR-1 is loaded onto meiotic chromosomes in diakinesis and is enriched on bivalents until meiosis I. Here we show that GFP::CPAR-1 signal loss from chromosomes precisely coincides with homolog segregation during anaphase I. This loss of GFP::CPAR-1 signal reflects proteolytic cleavage between GFP and the histone fold of CPAR-1, as CPAR-1::GFP, in which GFP is fused to the C-terminus of CPAR-1, does not exhibit any loss of GFP signal. A focused candidate screen implicated separase, the protease that initiates anaphase by cleaving the kleisin subunit of cohesin, in this cleavage reaction. Examination of the N-terminal tail sequence of CPAR-1 revealed a putative separase cleavage site and mutation of the signature residues in this site eliminated the cleavage reaction, as visualized by retention of GFP::CPAR-1 signal on separating homologous chromosomes at the metaphase-anaphase transition of meiosis I. Neither cleaved nor uncleavable CPAR-1 were centromere-localized in mitosis and instead localized throughout chromatin, indicating that centromere activity has not been retained in CPAR-1. Although the functions of CPAR-1 and of its separase-dependent cleavage remain to be elucidated, this effort reveals a new substrate of separase and provides an in vivo biosensor to monitor separase activity at the onset of meiosis I anaphase.
Ludwig Institute for Cancer Research & Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California, United States of America.
Joost
J
Monen
Ludwig Institute for Cancer Research & Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California, United States of America.
Neil
N
Hattersley
Ludwig Institute for Cancer Research & Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California, United States of America.
Andrew
A
Muroyama
Ludwig Institute for Cancer Research & Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California, United States of America.
Deanna
D
Stevens
Ludwig Institute for Cancer Research & Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California, United States of America.
Karen
K
Oegema
Ludwig Institute for Cancer Research & Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California, United States of America.
Arshad
A
Desai
D000595:Amino Acid Sequence; D000705:Anaphase; D000818:Animals; D001324:Autoantigens / Q000378:metabolism*; D017173:Caenorhabditis elegans / Q000166:cytology* / Q000235:genetics / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry* / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism; D002503:Centromere / Q000378:metabolism; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D020090:Chromosome Segregation; D002875:Chromosomes / Q000378:metabolism; D020440:Gene Duplication; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D008540:Meiosis; D008677:Metaphase; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D064247:Separase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D001324:Autoantigens; C000606335:CPAR-1 protein, C elegans; D029742:Caenorhabditis elegans Proteins; D018797:Cell Cycle Proteins; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone; D006657:Histones; D049452:Green Fluorescent Proteins; D064247:Separase