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Characterization of vertebrate cohesin complexes and their regulation in prophase.

Sumara I, Vorlaufer E, Gieffers C, Peters BH, Peters JM - J. Cell Biol. (2000)

Bottom Line: SA1 is also a subunit of 14S cohesin in Xenopus.The bulk of SA1- and SA2-containing complexes and PDS5 are chromatin-associated until they become soluble from prophase to telophase.These results suggest that vertebrate cohesins are regulated by a novel prophase pathway which is distinct from the APC pathway that controls cohesins in yeast.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Molecular Pathology (IMP), A-1030 Vienna, Austria.

ABSTRACT
In eukaryotes, sister chromatids remain connected from the time of their synthesis until they are separated in anaphase. This cohesion depends on a complex of proteins called cohesins. In budding yeast, the anaphase-promoting complex (APC) pathway initiates anaphase by removing cohesins from chromosomes. In vertebrates, cohesins dissociate from chromosomes already in prophase. To study their mitotic regulation we have purified two 14S cohesin complexes from human cells. Both complexes contain SMC1, SMC3, SCC1, and either one of the yeast Scc3p orthologs SA1 and SA2. SA1 is also a subunit of 14S cohesin in Xenopus. These complexes interact with PDS5, a protein whose fungal orthologs have been implicated in chromosome cohesion, condensation, and recombination. The bulk of SA1- and SA2-containing complexes and PDS5 are chromatin-associated until they become soluble from prophase to telophase. Reconstitution of this process in mitotic Xenopus extracts shows that cohesin dissociation does neither depend on cyclin B proteolysis nor on the presence of the APC. Cohesins can also dissociate from chromatin in the absence of cyclin-dependent kinase 1 activity. These results suggest that vertebrate cohesins are regulated by a novel prophase pathway which is distinct from the APC pathway that controls cohesins in yeast.

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Fractionation of human cohesin complexes by sucrose density gradient centrifugation. (A) Characterization of SA1 and SA2 antibodies. (Left) PhosphorImager scan of in vitro–translated 35S-labeled human SA1 and SA2 (IVT-SA1, IVT-SA2) separated by SDS-PAGE. Other panels, control rabbit reticulocyte lysate (RRL), in vitro–translated SA1 and SA2, protein extracts (xt) from HeLa cells, and Xenopus interphase egg extracts, and SA2 (446) immunoprecipitates isolated from HeLa extracts (SA2 IP) were analyzed by SDS-PAGE and immunoblotting with specific SA1 (444) or SA2 (446) antibodies. (B) Sucrose gradient fractions containing proteins from logarithmically growing HeLa cells were analyzed by SDS-PAGE and immunoblotting with antibodies to the indicated proteins. SA1 and SA2 were detected with antibodies 444 and 446, respectively. Prot, proteasome. (C) Sucrose gradient fractions containing proteins from Xenopus interphase extract were analyzed by SDS-PAGE and immunoblotting with antibodies to the indicated proteins.
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Figure 1: Fractionation of human cohesin complexes by sucrose density gradient centrifugation. (A) Characterization of SA1 and SA2 antibodies. (Left) PhosphorImager scan of in vitro–translated 35S-labeled human SA1 and SA2 (IVT-SA1, IVT-SA2) separated by SDS-PAGE. Other panels, control rabbit reticulocyte lysate (RRL), in vitro–translated SA1 and SA2, protein extracts (xt) from HeLa cells, and Xenopus interphase egg extracts, and SA2 (446) immunoprecipitates isolated from HeLa extracts (SA2 IP) were analyzed by SDS-PAGE and immunoblotting with specific SA1 (444) or SA2 (446) antibodies. (B) Sucrose gradient fractions containing proteins from logarithmically growing HeLa cells were analyzed by SDS-PAGE and immunoblotting with antibodies to the indicated proteins. SA1 and SA2 were detected with antibodies 444 and 446, respectively. Prot, proteasome. (C) Sucrose gradient fractions containing proteins from Xenopus interphase extract were analyzed by SDS-PAGE and immunoblotting with antibodies to the indicated proteins.

Mentions: The Scc3p subunit of budding yeast cohesin complexes is homologous to a family of closely related mammalian nuclear proteins called stromal antigens (Toth et al. 1999). Mouse and human cDNAs encoding two different stromal antigens (SA1–SA2) have been described (Carramolino et al. 1997), but the function of these proteins is unknown. To test whether they are cohesin subunits, we generated a panel of antibodies against human SA1 and SA2. In immunoblot experiments, the antibody 444 raised against a peptide of SA1 reacted specifically with in vitro–translated SA1 and with a 150-kD protein of similar electrophoretic mobility in extracts from HeLa cells and from Xenopus eggs, but not with in vitro–translated SA2 (Fig. 1 A). Antibody 446 raised against a peptide of SA2 reacted specifically with the human 140-kD SA2 protein (Fig. 1 A), whereas two other antibodies (445 and 447) recognized both SA1 and SA2 (data not shown).


Characterization of vertebrate cohesin complexes and their regulation in prophase.

Sumara I, Vorlaufer E, Gieffers C, Peters BH, Peters JM - J. Cell Biol. (2000)

Fractionation of human cohesin complexes by sucrose density gradient centrifugation. (A) Characterization of SA1 and SA2 antibodies. (Left) PhosphorImager scan of in vitro–translated 35S-labeled human SA1 and SA2 (IVT-SA1, IVT-SA2) separated by SDS-PAGE. Other panels, control rabbit reticulocyte lysate (RRL), in vitro–translated SA1 and SA2, protein extracts (xt) from HeLa cells, and Xenopus interphase egg extracts, and SA2 (446) immunoprecipitates isolated from HeLa extracts (SA2 IP) were analyzed by SDS-PAGE and immunoblotting with specific SA1 (444) or SA2 (446) antibodies. (B) Sucrose gradient fractions containing proteins from logarithmically growing HeLa cells were analyzed by SDS-PAGE and immunoblotting with antibodies to the indicated proteins. SA1 and SA2 were detected with antibodies 444 and 446, respectively. Prot, proteasome. (C) Sucrose gradient fractions containing proteins from Xenopus interphase extract were analyzed by SDS-PAGE and immunoblotting with antibodies to the indicated proteins.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169443&req=5

Figure 1: Fractionation of human cohesin complexes by sucrose density gradient centrifugation. (A) Characterization of SA1 and SA2 antibodies. (Left) PhosphorImager scan of in vitro–translated 35S-labeled human SA1 and SA2 (IVT-SA1, IVT-SA2) separated by SDS-PAGE. Other panels, control rabbit reticulocyte lysate (RRL), in vitro–translated SA1 and SA2, protein extracts (xt) from HeLa cells, and Xenopus interphase egg extracts, and SA2 (446) immunoprecipitates isolated from HeLa extracts (SA2 IP) were analyzed by SDS-PAGE and immunoblotting with specific SA1 (444) or SA2 (446) antibodies. (B) Sucrose gradient fractions containing proteins from logarithmically growing HeLa cells were analyzed by SDS-PAGE and immunoblotting with antibodies to the indicated proteins. SA1 and SA2 were detected with antibodies 444 and 446, respectively. Prot, proteasome. (C) Sucrose gradient fractions containing proteins from Xenopus interphase extract were analyzed by SDS-PAGE and immunoblotting with antibodies to the indicated proteins.
Mentions: The Scc3p subunit of budding yeast cohesin complexes is homologous to a family of closely related mammalian nuclear proteins called stromal antigens (Toth et al. 1999). Mouse and human cDNAs encoding two different stromal antigens (SA1–SA2) have been described (Carramolino et al. 1997), but the function of these proteins is unknown. To test whether they are cohesin subunits, we generated a panel of antibodies against human SA1 and SA2. In immunoblot experiments, the antibody 444 raised against a peptide of SA1 reacted specifically with in vitro–translated SA1 and with a 150-kD protein of similar electrophoretic mobility in extracts from HeLa cells and from Xenopus eggs, but not with in vitro–translated SA2 (Fig. 1 A). Antibody 446 raised against a peptide of SA2 reacted specifically with the human 140-kD SA2 protein (Fig. 1 A), whereas two other antibodies (445 and 447) recognized both SA1 and SA2 (data not shown).

Bottom Line: SA1 is also a subunit of 14S cohesin in Xenopus.The bulk of SA1- and SA2-containing complexes and PDS5 are chromatin-associated until they become soluble from prophase to telophase.These results suggest that vertebrate cohesins are regulated by a novel prophase pathway which is distinct from the APC pathway that controls cohesins in yeast.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Molecular Pathology (IMP), A-1030 Vienna, Austria.

ABSTRACT
In eukaryotes, sister chromatids remain connected from the time of their synthesis until they are separated in anaphase. This cohesion depends on a complex of proteins called cohesins. In budding yeast, the anaphase-promoting complex (APC) pathway initiates anaphase by removing cohesins from chromosomes. In vertebrates, cohesins dissociate from chromosomes already in prophase. To study their mitotic regulation we have purified two 14S cohesin complexes from human cells. Both complexes contain SMC1, SMC3, SCC1, and either one of the yeast Scc3p orthologs SA1 and SA2. SA1 is also a subunit of 14S cohesin in Xenopus. These complexes interact with PDS5, a protein whose fungal orthologs have been implicated in chromosome cohesion, condensation, and recombination. The bulk of SA1- and SA2-containing complexes and PDS5 are chromatin-associated until they become soluble from prophase to telophase. Reconstitution of this process in mitotic Xenopus extracts shows that cohesin dissociation does neither depend on cyclin B proteolysis nor on the presence of the APC. Cohesins can also dissociate from chromatin in the absence of cyclin-dependent kinase 1 activity. These results suggest that vertebrate cohesins are regulated by a novel prophase pathway which is distinct from the APC pathway that controls cohesins in yeast.

Show MeSH
Related in: MedlinePlus