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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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SA1 and SA2 are subunits of two distinct human 14S cohesin complexes. (A) Low-speed supernatant of extracts from logarithmically growing HeLa cells or Xenopus interphase egg extracts were analyzed by immunoprecipitation (IP) with either preimmune (P) or immune antibodies (I) against SA1 (444) or SA2 (446) and analyzed by SDS-PAGE and Western blotting (WB) with antibodies to the indicated proteins. (B) HeLa extracts were immunodepleted as in A and the resulting supernatant fractions were analyzed by SDS-PAGE and immunoblotting with SA1 (444) and SA2 (446) antibodies.
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Figure 2: SA1 and SA2 are subunits of two distinct human 14S cohesin complexes. (A) Low-speed supernatant of extracts from logarithmically growing HeLa cells or Xenopus interphase egg extracts were analyzed by immunoprecipitation (IP) with either preimmune (P) or immune antibodies (I) against SA1 (444) or SA2 (446) and analyzed by SDS-PAGE and Western blotting (WB) with antibodies to the indicated proteins. (B) HeLa extracts were immunodepleted as in A and the resulting supernatant fractions were analyzed by SDS-PAGE and immunoblotting with SA1 (444) and SA2 (446) antibodies.

Mentions: When we separated HeLa cell extracts by anion exchange and gel filtration chromatography, both SA1 and SA2 cofractionated with the known subunits of 14S cohesin, SMC1, SMC3, and SCC1 (data not shown). In density gradient centrifugation experiments, SMC1 and SMC3 sedimented as 9S and 14S cohesin complexes, as previously shown for their Xenopus orthologs (Losada et al. 1998). SA2 cosedimented entirely and SA1 in part with SMC1, SMC3, and SCC1 (Fig. 1 B), suggesting that they may be subunits of 14S cohesin. To test this hypothesis we performed coimmunoprecipitation experiments that were analyzed by immunoblotting (Fig. 2 A, left). Both the SA1-specific antibody 444 and the SA2 antibody 446 coprecipitated SCC1, SMC1, and SCC3. However, the SA1 antibodies did not precipitate SA2 and the SA2 antibodies did not precipitate SA1. Likewise, SA1 antibodies depleted SA1 but not SA2, and SA2 antibodies depleted SA2 but not SA1 from cell extracts (Fig. 2 B). These results indicate that SA1 and SA2 assemble with SMC1, SMC3, and SCC1 into two distinct types of 14S cohesin complexes that contain either SA1 or SA2. In density gradient centrifugation experiments, SA1 was also found in fractions that contain 9S cohesin (Fig. 1 B), but coimmunoprecipitation experiments indicated that SA1 is not associated with SMC1 and SMC3 in these fractions (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)

SA1 and SA2 are subunits of two distinct human 14S cohesin complexes. (A) Low-speed supernatant of extracts from logarithmically growing HeLa cells or Xenopus interphase egg extracts were analyzed by immunoprecipitation (IP) with either preimmune (P) or immune antibodies (I) against SA1 (444) or SA2 (446) and analyzed by SDS-PAGE and Western blotting (WB) with antibodies to the indicated proteins. (B) HeLa extracts were immunodepleted as in A and the resulting supernatant fractions were analyzed by SDS-PAGE and immunoblotting with SA1 (444) and SA2 (446) antibodies.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169443&req=5

Figure 2: SA1 and SA2 are subunits of two distinct human 14S cohesin complexes. (A) Low-speed supernatant of extracts from logarithmically growing HeLa cells or Xenopus interphase egg extracts were analyzed by immunoprecipitation (IP) with either preimmune (P) or immune antibodies (I) against SA1 (444) or SA2 (446) and analyzed by SDS-PAGE and Western blotting (WB) with antibodies to the indicated proteins. (B) HeLa extracts were immunodepleted as in A and the resulting supernatant fractions were analyzed by SDS-PAGE and immunoblotting with SA1 (444) and SA2 (446) antibodies.
Mentions: When we separated HeLa cell extracts by anion exchange and gel filtration chromatography, both SA1 and SA2 cofractionated with the known subunits of 14S cohesin, SMC1, SMC3, and SCC1 (data not shown). In density gradient centrifugation experiments, SMC1 and SMC3 sedimented as 9S and 14S cohesin complexes, as previously shown for their Xenopus orthologs (Losada et al. 1998). SA2 cosedimented entirely and SA1 in part with SMC1, SMC3, and SCC1 (Fig. 1 B), suggesting that they may be subunits of 14S cohesin. To test this hypothesis we performed coimmunoprecipitation experiments that were analyzed by immunoblotting (Fig. 2 A, left). Both the SA1-specific antibody 444 and the SA2 antibody 446 coprecipitated SCC1, SMC1, and SCC3. However, the SA1 antibodies did not precipitate SA2 and the SA2 antibodies did not precipitate SA1. Likewise, SA1 antibodies depleted SA1 but not SA2, and SA2 antibodies depleted SA2 but not SA1 from cell extracts (Fig. 2 B). These results indicate that SA1 and SA2 assemble with SMC1, SMC3, and SCC1 into two distinct types of 14S cohesin complexes that contain either SA1 or SA2. In density gradient centrifugation experiments, SA1 was also found in fractions that contain 9S cohesin (Fig. 1 B), but coimmunoprecipitation experiments indicated that SA1 is not associated with SMC1 and SMC3 in these fractions (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