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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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Purification of human cohesin complexes. (A) Immunoprecipitates (IP) obtained with either SA1/2 (447) antibodies or as a control with CDC27 antibodies (APC IP) from low-speed supernatant of extracts from logarithmically growing HeLa cells were analyzed by SDS-PAGE and silver staining. The positions of cohesin subunits, p85 and APC subunits as determined by immunoblotting are indicated. (B) Proteins were eluted with antigenic peptides from cohesin immunoprecipitates obtained as in A and further separated by sucrose density gradient centrifugation. Fractions 5–8 were analyzed by SDS-PAGE and immunoblotting with antibodies to the indicated proteins. (C) Sucrose density gradient fractions from the experiment shown in B were analyzed by SDS-PAGE and silver staining.
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Figure 3: Purification of human cohesin complexes. (A) Immunoprecipitates (IP) obtained with either SA1/2 (447) antibodies or as a control with CDC27 antibodies (APC IP) from low-speed supernatant of extracts from logarithmically growing HeLa cells were analyzed by SDS-PAGE and silver staining. The positions of cohesin subunits, p85 and APC subunits as determined by immunoblotting are indicated. (B) Proteins were eluted with antigenic peptides from cohesin immunoprecipitates obtained as in A and further separated by sucrose density gradient centrifugation. Fractions 5–8 were analyzed by SDS-PAGE and immunoblotting with antibodies to the indicated proteins. (C) Sucrose density gradient fractions from the experiment shown in B were analyzed by SDS-PAGE and silver staining.

Mentions: The association of SA1 and SA2 with human cohesin subunits was further confirmed by analyzing the protein composition of SA1/SA2 immunoprecipitates by silver staining. Extracts from logarithmically growing HeLa cells were immunoprecipitated with the antibody 447, which recognizes both SA1 and SA2. After elution of bound proteins with either buffers of low pH (Fig. 3 A) or the antigenic peptide (Fig. 3 C) we observed protein bands corresponding to 160, 140–150, 120, and 85 kD, with the 140–150-kD band often appearing as a doublet (Fig. 3 A). Immunoblot experiments suggested that the 160-kD band contained SMC1, the 140–150-kD doublet SMC3 and SA2 in the lower band and SA1 in the upper band, and the 120-kD band contained SCC1 (Fig. 3 B). None of these proteins could be precipitated with antibodies to other protein complexes such as the APC (Fig. 3 A) or with control immunoglobulins, suggesting that their coprecipitation with SA1 and SA2 is specific.


Characterization of vertebrate cohesin complexes and their regulation in prophase.

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

Purification of human cohesin complexes. (A) Immunoprecipitates (IP) obtained with either SA1/2 (447) antibodies or as a control with CDC27 antibodies (APC IP) from low-speed supernatant of extracts from logarithmically growing HeLa cells were analyzed by SDS-PAGE and silver staining. The positions of cohesin subunits, p85 and APC subunits as determined by immunoblotting are indicated. (B) Proteins were eluted with antigenic peptides from cohesin immunoprecipitates obtained as in A and further separated by sucrose density gradient centrifugation. Fractions 5–8 were analyzed by SDS-PAGE and immunoblotting with antibodies to the indicated proteins. (C) Sucrose density gradient fractions from the experiment shown in B were analyzed by SDS-PAGE and silver staining.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Purification of human cohesin complexes. (A) Immunoprecipitates (IP) obtained with either SA1/2 (447) antibodies or as a control with CDC27 antibodies (APC IP) from low-speed supernatant of extracts from logarithmically growing HeLa cells were analyzed by SDS-PAGE and silver staining. The positions of cohesin subunits, p85 and APC subunits as determined by immunoblotting are indicated. (B) Proteins were eluted with antigenic peptides from cohesin immunoprecipitates obtained as in A and further separated by sucrose density gradient centrifugation. Fractions 5–8 were analyzed by SDS-PAGE and immunoblotting with antibodies to the indicated proteins. (C) Sucrose density gradient fractions from the experiment shown in B were analyzed by SDS-PAGE and silver staining.
Mentions: The association of SA1 and SA2 with human cohesin subunits was further confirmed by analyzing the protein composition of SA1/SA2 immunoprecipitates by silver staining. Extracts from logarithmically growing HeLa cells were immunoprecipitated with the antibody 447, which recognizes both SA1 and SA2. After elution of bound proteins with either buffers of low pH (Fig. 3 A) or the antigenic peptide (Fig. 3 C) we observed protein bands corresponding to 160, 140–150, 120, and 85 kD, with the 140–150-kD band often appearing as a doublet (Fig. 3 A). Immunoblot experiments suggested that the 160-kD band contained SMC1, the 140–150-kD doublet SMC3 and SA2 in the lower band and SA1 in the upper band, and the 120-kD band contained SCC1 (Fig. 3 B). None of these proteins could be precipitated with antibodies to other protein complexes such as the APC (Fig. 3 A) or with control immunoglobulins, suggesting that their coprecipitation with SA1 and SA2 is specific.

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