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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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PDS5 is found in association with SA1- and SA2-containing cohesin complexes. (A) Low-speed supernatant of extracts from logarithmically growing HeLa cells was analyzed by immunoprecipitation with either preimmune (P) or immune antibodies (I) to PDS5. Immunoprecipitates (IP) and supernatants (SUP) were analyzed by SDS-PAGE and Western blotting with antibodies to the indicated proteins. (B) HeLa extracts prepared as in A were analyzed by immunoprecipitation with SA1, SA2, or with nonspecific control (MOCK) antibodies and the immunoprecipitates (IP), extracts before immunoprecipitation (Input) and supernatants (Sup) were analyzed by SDS-PAGE and Western blotting with antibodies to PDS5. (C) HeLa extracts prepared as in A were analyzed by immunoprecipitation with either preimmune (P) or immune (I) SA2 antibodies (446). After washing with buffers containing either 150, 250, or 500 mM NaCl, the immunoprecipitates were analyzed by SDS-PAGE and Western blotting with antibodies to the indicated proteins. (D) Xenopus interphase extracts were analyzed by immunoprecipitation with either nonspecific (IP-contr), Xenopus PDS5 (IP-PDS5), or SA1 (IP-SA1) antibodies. The precipitates were analyzed by immunoblotting with PDS5, SA1, and SCC1 antibodies. After immunoprecipitation with control (xtΔcontr), PDS5 (xtΔPDS5), or SA1 (xtΔSA1) antibodies the resulting supernatants were analyzed side by side. PDS5 antibody 648 was used for the IP and 647 for immunoblotting.
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Figure 4: PDS5 is found in association with SA1- and SA2-containing cohesin complexes. (A) Low-speed supernatant of extracts from logarithmically growing HeLa cells was analyzed by immunoprecipitation with either preimmune (P) or immune antibodies (I) to PDS5. Immunoprecipitates (IP) and supernatants (SUP) were analyzed by SDS-PAGE and Western blotting with antibodies to the indicated proteins. (B) HeLa extracts prepared as in A were analyzed by immunoprecipitation with SA1, SA2, or with nonspecific control (MOCK) antibodies and the immunoprecipitates (IP), extracts before immunoprecipitation (Input) and supernatants (Sup) were analyzed by SDS-PAGE and Western blotting with antibodies to PDS5. (C) HeLa extracts prepared as in A were analyzed by immunoprecipitation with either preimmune (P) or immune (I) SA2 antibodies (446). After washing with buffers containing either 150, 250, or 500 mM NaCl, the immunoprecipitates were analyzed by SDS-PAGE and Western blotting with antibodies to the indicated proteins. (D) Xenopus interphase extracts were analyzed by immunoprecipitation with either nonspecific (IP-contr), Xenopus PDS5 (IP-PDS5), or SA1 (IP-SA1) antibodies. The precipitates were analyzed by immunoblotting with PDS5, SA1, and SCC1 antibodies. After immunoprecipitation with control (xtΔcontr), PDS5 (xtΔPDS5), or SA1 (xtΔSA1) antibodies the resulting supernatants were analyzed side by side. PDS5 antibody 648 was used for the IP and 647 for immunoblotting.

Mentions: When HeLa cell extracts were immunoprecipitated with PDS5 antibodies all known cohesin subunits, including SA1 and SA2, could be detected in the precipitates by immunoblotting, whereas no cohesins could be detected in control precipitates obtained with preimmune immunoglobulins (Fig. 4 A). Conversely, SA1 and SA2 antibodies were able to coprecipitate PDS5 (Fig. 4 B). PDS5 antibodies were not able, however, to deplete cohesin subunits from HeLa extracts, although the majority of PDS5 was removed under these conditions (Fig. 4 A). Likewise, PDS5 could not be immunodepleted with SA1 and SA2 antibodies (Fig. 4 B), suggesting that only small portions of 14S cohesin and PDS5 are bound to each other. Consistent with this possibility, we found in sucrose gradient centrifugation experiments that only small amounts of PDS5 could be detected in 14S cohesin fractions in long immunoblot exposures (data not shown), whereas the majority of PDS5 sedimented at 9S, i.e., less far than 14S cohesin subunits (Fig. 1 B). PDS5 did not coimmunoprecipitate with SMC1/3 from the 9S fraction (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)

PDS5 is found in association with SA1- and SA2-containing cohesin complexes. (A) Low-speed supernatant of extracts from logarithmically growing HeLa cells was analyzed by immunoprecipitation with either preimmune (P) or immune antibodies (I) to PDS5. Immunoprecipitates (IP) and supernatants (SUP) were analyzed by SDS-PAGE and Western blotting with antibodies to the indicated proteins. (B) HeLa extracts prepared as in A were analyzed by immunoprecipitation with SA1, SA2, or with nonspecific control (MOCK) antibodies and the immunoprecipitates (IP), extracts before immunoprecipitation (Input) and supernatants (Sup) were analyzed by SDS-PAGE and Western blotting with antibodies to PDS5. (C) HeLa extracts prepared as in A were analyzed by immunoprecipitation with either preimmune (P) or immune (I) SA2 antibodies (446). After washing with buffers containing either 150, 250, or 500 mM NaCl, the immunoprecipitates were analyzed by SDS-PAGE and Western blotting with antibodies to the indicated proteins. (D) Xenopus interphase extracts were analyzed by immunoprecipitation with either nonspecific (IP-contr), Xenopus PDS5 (IP-PDS5), or SA1 (IP-SA1) antibodies. The precipitates were analyzed by immunoblotting with PDS5, SA1, and SCC1 antibodies. After immunoprecipitation with control (xtΔcontr), PDS5 (xtΔPDS5), or SA1 (xtΔSA1) antibodies the resulting supernatants were analyzed side by side. PDS5 antibody 648 was used for the IP and 647 for immunoblotting.
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Figure 4: PDS5 is found in association with SA1- and SA2-containing cohesin complexes. (A) Low-speed supernatant of extracts from logarithmically growing HeLa cells was analyzed by immunoprecipitation with either preimmune (P) or immune antibodies (I) to PDS5. Immunoprecipitates (IP) and supernatants (SUP) were analyzed by SDS-PAGE and Western blotting with antibodies to the indicated proteins. (B) HeLa extracts prepared as in A were analyzed by immunoprecipitation with SA1, SA2, or with nonspecific control (MOCK) antibodies and the immunoprecipitates (IP), extracts before immunoprecipitation (Input) and supernatants (Sup) were analyzed by SDS-PAGE and Western blotting with antibodies to PDS5. (C) HeLa extracts prepared as in A were analyzed by immunoprecipitation with either preimmune (P) or immune (I) SA2 antibodies (446). After washing with buffers containing either 150, 250, or 500 mM NaCl, the immunoprecipitates were analyzed by SDS-PAGE and Western blotting with antibodies to the indicated proteins. (D) Xenopus interphase extracts were analyzed by immunoprecipitation with either nonspecific (IP-contr), Xenopus PDS5 (IP-PDS5), or SA1 (IP-SA1) antibodies. The precipitates were analyzed by immunoblotting with PDS5, SA1, and SCC1 antibodies. After immunoprecipitation with control (xtΔcontr), PDS5 (xtΔPDS5), or SA1 (xtΔSA1) antibodies the resulting supernatants were analyzed side by side. PDS5 antibody 648 was used for the IP and 647 for immunoblotting.
Mentions: When HeLa cell extracts were immunoprecipitated with PDS5 antibodies all known cohesin subunits, including SA1 and SA2, could be detected in the precipitates by immunoblotting, whereas no cohesins could be detected in control precipitates obtained with preimmune immunoglobulins (Fig. 4 A). Conversely, SA1 and SA2 antibodies were able to coprecipitate PDS5 (Fig. 4 B). PDS5 antibodies were not able, however, to deplete cohesin subunits from HeLa extracts, although the majority of PDS5 was removed under these conditions (Fig. 4 A). Likewise, PDS5 could not be immunodepleted with SA1 and SA2 antibodies (Fig. 4 B), suggesting that only small portions of 14S cohesin and PDS5 are bound to each other. Consistent with this possibility, we found in sucrose gradient centrifugation experiments that only small amounts of PDS5 could be detected in 14S cohesin fractions in long immunoblot exposures (data not shown), whereas the majority of PDS5 sedimented at 9S, i.e., less far than 14S cohesin subunits (Fig. 1 B). PDS5 did not coimmunoprecipitate with SMC1/3 from the 9S fraction (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