Human cohesin subunit SMC1 interacts with Rae1 during mitosis. IP from mitotic HeLa cell extracts with anti-Rae1, anti-SMC1, or nonspecific rabbit antibodies (IgG) were analyzed by SDS/PAGE, followed by immunoblotting with Rae1, SMC1, SMC3, Nup98 or tubulin (DM1A) antibodies. In lanes marked “2% input,” 5 μl of 250 μl of extract that was used per IP was analyzed directly (A and B). Anti-Rae1 IP was washed with 2.0 M guanidine·HCl before being analyzed by SDS/PAGE and immunoblotting with anti-SMC1 or -SMC3 antibodies (C).

Mapping of the SMC1 domain interacting with Rae1. Schematic drawing of the hinge-like structure known to be formed by the two cohesin subunits, SMC1 and SMC3; purple stars indicates the location of the two Ser residues that have been reported to be phosphorylated by ATM (A Upper). Domain structure of SMC1 and the constructs 1–5 covering the entire length of SMC1; numbers refer to amino acid residues (A Lower). Each of the five untagged SMC1 fragments (see above) and Flag-tagged Rae1 were synthesized in ³⁵S methionine containing reticulocyte lysate followed by pulldown using anti-Flag coated beads. Pulldown samples were resolved in a 4–20% SDS-polyacrylamide gradient gel and visualized by autoradiography. Note that Rae1 pulled down SMC1 fragment 5, containing residues 947-1233 (A Lower; B, lane 5). Numbers on left indicate molecular mass markers in kilodaltons. Further mapping of SMC1–5; three constructs (5A-C) representing residues 947-1100, 947-1025, and 947–967 were made; note that all three contain Ser⁹⁵⁷ and Ser⁹⁶⁶, indicated in red (C). Fragments 5A-C were cosynthesized with Flag-Rae1 as in B and pulldowns were analyzed by SDS/PAGE by using an 18% acrylamide gel; arrows indicate pulled down fragments 5A, 5B, and 5C (D). In vivo expression of HA-tagged SMC1–5B and SMC1–5C and empty HA vector in HeLa cells, followed by anti-HA IP and immunoblotting with anti-Rae1 (E).

Colocalization of SMC1 and Rae1 at the spindle pole. Confocal microscopy of mitotic HeLa cells that were fixed and permeabilized with cold methanol and 0.3% digitonin and costained with anti-SMC1 (red) and -tubulin (green) (A) or anti-SMC1 (green) and -Rae1 (red) (B). DNA was visualized by using DAPI (blue). Confocal microscopy of asters that were formed in vitro from mitotic HeLa cell extracts and that were visualized by anti-tubulin (green) and anti-Rae1 (red) (C) or anti-tubulin (green) and -SMC1 (red) (D). (Scale bar, 5 μm.)

Phosphorylation of Ser⁹⁵⁷ and Ser⁹⁶⁶ of SMC1 stimulate binding to Rae1. The kinase inhibitor LY294002 was added at the indicated concentrations to the reticulocyte system that was programmed with Rae1-Flag and full length SMC1 cDNA; Rae1-Flag pulldowns were analyzed by SDS/PAGE (18% acryalmide gel) and autoradiography (A). (B) As in A, except that the incubations contained the indicated SMC1 wild type or mutant constructs. HeLa cells were cotransfected with Rae1-HA and Myc-SMC1-WT, Myc-SMC1-S957A, Myc-SMC1-S966A, or Myc-SMC1- DBA (double S957A and S966A) and treated with nocodazole (2); anti-Myc IPs of lysates were analyzed by SDS/PAGE and immunoblotted with HA antibodies (C Upper). In a control, cotransfected cell lysates were blotted with anti-HA and -Myc antibodies (C Lower); aster formed in vitro and stained with tubulin antibodies (green) and ATM antibodies (red) (D). (E) As in D but using SMC1 antibodies (green) and ATM antibodies (red).