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Identification of Subunits of the Anaphase-Promoting Complex of Saccharomyces cerevisiae

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Science  15 Nov 1996:
Vol. 274, Issue 5290, pp. 1201-1204
DOI: 10.1126/science.274.5290.1201

Figures

  • Fig. 1.

    Defective proteolysis and ubiquitination of mitotic cyclins in the cdc26-519 and apc1-1 mutants. (A) Accumulation of Clb2p in G1-arrested apc1-1 cells. Wild-type (K5137) and apc1-1 cells (K6221) of the genotype MATa GALp-CLB2-HA3 bar1 were arrested in G1 with α-factor in raffinose medium at 23°C. Cells were transferred to raffinose-galactose medium containing α-factor at 35°C. Samples for immunoblotting and analysis of cellular DNA content were withdrawn at the indicated time points. (B) Ubiquitin-conjugating activity in extracts from a wild-type (K5518) (WT) and two cdc26-519 strains (K6200 and YWZ135). Strains (MATa pep4 bar1) were arrested in G1 with α-factor at 25°C and shifted to 37°C for 90 min. Extracts were incubated for 5 min with an adenosine triphosphate-regenerating system and the indicated HA3-tagged cyclin substrate as described (8). Clb2ΔDBp lacks the destruction box (2). Cyclin-ubiquitin conjugates were detected by immunoblotting with an antibody to HA. (C) Cyclin ubiquitination in extracts from G1-arrested wild-type (K1771) and apc1-1 cells (K6199). Strains were arrested with α-factor at 23°C and shifted to 37°C for 40 min. Molecular sizes are indicated on the left (in kilodaltons).

  • Fig. 2.

    Coimmmunoprecipitation of Apc1p and Cdc26p with other subunits of the APC. (A) Association of Cdc26p and Apc1p with Cdc16p, and self-association of Apc1p. Extracts were prepared from strains expressing Myc- and HA-tagged proteins as indicated and subjected to immunoprecipitations with the antibody to HA (24). Epitope-tagged proteins in the extracts and the immunoprecipitates (Anti-HA IP) were detected by immunoblotting with the antibodies 12CA5 (Anti-HA) or 9E10 (Anti-Myc). (B) Subunits of the yeast APC. Cells of several strains, each expressing a different Myc-tagged protein, were labeled with 35S-methionine and 35S-cysteine. Extracts were prepared and subjected to immunoprecipitations with the antibody 9E10 (anti-Myc) (24). Bound proteins were detected by fluorography. A protein whose precipitation does not depend on the Myc epitope tag is marked with an asterisk.

  • Fig. 3.

    Cosedimentation of Apc1p, Cdc16p, and Cdc26p as a 36S particle. (A) An extract from an APC1-HA3 CDC16-myc6 strain (K6190) was separated in a 10 to 35% glycerol gradient, and fractions were analyzed by immunoblotting (25). Cim5p, a subunit of the 19S proteasome-activator complex, and fatty acid synthetase (FAS, 40.6S) were detected with polyclonal rabbit antisera. (B) Extracts from CDC16-HA3 CSE1-myc9 (K6184) and CDC16-HA3 CDC26-myc9 (K6323) strains were separated on parallel gradients as described in (A).

  • Fig. 4.

    Nuclear localization of subunits of the yeast APC. Control cells (No Myc, K1534) and cells containing CDC16-myc6 (K6180), CDC26-myc9 (K6322), or APC1-myc18 (K6329) were fixed, and Myc-tagged proteins were detected by indirect immunofluorescence (26). DNA was stained with 4′,6′-diamidino-2-phenylindole (DAPI).

  • Fig. 5.

    Defective anaphase in the apc1-1 mutant. Small, unbudded G1 cells were isolated by centrifugal elutriation from a wild-type (K699) and an apc1-1 strain (K5717) grown at 25°C. Cells were released into fresh medium at 37°C, and samples were withdrawn at the indicated time points (28). (A) Percentage of budded cells (□), of cells containing a short spindle in an undivided nucleus (ˆ), and of cells with separated chromosomes and an elongated spindle (late anaphase-telophase) (•). (B) Distribution of the DNA content. (C) Clb2 protein and Clb2p-associated kinase activity. A clb2 deletion strain (K1890) was used for negative controls, and Swi6p was detected as a loading control. (D) Defective Clb2p destruction and lack of astral microtubules in apc1-1 cells. A wild-type (K6208) and an apc1-1 (K6131) strain, both containing CLB2-myc12, were grown on plates at 25°C to obtain unbudded G1 cells. Strains were inoculated into liquid medium at 37°C, and cells were fixed after 3 hours. Tubulin (green) and Clb2-Myc12p (red) were detected by indirect immunofluorescence (26), and DNA was stained with DAPI (blue). Nom, Nomarski optics.

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