Doc1 mediates the activity of the anaphase-promoting complex by contributing to substrate recognition

Abstract

The anaphase-promoting complex (APC) is a multisubunit E3 ubiquitin ligase that targets specific cell cycle-related proteins for degradation, regulating progression from metaphase to anaphase and exit from mitosis. The APC is regulated by binding of the coactivator proteins Cdc20p and Cdh1p, and by phosphorylation. We have developed a purification strategy that allowed us to purify the budding yeast APC to near homogeneity and identify two novel APC-associated proteins, Swm1p and Mnd2p. Using an in vitro ubiquitylation system and a native gel binding assay, we have characterized the properties of wild-type and mutant APC. We show that both the D and KEN boxes contribute to substrate recognition and that coactivator is required for substrate binding. APC lacking Apc9p or Doc1p/Apc10 have impaired E3 ligase activities. However, whereas Apc9p is required for structural stability and the incorporation of Cdc27p into the APC complex, Doc1p/Apc10 plays a specific role in substrate recognition by APC-coactivator complexes. These results imply that Doc1p/Apc10 may play a role to regulate the binding of specific substrates, similar to that of the coactivators.

Fig. 1. Purification of the intact APC and identification of subunits. (A) Analysis of the APC purification from CDC16-TAP yeast by silver-stained SDS–PAGE. Cdc16-TAP binds to IgG–Sepharose and is eluted by cleavage with TEV protease (eluate, lane 1). The eluate is bound to calmodulin Sepharose (flow through, lane 2), and eluted with EGTA (elution fractions, lanes 3–5). Proteins identified by MALDI-TOF mass spectrometry are labelled. p90 is a Cdc16p degradation product, p70 is the Hsp70 family heat shock protein SSA2, p55 is Mnd2p, and Apc13p is Swm1p. (B) Identification of Apc13p and p55. Silver-stained SDS–PAGE analysis of APC purifications from CDC16-TAP Δswm1, CDC16-TAP Δmnd2, SWM1-TAP, MND2-TAP and a negative-control yeast strain with no tag.

Fig. 2. In vitro activity assays of purified APC. APC was incubated with E2 (Ubc4p), ubiquitin, ATP, ³⁵S-labelled substrate and one of the co-activators Cdc20p or Cdh1p. (A) APC ubiquitylation assays with the substrates Pds1p and Clb2p. The addition of polyubiquitin chains onto ³⁵S-labelled Pds1p or Clb2p results in the appearance of high molecular weight smears. Where neither Cdc20p nor Cdh1p were added, an equivalent volume of reticulocyte lysate was added as a negative control. (B) Time courses of the activities of APC with Cdc20p (APC^Cdc20) and APC with Cdh1p (APC^Cdh1) towards the substrate [³⁵S]Pds1p. Samples were taken at the indicated time points and added to gel loading buffer.

Fig. 3. Ubiquitylation of substrates containing D and KEN box mutations. APC ubiquitylation assays were performed on ³⁵S-labelled (A) Pds1p and (B) Clb2p mutants. Pds1^dbm, Pds1^dkbm, Clb2^dbm and Clb2^dkbm contained D box mutations, and Pds1^kbm, Pds1^dkbm, Clb2^kbm and Clb2^dkbm contained KEN box mutations.

Fig. 4. Analysis of the interactions between the APC and its coactivators. (A and B) An increasing amount of purified APC was added to IVT-produced (A) [³⁵S]Cdh1p or (B) [³⁵S]His₆-Cdc20p, run on a 5.25% native gel and visualized by autoradiography. The positions of free Cdh1p/Cdc20p and Cdh1p/Cdc20p in complex with APC or CCT are marked. The positions of APC and CCT migration were verified using Coomassie Blue staining. Lane 1 contains no APC. (C) APC and CCT band-shifts using specific antibodies. [³⁵S]His₆-Cdh1p was run on a native gel in the presence or absence of APC and an antibody to Cdc27, Cdc16 or CCT. The band corresponding to APC^Cdh1 is shifted upon the addition of Cdc27 or Cdc16 antibody (lanes 2 and 3), whereas the CCT band is shifted upon the addition of a CCT antibody (lanes 5 and 6). CCT shifted with a CCT antibody runs at the same position as the APC. (D) Analysis of the migration of purified APC using Coomassie Blue-stained native gel analysis. [³⁵S]His₆-Cdh1p, in the presence or absence of APC, was analysed on the same gel to compare the positions of pure APC and the APC–[³⁵S]Cdh1p complex. (E) Native gel analysis of Cdh1p mutants. The [³⁵S]Cdh1p mutants were produced using IVT, run on a native gel with and without the addition of APC and analysed using autoradiography. The positions of CCT and APC are shown. Cdh1p mutants have N-terminal His₆ tags. Cdh1^ΔIR contains a deletion of the C-terminal IR motif, whereas Cdh1^IK contains a lysine substitution of the C-terminal arginine. TLE2 and PR55 are negative control WD40 repeat-containing proteins. (F) APC ubiquitylation assays using mutant Cdh1p and Cdc20p with the substrates [³⁵S]Clb2p and [³⁵S]Pds1p. The control lanes contain reticulocyte lysate in place of coactivator. Cdc20^IA contains an alanine substitution of the C-terminal arginine.

Fig. 5. Analysis of APC-substrate interactions. (A) ³⁵S-labelled Clb2p, His₆-Hsl1^667–872 or His₆-Hsl1^{667–872 dkbm} were mixed with APC in the presence or absence of His₆-Cdh1p, run on a native gel and analysed by autoradiography. Antibodies to Cdc27p or Cdc16p, which retard the migration of the APC, were added to some samples. [³⁵S]His₆-Cdh1 with or without APC was included as a marker for the position of APC. (B) ³⁵S-labelled Clb2p (Clb2^wt), Clb2p with a D box mutation (Clb2^dbm), Clb2p with a KEN box mutation (Clb2^kbm) or Clb2p with both D and KEN box mutations (Clb2^dkbm) were mixed with APC. Samples were run on a native gel in the presence or absence of His₆-Cdh1p, then analysed by autoradiography.

Fig. 6. Purifications and ubiquitylation activities of APC^Δapc9 and APC^Δcdc26. (A) Silver-stained SDS–PAGE analysis of APC purifications from CDC16-TAP (wild-type), CDC16-TAP Δapc9 and CDC16-TAP Δcdc26 yeast strains. Arrows indicate the positions of deleted or missing subunits. (B) Ubiquitylation activities of APC^Δapc9 and APC^Δcdc26. Ten-fold more APC^Δapc9 was added because it had no apparent activity at lower concentrations. (C) Native gel analysis of Cdh1p binding to APC^Δapc9. [³⁵S]His₆-Cdh1p was mixed with APC or APC^Δapc9, with or without the readdition of Apc9p and/or Cdc27p produced in IVT.

Fig. 7. Purification and ubiquitylation activity of APC^Δdoc1. (A) Silver-stained SDS–PAGE analysis of APC purifications from CDC16-TAP (wild-type) and CDC16-TAP Δdoc1 yeast strains. An arrow indicates the position of the deleted Doc1p subunit. M = molecular weight markers. (B and C) Ubiquitylation activity of APC^Δdoc1 with the substrates (B) [³⁵S]Clb2, [³⁵S]His₆-Hsl1^667–872 and (C) [³⁵S]Pds1p. Doc1p produced in IVT or Doc1p purified from E.coli was added where indicated. The lower Hsl1 band is most probably an internal initiation product that does not contain the N-terminal tag.

Fig. 8. Binding studies on APCΔ^doc1. Native gels were used to analyse protein–protein interactions between (A) [³⁵S]His₆-Doc1p and APC or APC^Δdoc1; (B) [³⁵S]His₆-Cdh1p and APC or APC^Δdoc1, in the presence or absence of Doc1p purified from E.coli; (C) [³⁵S]His₆-Cdc20p and APC or APC^Δdoc1; (D) [³⁵S]Clb2p or [³⁵S]His₆-Hsl1^667–872 and APC or APC^Δdoc1, in the presence or absence of Doc1p purified from E.coli and His₆-Cdh1p. APC^Δdoc1 sometimes runs slightly faster than APC (C).