Mechanisms of pseudosubstrate inhibition of the anaphase promoting complex by Acm1 - PubMed (original) (raw)
Mechanisms of pseudosubstrate inhibition of the anaphase promoting complex by Acm1
Janet L Burton et al. EMBO J. 2011.
Abstract
The anaphase promoting complex (APC) is a ubiquitin ligase that promotes the degradation of cell-cycle regulators by the 26S proteasome. Cdc20 and Cdh1 are WD40-containing APC co-activators that bind destruction boxes (DB) and KEN boxes within substrates to recruit them to the APC for ubiquitination. Acm1 is an APC(Cdh1) inhibitor that utilizes a DB and a KEN box to bind Cdh1 and prevent substrate binding, although Acm1 itself is not a substrate. We investigated what differentiates an APC substrate from an inhibitor. We identified the Acm1 A-motif that interacts with Cdh1 and together with the DB and KEN box is required for APC(Cdh1) inhibition. A genetic screen identified Cdh1 WD40 domain residues important for Acm1 A-motif interaction and inhibition that appears to reside near Cdh1 residues important for DB recognition. Specific lysine insertion mutations within Acm1 promoted its ubiquitination by APC(Cdh1) whereas lysine removal from the APC substrate Hsl1 converted it into a potent APC(Cdh1) inhibitor. These findings suggest that tight Cdh1 binding combined with the inaccessibility of ubiquitinatable lysines contributes to pseudosubstrate inhibition of APC(Cdh1).
Conflict of interest statement
The authors declare that they have no conflict of interest.
Figures
Figure 1
The ‘A-motif’ in Acm1-N128 is important for Cdh1 binding. (A) Schematic of the alanine-scanning mutagenesis performed between amino acids 65 and 128 of Acm1-N128. The KEN box and DB3 were mutated to alanines (mkb and mdb3) in all alanine-scanning mutants. The amino-acid changes for each of the mutants are shown. (B) Two-hybrid interactions between Cdh1-Δ200 and Acm1-N128 alanine-scanning mutants A–I. Mutant A combined with mdb3 and mkb disrupted the Cdh1–Acm1 two-hybrid interaction. No other alanine-scanning mutant had any effect on the interaction. (C) The A-motif, KEN box and DB3 each contributes to Cdh1 binding in vitro. Control or full-length 6xHis–Cdh1-containing beads were incubated with the indicated MBP–Acm1-N128 proteins or with MBP–Acm1-C81. Acm1 binding was detected by immunoblotting with anti-MBP antibodies. Acm1-C81 and Acm1-N128 with the AA (E65A, E66A), mdb3 and mkb mutations resulted in undetectable Cdh1 binding. Fifty percent of the input to each binding reaction is shown (right panel).
Figure 2
A carboxyl-terminal motif within full-length Acm1 collaborates with the A-motif in Cdh1 binding and inhibition. (A) The A-motif is important for inhibition of APCCdh1 function by Acm1-N128 in vivo. Cells expressed an Hsl1-mkb–Ura3 fusion protein, which allows cell growth in the absence of uracil only when APCCdh1 activity is low. Cells also expressed WT or Mut. A versions of full-length Acm1 (top half) or of Acm1-N128 (bottom half). Cells were grown in the presence (left) or absence (right) of uracil. (B) Yeast two-hybrid analysis of Cdh1 interaction with full-length (top half) and N128 (bottom half) forms of Acm1. Mutation of the A-motif together with mutations in the KEN box and DB3 disrupted the interaction of Acm1-N128, but not of full-length Acm1, with Cdh1-Δ200. (C) Mutant K, and to a lesser extent mutant J, collaborates with mutant A to disrupt APCCdh1 inhibition. Cells expressed full-length WT Acm1, or Acm1 containing alanine-scanning mutant A, mutant J (186SRSTDD → 186AAAAAA), mutant K (196KVVRK → 196AVVAA), or the AJ or AK double mutants in the Hsl1-mkb–Ura3 reporter strain used in (A). Cells were grown in the presence or absence of uracil. (D) The A-motif, the K-motif, DB3 and the KEN box all participate in the binding of full-length Acm1 to Cdh1. Top panel, control (lanes 1 and 9) or 6xHis–Cdh1-containing beads (lanes 2–8) were incubated with the indicated MBP–Acm1 full-length fusion proteins and bound proteins were detected by immunoblotting with anti-MBP antibodies. Bottom panel shows 50% of the input used in each binding assay. (E) The A-motif is required for APCCdh1 inhibition in a ubiquitination assay in vitro. Cdh1 and the APC were pre-incubated with 1.0 μg of the indicated full-length Acm1 proteins before the addition of the APC substrate 35S-MBP–Hsl1. Unmodified and modified species were visualized by fluorography. Lower panel, a short exposure of the unmodified substrate.
Figure 3
Isolation of Cdh1 mutants that no longer recognize the A-motif of Acm1. (A) Two-hybrid analysis of the interaction of WT or D12 forms of Cdh1-Δ200 with Hsl1, WT Acm1 or Acm1-Mut. A. The Cdh1-Δ200 D12 mutant disrupted interaction with Hsl1 and Acm1-Mut. A but not with WT Acm1. (B) Two-hybrid analysis of the interaction of Cdh1-Δ200 WD40 mutants with Acm1-mdb1mdb3mkb, WT Acm1 and WT Hsl1. The A534V single mutation from clone #7 and the D262V mutation from clone #11 disrupted the Cdh1–A-motif interaction. (C) Cdh1 mutants are resistant to Acm1 in vivo. Cells carrying _GAL-CDH1_-m11 cannot grow in the presence of galactose, which induces expression of this constitutively active form of Cdh1. Overexpression of Acm1 suppressed the toxicity of Cdh1-m11, but not of the Cdh1-m11-A534V or -D262V mutants, indicating that these Cdh1 mutants were resistant to Acm1-mediated inhibition. (D) modelling of the yeast Cdh1 WD40 domain based on the known structures of other WD40 containing proteins. The 7-blade β-sheet propeller structure is shown with WD40 residues on the first and seventh blades important for DB interaction shown in red (top, D1: L255, P258; lower, D2: G535, D536). Amino-acid residues whose mutation disrupts the Cdh1–Acm1–A-motif interaction (with the exception of V388) are shown: P251, yellow; D262, green; S267 and L523, grey; V388, light blue; E485, orange; Y492 and L521, white; G513, pink; H514, hot pink; A534, blue; D526, cyan.
Figure 4
Ubiquitination of WT and mutant MBP–Acm1 in vitro. Ubiquitination reactions using WT or the indicated mutant forms of MBP–Acm1 were performed in the presence (+) or absence (−) of Cdh1, as indicated. Modified and unmodified MBP–Acm1 were detected by immunoblotting with anti-MBP antibodies. (A) Ubiquitination of MBP–Acm1-N128-mdb3mkb depends on DB1. Lower panel, a shorter exposure of the unmodified substrate. (B) Mutation of the A-motif does not convert Acm1 into an APC substrate. The AA mutations eliminate ubiquitination of Acm1-N128-mdb3mkb by eliminating the interaction with Cdh1. (C) Introduction of lysine residues converts Acm1 into an APCCdh1 substrate. Lysines or arginines replaced asparagines at positions 83 and 84 in the K83 K84 and R83 R84 mutants. The lower panel shows a quantitation of the amount of modified Acm1 in each reaction.
Figure 5
Removal of lysine residues can convert Hsl1 from an APC substrate into an APC inhibitor. (A) Hsl1 ‘K-less’ inhibits the APCCdh1 in vivo. WT Acm1, WT Hsl1, K-less Hsl1 or K-less Hsl1-mdb were expressed in cells expressing an Hsl1-mkb–Ura3 reporter, which allows cell growth in the absence of added uracil when APCCdh1 activity is low. Only Acm1 and Hsl1-K-less inhibited Cdh1 to allow cell growth. (B) Hsl1-K-less is a potent APCCdh1 inhibitor in vitro. The indicated amounts of Acm1 or Hsl1-K-less were pre-incubated with APCCdh1 before the addition of 35S-MBP–Hsl1 and other ubiquitination assay components. Modified and unmodified MBP–Hsl1 were visualized by autoradiography. Note that lower concentrations of Hsl1-K-less were utilized in the lower right panel.
References
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- Burton JL, Tsakraklides V, Solomon MJ (2005) Assembly of an APC-Cdh1-substrate complex is stimulated by engagement of a destruction box. Mol Cell 18: 533–542 - PubMed
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