Abstract
The anaphase-promoting complex/cyclosome (APC/C) is a large, multisubunit ubiquitin ligase involved in regulation of cell division. APC/C substrate specificity arises from binding of short degron motifs in its substrates to transient activator subunits, Cdc20 and Cdh1. The destruction box (D-box) is the most common APC/C degron and plays a crucial role in substrate degradation by linking the activator to the Doc1/Apc10 subunit of core APC/C to stabilize the active holoenzyme and promote processive ubiquitylation. Degrons are also employed as pseudosubstrate motifs by APC/C inhibitors, and pseudosubstrates must bind their cognate activators tightly to outcompete substrate binding while blocking their own ubiquitylation. Here we examined how APC/C activity is suppressed by the small pseudosubstrate inhibitor Acm1 from budding yeast (Saccharomyces cerevisiae). Mutation of a conserved D-box converted Acm1 into an efficient ABBA (cyclin A, BubR1, Bub1, Acm1) motif-dependent APC/CCdh1 substrate in vivo, suggesting that this D-box somehow inhibits APC/C. We then identified a short conserved sequence at the C terminus of the Acm1 D-box that was necessary and sufficient for APC/C inhibition. In several APC/C substrates, the corresponding D-box region proved to be important for their degradation despite poor sequence conservation, redefining the D-box as a 12-amino acid motif. Biochemical analysis suggested that the Acm1 D-box extension inhibits reaction processivity by perturbing the normal interaction with Doc1/Apc10. Our results reveal a simple, elegant mode of pseudosubstrate inhibition that combines high-affinity activator binding with specific disruption of Doc1/Apc10 function in processive ubiquitylation.
Highlights
The anaphase-promoting complex/cyclosome (APC/C) is a large, multisubunit ubiquitin ligase involved in regulation of cell division
We describe a simple and novel mechanism of APC/C pseudosubstrate inhibition by Acm1 that combines high-affinity activator binding via multiple degron motifs with a unique destruction box (D-box) C terminus that disrupts the coreceptor function of Doc1/Apc10
The central region, which is responsible for Cdh1 inhibition, contains an ABBA motif, KENbox, KEN-like NEN sequence, and D-box that all interact with Cdh1 directly and are responsible for the high-affinity Acm1– Cdh1 interaction [3, 21, 22, 25]
Summary
Acm contains several conserved sequences that match consensus APC/C degrons (Fig. 1A). D-box and ABBA motif mutant was stable, consistent with our previous observation that the ABBA motif is a functional Cdh degron in budding yeast [9] These results are inconsistent with the high-affinity binding hypothesis and instead reveal that an intact D-box in the central inhibitory domain is required, paradoxically, for Acm to evade efficient APC/CCdh1-mediated degradation. Replacement of the DBE with the corresponding Hsl sequence or with a stretch of alanines (acm1ala dbe) impaired complementation, similar to the acm1RXXL3AXXA mutant (Fig. 2E) These DBE mutations reduced the ability of Acm to inhibit APC/CCdh1-mediated degradation of the substrate Pds in our G1 stability assay (Fig. 2F), likely because of the lower levels resulting from more rapid turnover. To test whether the Acm DBE is sufficient for evasion of APC/CCdh1-mediated degradation, we performed the reciprocal sequence swap experiment, in which the DBE sequence from Acm replaced the corresponding sequence in several
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