Unique D box and KEN box sequences limit ubiquitination of Acm1 and promote pseudosubstrate inhibition of the anaphase-promoting complex

Abstract

The anaphase-promoting complex (APC) regulates cell division in eukaryotes by targeting specific proteins for destruction. APC substrates generally contain one or more short degron sequences that help mediate their recognition and poly-ubiquitination by the APC. The most common and well characterized degrons are the destruction box (D box) and the KEN box. The budding yeast Acm1 protein, an inhibitor of Cdh1-activated APC (APC(Cdh1)) also contains several conserved D and KEN boxes, and here we report that two of these located in the central region of Acm1 constitute a pseudosubstrate sequence required for APC(Cdh1) inhibition. Acm1 interacted with and inhibited substrate binding to the WD40 repeat domain of Cdh1. Combined mutation of the central D and KEN boxes strongly reduced both binding to the Cdh1 WD40 domain and APC(Cdh1) inhibition. Despite this, the double mutant, but not wild-type Acm1, was poly-ubiquitinated by APC(Cdh1) in vitro. Thus, unlike substrates in which D and KEN boxes promote ubiquitination, these same elements in the central region of Acm1 prevent ubiquitination. We propose that this unique property of the Acm1 degron sequences results from an unusually high affinity interaction with the substrate receptor site on the WD40 domain of Cdh1 that may serve both to promote APC inhibition and protect Acm1 from destruction.

FIGURE 1.

Acm1 is a general inhibitor of APC^Cdh1, independent of CDK phosphorylation and 14-3-3 protein binding. A, APC^Cdh1-catalyzed ubiquitination of [³⁵S]methionine-labeled substrates Clb2, Hsl1^667–872, and Fin1^1–152 was assayed in the absence or presence of 125 nm recombinant His₆-Acm1 purified from E. coli as described under “Experimental Procedures.” Reaction products (ubiquitin conjugates) indicated by the bracket are detected based on reduced mobility during SDS-PAGE. B, inhibition of APC^Cdh1-catalyzed ubiquitination of Clb2, Hsl1^667–872, and Pds1 was measured as in A as a function of recombinant His₆-Acm1 concentration. NC is a negative control lacking APC. Reaction products are labeled “Ubiq. Conj.” C, 10-fold serial dilutions of strain YKA247 expressing the indicated proteins from the GAL1 promoter on centromeric plasmids were spotted on rich media plates containing either glucose or galactose as the carbon source and grown for several days at 30 °C.

FIGURE 2.

Acm1 contains substrate-like degron sequences that have been conserved during evolution. A, sections of Acm1 containing conserved D box (RXXL) and KEN box motifs from 6 Saccharomyces species aligned with ClustalW are shown. Consensus residues are highlighted in gray. Note that D box 2 is not conserved. B, a similar alignment of Acm1 orthologs from more distantly related budding yeasts with S. cerevisiae Acm1, illustrating conservation of D box 1, D box 3, and the KEN box. In both panels the asterisk indicates an invariant residue, “:” is a conservative substitution, and “.” is a semi-conservative substitution.

FIGURE 3.

Central D box and KEN box sequences in Acm1 are required for high affinity binding to the Cdh1 WD40 domain. A, yeast strain YKA294 expressing endogenous 3FLAG-Cdh1 and containing centromeric plasmids expressing wild-type or the indicated mutant 3HA-Acm1 proteins from the ACM1 promoter were grown to mid-log phase. An anti-FLAG IP was performed from cell extracts and co-purification of 3HA-tagged protein monitored by anti-HA immunoblotting. Cdc28 is a loading control. B, the same procedure as in panel A using strain YKA226 expressing endogenous 3HA-Acm1 and containing a centromeric plasmid expressing 3FLAG-tagged N-terminal (amino acids 1–249) or C-terminal (amino acids 241–566) domains of Cdh1 expressed from the ADH promoter. C, yeast strain YKA257 expressing endogenous 3HA-Hsl1 in an acm1Δ background was transformed with empty control plasmids (lane 1), a centromeric plasmid expressing 3FLAG-Cdh1WD40 from the ADH promoter (lane 2), or the same 3FLAG-Cdh1WD40 plasmid plus a centromeric plasmid expressing 3HA-Acm1 from the GAL1 promoter. Cells were grown to mid-log phase in raffinose-containing medium, then 2% galactose added and cells harvested after 2 h and subjected to α-FLAG co-IP. D, the same experiment described in panel A, except strain YKA247 contained centromeric plasmids expressing 3FLAG-Cdh1WD40 (amino acids 241–566 only) from the ADH promoter and the indicated 3HA-Acm1 variant from the ACM1 promoter. Also, NaCl concentration in the co-IP buffer was increased from 100 mm to 400 mm. G6PD is a loading control. NC is a control lacking a 3HA-tagged Acm1 protein. E, synchronous cultures of strain YKA254 harboring centromeric plasmids expressing wild-type or the indicated mutant 3HA-Acm1 protein from the ACM1 promoter were obtained by G₁α-factor arrest and then released into fresh medium. Samples taken at the indicated time points were analyzed by anti-HA, anti-Clb2, and anti-G6PD (loading control) immunoblotting, and α-factor was added back at 60 min to re-arrest cells in the subsequent G₁. cyc, asynchronous cycling cells. F, the stability of HA-Acm1 and the HA-Acm1-db1/db3/ken mutant were assessed by promoter shutoff in α-factor-arrested G₁ YKA150 cells. After arrest, expression was induced with galactose for 2 h and quenched by addition of glucose and cycloheximide (time 0). The level of each protein at the indicated time points was monitored by anti-HA immunoblotting. G6PD is a loading control.

FIGURE 4.

The central D box and KEN box in Acm1 are required for full inhibition of APC^Cdh1 activity. A, 10-fold serial dilutions of strain YKA247 expressing the indicated proteins from the GAL1 promoter on centromeric plasmids were spotted on rich media plates containing either glucose or galactose and grown for several days at 30 °C. The α-HA immunoblot on the right compares the level of galactose-induced overexpression of each of the Acm1 variants. B, APC-catalyzed ubiquitination of Clb2 was assayed in the absence or presence of 500 nm wild-type (wt) recombinant His₆-Acm1 or the indicated Acm1 mutants. Relative activity was obtained from the total ubiquitin conjugate signal (bracket). C, same as B except Hsl1^667–872 was used as the substrate. D, the same quantities of the indicated recombinant His₆-Acm1 proteins used in B and C were compared by anti-His₆ immunoblot to demonstrate equivalent concentrations. E, inhibition of APC-catalyzed ubiquitination of Clb2 was measured as a function of inhibitor concentration for wild-type (wt) His₆-Acm1 and the db3/ken and db1 mutants. F, identical to E except Hsl1^667–872 was used as the substrate.

FIGURE 5.

The central D box and KEN box restrict poly-ubiquitination of Acm1 by APC^Cdh1. A, full-length Acm1 (amino acids 1–209) and N- and C-terminal truncated forms were synthesized and ³⁵S-labeled by in vitro coupled transcription/translation and used as substrates in an APC^Cdh1 ubiquitination assay. B, products of APC^Cdh1-catalyzed reactions using ubiquitin and methylated ubiquitin (me-Ubiquitin) were compared for the ³⁵S-labeled substrates Acm1, Clb2, and Hsl1^667–872. Arrows indicate the unmodified substrates. C, ubiquitination of Acm1 and Acm1-db1 by APC^Cdh1 were compared. The dominant mono-ubiquitin product (mono-ub) is indicated. The percentage of substrate converted to mono-ubiquitin conjugate was determined with Image-QuaNT. D, products of APC^Cdh1-catalyzed reactions using ubiquitin and methyl-ubiquitin were compared for the ³⁵S-labeled substrates wild-type (wt) Acm1 and the Acm1-db3/ken mutant. E, ubiquitination of wild-type Acm1, Acm1-db3/ken, and Acm1-db1/db3/ken by APC^Cdh1 were compared. For Acm1-db3/ken, the dependence of poly-ubiquitination on yeast Ubc4 and Cdh1 are also shown. F, ubiquitination of full-length Acm1-(1–209) and the wild-type and db3/ken forms of an Acm1 fragment encompassing residues 42–177 by APC^Cdh1 was compared. Arrows point to unmodified substrate. Note the complete conversion of the Acm1^42–177 db3/ken substrate to poly-ubiquitin conjugates.