Biochemical, biophysical, and functional characterisation of the E3 ubiquitin ligase APC/C regulator CDC20 from Arabidopsis thaliana

Abstract

The Anaphase Promoting Complex (APC/C), a large cullin-RING E3-type ubiquitin ligase, constitutes the ultimate target of the Spindle Assembly Checkpoint (SAC), an intricate regulatory circuit that ensures the high fidelity of chromosome segregation in eukaryotic organisms by delaying the onset of anaphase until each chromosome is properly bi-oriented on the mitotic spindle. Cell-division cycle protein 20 homologue (CDC20) is a key regulator of APC/C function in mitosis. The formation of the APC/C^CDC20 complex is required for the ubiquitination and degradation of select substrates, which is necessary to maintain the mitotic state. In contrast to the roles of CDC20 in animal species, little is known about CDC20 roles in the regulation of chromosome segregation in plants. Here we address this gap in knowledge and report the expression in insect cells; the biochemical and biophysical characterisation of Arabidopsis thaliana (AtCDC20) WD40 domain; and the nuclear and cytoplasmic distribution of full-length AtCDC20 when transiently expressed in tobacco plants. We also show that most AtCDC20 degrons share a high sequence similarity to other eukaryotes, arguing in favour of conserved degron functions in AtCDC20. However, important exceptions were noted such as the lack of a canonical MAD1 binding motif; a fully conserved RRY-box in all six AtCDC20 isoforms instead of a CRY-box motif, and low conservation of key residues known to be phosphorylated by BUB1 and PLK1 in other species to ensure a robust SAC response. Taken together, our studies provide insights into AtCDC20 structure and function and the evolution of SAC signalling in plants.

Keywords: APC/C regulation; Arabidopsis thaliana; CDC20; Spindle Assembly Checkpoint (SAC); cell division; chromosome segregation; mitosis.

FIGURE 1

(A) Amino acid sequence alignment of the six AtCDC20 isoforms reported to date. The assignation of secondary structure elements was based on the 3D model structure of the AtCDC20.1 WD40 domain. The alignment was created with Clustal Omega (Sievers et al., 2011) and visualised with EsPript (Robert and Gouet, 2014). (B) Domain organisation and distribution of SLiMs of full length AtCDC20. The boxes indicate the known functions of the CDC20 SLiMs in the SAC. MIM refers to the MAD2-interacting motif. Plausible functions of other potential motifs of AtCDC20 are indicated with a question mark.

FIGURE 2

Western blot showing the time-course expression of AtCDC20.1 WD40 in Sf9 (A) and T.ni (B) cells. In each case, three different MOIs, 0.1, 1.0, and 5.0, were tested. Non-infected cells (e.g., mock) were used as a control.

FIGURE 3

(A) AtCDC20.1 WD40 purification by IMAC and after SEC. In both cases, the eluted fractions were compared against the Precision Protein Plus Molecular Weight Marker (M). (B) The SEC-MALS profiles confirmed AtCDC20.1 WD40 is a globular protein domain of ca. 40 kDa. (C) Far-UV spectra of AtCDC20.1 WD40 at pH 4.0 (○), 6.0 (●), 8.0 (□), and 9.0 (◊). Inset: CD thermal denaturation profile of AtCDC20.1 WD40 recorded at 218 nm (Tm = 56 Celsius). Protein sample prepared in 40 mM sodium phosphate pH 8.0.

FIGURE 4

Predicted AtCDC20.1 WD40 3D structure. (A) Two views of the AtCDC20.1 3D structure model in a ribbon representation. The model was generated by comparative modelling using Phyre2 (Kelley et al., 2015). (B) Structure superposition of AtCDC20.1 WD40 structure model (cyan) with human CDC20 WD40 (green, PDB ID 4GGC), and CDH1 WD40 from budding yeast (purple, PDB ID 4BH6) reveals a high 3D structure conservation of CDC20 from various species and yeast CDH1. (C) Structure superposition of CDH1 in complex with an ACM1 fragment containing the KEN-box and ABBA motifs (shown in yellow, PDB ID 4BH6) and CDC20 from human (shown in green, PDB ID 4GGC) and the A. thaliana 3D structure model shows the predicted mode of interaction of AtCDC20.1 with interaction partners of this protein that contain these motifs and mediate SAC signalling. The AtCDC20.1 residues predicted to bind the ABBA motif are shown in red in stick and ball representation, while those predicted to bind the KEN-box are highlight in orange. (D). Cartoon illustrating the mapping of the CRY/RRY motif (shown in purple) and a simplified view of the regions containing the residues that are implicated in binding the KEN-box (green) and ABBA motif (red) of CDC20 interaction partners. The binding of AtCDC20.1 to the latter SLiMs is anticipated to be critical for the proper segregation of chromosomes upon cell division in A. thaliana.

FIGURE 5

AtCDC20.1 subcellular localisation. AtCDC20-GFP together with suborganellar markers is transiently expressed in tobacco leaf epidermal cells via Agrobacterium-mediated gene expression. AtCDC20.1-GFP is co-expressed with the ER lumenal marker RFP-HDEL and visualised on the nucleus (A), showing labelling in the nucleoplasm and ER (B). AtCDC20.1-GFP co-localises with free RFP labelling the cytosol (C). Example images from n = 3 with at least 5 technical replicas each. Size bars = 5 µm.