Fission yeast Dma1 requires RING domain dimerization for its ubiquitin ligase activity and mitotic checkpoint function

Abstract

In fission yeast (Schizosaccharomyces pombe), the E3 ubiquitin ligase Dma1 delays cytokinesis if chromosomes are not properly attached to the mitotic spindle. Dma1 contains a C-terminal RING domain, and we have found that the Dma1 RING domain forms a stable homodimer. Although the RING domain is required for dimerization, residues in the C-terminal tail are also required to help form or stabilize the dimeric structure because mutation of specific residues in this region disrupts Dma1 dimerization. Further analyses showed that Dma1 dimerization is required for proper localization at spindle pole bodies and the cell division site, E3 ligase activity, and mitotic checkpoint function. Thus, Dma1 forms an obligate dimer via its RING domain, which is essential for efficient transfer of ubiquitin to its substrate(s). This study further supports the mechanistic paradigm that many RING E3 ligases function as RING dimers.

FIGURE 1.

Dma1 self-associates in vivo. A, co-immunoprecipitation of Dma1-3-V5 and Dma1-3-FLAG from diploid cells. IP, immunoprecipitation; WB, Western blot. B, yeast two-hybrid interactions using full-length (F/L) Dma1 and Dma1 fragments as bait and prey. C, schematics of Dma1 fragments that were tested for yeast two-hybrid interaction and summary of their interactions. ++, interaction comparable with the wild type; −, no interaction detected; NT, not tested.

FIGURE 2.

Dma1 RING domain preferentially forms a dimer in vitro. A, His₆-Dma1(187–end) was affinity-purified on His·Bind resin followed by gel filtration. Fractions 15–20 were combined and concentrated for SVAU. B, expected sizes of Dma1 oligomers. C, SVAU analysis of His₆-Dma1(187–end) in 150 mm NaCl. s values, determined molecular masses, and percent abundance are given for each indicated peak. RMSD, root mean square deviation. D, SVAU analysis of His₆-Dma1(187–end) in 500 mm NaCl. s values, determined molecular masses, and percent abundance are given for each indicated peak.

FIGURE 3.

Phe-206, Leu-241, and Val-245 are critical for Dma1 dimerization. A, ClustalW alignment of the RING domain and C-terminal flanking residues from S. cerevisiae (Sc.c.) Dma1 (YNL116W) and Dma2 (YHR115C) and Schizosaccharomyces Dma1 homologs: S. pombe (Sz.p.; SPAC17G8.10c), Schizosaccharomyces japonicus (Sz.j.; SJAG02169.4), Schizosaccharomyces octosporus (Sz.o.; SOCG04269.5), and Schizosaccharomyces cryophilus (Sz.c.; SPOG00270.3). Amino acid numbers correspond to S. pombe Dma1 amino acid positions. Conserved residues are highlighted, and asterisks indicate amino acids that were tested for involvement in self-interaction. The dashed line indicates the region of Dma1 that was dispensable for self-interaction in yeast two-hybrid experiments. The gray bar underlines the C-terminal flanking residues that were required for self-interaction in two-hybrid experiments. The black bar underlines the core RING residues. B, yeast two-hybrid interactions. All point mutations were made in the Dma1(187–end) fragment and used as both bait and prey. C, summary of point mutations that were tested for two-hybrid interaction. ++, interaction comparable with the wild type; +, interaction weaker than the wild type; −, no interaction detected. RF, RING finger. D, co-immunoprecipitation experiments from dma1-3-V5/dma1-3-FLAG, dma1(L241A,V245A)-3-V5/dma1(L241A,V245A)-3-FLAG, and dma1(F206A)-3-V5/dma1(F206A)-3-FLAG diploid cells. IP, immunoprecipitation; WB, Western blot.

FIGURE 4.

Monomeric Dma1 exhibits defective intracellular localization. A, protein levels of Dma1-GFP, Dma1(L241A,V245A)-GFP, and Dma1(F206A)-GFP. The Cdc2 blot is shown as a protein loading control. WB, Western blot; IP, immunoprecipitation. B, Dma1-GFP, Dma1(L241A,V245A)-GFP, or Dma1(F206A)-GFP was imaged with Sid4-red fluorescent protein (RFP; magenta) in live cells. Scale bar = 5 μm. C, Dma1(R64A,L241A,V245A)-GFP localization. Scale bar = 5 μm. D, representative live cell image of Dma1(L241A,V245A)-GFP with Cdc7-mCherry₃ (n = five cells). Scale bar = 5 μm. E, pREP42-GFP-dma1(L241A,V245A) was overproduced in dma1Δ cells, and the cells were imaged live. Scale bar = 5 μm. BF, bright field.

FIGURE 5.

Dimerization of Dma1 is essential for its checkpoint function and E3 ubiquitin ligase activity. A, checkpoint assay with nda3-KM311 dma1⁺, nda3-KM311 dma1Δ, nda3-KM311 dma1(L241A,V245A), and nda3-KM311 dma1(F206A) cells. Cells were synchronized in S-phase with hydroxyurea and released at 18 °C to activate the spindle checkpoint. Septation indices were measured at 0 and 7 h (n = 3). *, p < 0.05 compared with nda3-KM311 dma1⁺. B, representative images of cells from the 7-h time point for each strain indicated stained with DAPI and methyl blue. Scale bar = 10 μm. C, in vitro ubiquitination assay. Dma1-GFP, Dma1(L241A,V245A)-GFP, and Dma1(F206A)-GFP were immunoprecipitated and incubated with an E1 activating enzyme, ATP, and ubiquitin with (+) or without (−) the E2 conjugating enzyme complex Ubc13-Uev1. Ubiquitinated proteins were detected by immunoblotting with anti-GFP antibody. WB, Western blot. D, Sid4 ubiquitination in vivo. Sid4 was immunoprecipitated with anti-Sid4 antibody, treated with λ-protein phosphatase (λ-ppase) to visualize the ubiquitin ladder more clearly, and resolved by SDS-PAGE. Ubiquitinated proteins were detected by immunoblotting with anti-Sid4 serum.