Skip to main content
. 2023 Nov 10;19(11):e1011026. doi: 10.1371/journal.pgen.1011026

Fig 6. Fusion of a putative NES from human TRIP13 restores checkpoint function to Pch2-nes4A.

Fig 6

(A) Schematic representation of the S. cerevisiae Pch2 protein (ScPch2) and the human ortholog (HsTRIP13) indicating the characteristic AAA+ ATPase motifs, the conserved N-terminal domain (NTD) and the extended N-terminal domain exclusive of yeast Pch2 (NTDEXT). The positions and sequences of the putative NESs analyzed in this work are depicted (purple boxes). (B) Fluorescence microscopy images of the localization of GFP-Pch2, GFP-Pch2-nes4A, GFP-NESTRIP13-Pch2-nes4A, and GFP-nes7ATRIP13-Pch2-nes4A in zip1Δ cells. Images were taken 16 h after meiotic induction. Representative cells are shown. A schematic representation of the different GFP-Pch2 versions is also shown on top. Scale bar, 2 μm. The strain is DP1405 (zip1Δ pch2Δ) transformed with the centromeric plasmids pSS393 (GFP-PCH2), pSS459 (GFP-pch2-nes4A), pSS472 (GFP-NESTRIP13-pch2-nes4A) and pSS474 (GFP-nes7ATRIP13-pch2-nes4A). (C) Quantification of the ratio of nuclear (including nucleolar) to cytoplasmic GFP fluorescent signal for the experiment shown in (B). Error bars, SD. (D) Time course analysis of meiotic nuclear divisions. The percentage of cells containing two or more nuclei is represented. Error bars: SD; n = 3. At least 300 cells were scored for each strain at every time point. Strains are: DP1625 (zip1Δ GFP-PCH2), DP1986 (zip1Δ GFP-pch2-nes4A), DP2025 (zip1Δ GFP-NESTRIP13-pch2-nes4A) and DP2033 (zip1Δ GFP-nes7ATRIP13-pch2-nes4A). (E) Western blot analysis of Hop1-T318 phosphorylation in the strains analyzed in (D) at the indicated time points in meiosis. Pgk1 was used as a loading control. The graph shows the quantification of Hop1-T318ph relative levels. (F) Pch2 structure modeled by AlphaFold. (G) Human TRIP13E253Q structure obtained from the Protein Data Bank (PDB 5VQ9) [42]. (H) AlphaFold predicted structure resulting from the fusion of NESTRIP13 to Pch2-nes4A. In (F-H), the N-terminal domains of Pch2 and TRIP13 are labeled in pink, the position of NESPch2 (or nesPch2-4A) is labeled in blue, and the position of the putative NESTRIP13 is labeled in orange.