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. Author manuscript; available in PMC: 2023 Oct 23.
Published in final edited form as: Cell Rep. 2023 Sep 1;42(9):113052. doi: 10.1016/j.celrep.2023.113052

Figure 4. Cdc14 binds and dephosphorylates Rim4 on BBSs.

Figure 4.

(A) IB analysis of cell lysates from cdc14-1 or Cdc14 (WT) strains at prophase I under permissive (23°C) or restrictive (30°C) conditions. Pgk1 served as a loading control.

(B) Quantification of EGFP-Rim4-p signals from (A) normalized to EGFP-Rim4 signals (IB, α-RRxS/T-p/α-EGFP).

(C) IB of whole-cell lysates with α-RRxS/T-p antibody and Ponceau S staining under the indicated conditions as in (A) but showing the whole gel images. Red asterisks indicate EGFP-Rim4-p positions.

(D) Quantification of IB signals between 20 and 150 kDa from (C), normalized by Ponceau S signals. Data are compared with the reference group (Cdc14 [WT] at 30°C) and presented as fold change.

(E) Representative IB image with the indicated antibodies, showing coIP of EGFP-Rim4 with Cdc14(C283S)-FLAG in prophase I cell lysates. Empty protein G beads served as a negative control.

(F) IB analysis showing recombinant FLAG-Cdc14 pulled down EGFP-Rim4 (pRIM4, EGFP-Rim4) from prophase I cell lysates, controlled by empty α-FLAG beads.

(G) Schematics of Rim4 variants with a WT or modified Cdc14 docking site (PxLm and PxLSic1) and Rim4 with the C-terminal 289 residues truncated (Rim4 [ΔC289]).

(H) Recombinant Cdc14-FLAG pulled down recombinant Rim4 variants using α-FLAG beads. Rim4-EGFP-His6 serves as a competitor. Red asterisk: Rim4(PxLm)-His6, black asterisk: Rim4(ΔC289)-His6. SDS-page with Cdc14-FLAG further separated from Rim4(ΔC289)-His6 is shown in (Figure S4E).

(I) Sporulation efficiency of the indicated Rim4 variants presented as tetrad percentage as in Figure 1C.

(J) IB analysis of prophase I cell lysates derived from the indicated EGFP-Rim4 variants using the indicated antibodies. Asterisks indicate non-specific bands.

(K) Quantification of EGFP-Rim4-p signals from (J) normalized to EGFP-Rim4 signals (IB, α-RRxS/T-p/α-EGFP).

(L) IB analysis of recombinant Bmh1/2-EGFP-His6 pulled down endogenous EGFP-Rim4 from prophase I cell lysates pre-incubated (30 min at 23°C) with Cdc14 (0.1 μg/μL) or mock treatment. The numbers listed below the images are the EGFP-Rim4-p signal normalized by the EGFP-Rim4 signal.

In (B), (D), (I), and (K), data are shown as mean ± SE (three independent experiments, unpaired Welch’s t test). *p < 0.05, **p < 0.01.