Fig 2: Miro GTPases interact physically with GBF1. (A,B) RPE1 cells were cotransfected with Venus or Venus-tagged GBF1 and either Myc or Myc-Miro1 (A) or Myc-Miro2 (B). Immunoprecipitations were carried out with GBP-beads. Cells lysates (Input) and immunoprecipitated proteins were analyzed by Western blotting using anti-GFP and anti-Myc antibodies. The input lanes represent 15% of lysate used in immunoprecipitation reactions. (C) RPE1 cells were cotransfected with Arf1-T31N-GFP, Arf1-Q71L-GFP or GFP and Myc-Miro2. GFP-tagged proteins were immunoprecipitated with magnetic GFP-Trap®_A. Cells lysates (Input) and immunoprecipitated proteins were analyzed by Western blotting using GFP- and Myc-antibodies. The input lanes represent 10% of lysate used in immunoprecipitation reactions. One representative experiment of three independent experiments is shown. Bands were quantified by a densitometric analysis performed using Image J software. Results are the mean of the ratio between the Myc and the GFP band values ± S.D. of the three independent experiments. *P = 0.05 versus control cells.

Fig 3: Neural activation induces a molecular change in CoRest-C.a Binding of CoRest-F proteins to the recombinant Rpd3 proteins was not altered by optogenetic activation. The flies carrying MBsw, UAS-CsChrimson, and UAS-CoRest-F::myc were fed RU, and illuminated with red light. The head extracts were pulled down with the recombinant Rpd3::HA, which was expressed in Sf9 insect cells. Two-sided Mann–Whitney U-test, P = 0.6475; n = 6. b Binding of Rpd3 proteins to the recombinant CoRest-F proteins was reduced by optogenetic activation, depending on CoRest-C. Rpd3::HA and CsChrimson were expressed by MBsw in WT flies or in flies carrying UAS-CoRest-C-miRNA, and the flies were illuminated with red light. The head extracts were pulled down with recombinant CoRest-F::myc, which was expressed in E. coli. (Left) Two-sided Mann–Whitney U-test, P = 0.0007, n = 5. (Right) Two-sided Mann–Whitney U-test, P = 0.7481, n = 4. c Binding of CoRest-C proteins to the recombinant Rpd3 proteins was increased by optogenetic activation, when the deacetylation activity of Rpd3 was inhibited. CoRest-C::myc and CsChrimson were expressed by MBsw, and the flies were illuminated with red light. The head extracts were pulled down with recombinant Rpd3::HA (WT proteins or proteins carrying H137A mutation), which was expressed in Sf9 insect cells. The indicated concentration of the HDAC inhibitor sodium butyrate (NaBu) was included in the reaction. (Left) One-way ANOVA (P = 0.0078; n = 6) followed by Tukey’s multiple comparisons (two sided) was performed. (Right) Two-sided Mann–Whitney U-test, P = 0.0369; n = 6. The antibodies for the indicated epitope tags were used in a western blot analysis. Data are represented as mean ± s.e.m. n.s., not significant, P > 0.05; *P < 0.05; ***P < 0.001, which is based on experimental replicates using different pooled samples, and the number is indicated as n above. Source data are provided as a Source Data file.

Fig 4: SUMOylation blocks Rts1 binding to Sgo1, and release of this interaction is important for stable biorientation. (A) SUMOylated Sgo1 has reduced binding affinity for Rts1. Recombinant V5-tagged Sgo1 was mixed with components of the SUMOylation pathway in the presence or absence of ATP. Anti-V5 antibody coupled to Protein G Dynabeads was added to the mixture, washed thoroughly, and incubated with extract from sgo1? (AM827) or sgo1? RTS1-9MYC (AMy8832) cells. Levels of Sgo1 and Rts1 bound to beads were probed by anti-V5 and anti-Myc Western blotting, respectively. IB, immunoblot. (B) Rts1 preferentially binds to unSUMOylated Sgo1. Rts1-9Myc was immunoprecipitated from sgo1? RTS1-9MYC (AMy8832) using anti-Myc antibody coupled to Protein G Dynabeads. Beads were incubated with in vitro SUMOylation reaction mixture containing Sgo1. Levels of Sgo1 and Rts1 bound to beads were probed by anti-V5 and anti-Myc Western blotting, respectively. (C and D) The kinetochore-proximal pool of Rts1 was increased in the absence of SUMOylation in metaphase. Strains used carried Rts1-GFP and Mtw1-tdTOMATO and were wild type (AMy29076), siz1? siz2? (AMy29074), and sgo1-4R (AMy29083). Measurements were made in shmooing cells (G1) or right before the emergence of two Mtw1-tdTOMATO kinetochore foci (metaphase). (C) Representative images of Rts1-GFP and Mtw1-tdTOMATO signals from the same z-positions. Images were acquired as described in Materials and methods. Scale bar = 1 µm. (D) Quantification of Rts1-GFP peak intensity that occupied the same area as the kinetochore (Mtw1-tdTOMATO). ImageJ was used to measure the intensities. For each cell, the area occupied by the kinetochore was selected by outlining the boundaries of Mtw1-tdTOMATO signals. The same area in the same z-slice was restore selected in the Rts1-GFP channel, the peak green value was measured, and background was subtracted. 48 cells from two independent experiments were analyzed for each strain. Red dots represent mean values, and error bars represent standard deviation. Statistics: one-tailed Student’s t test (**, P < 0.01; ****, P < 0.0001). (E–G) Forced interaction between Rts1 and Sgo1 destabilizes biorientation. Strains used carry CEN4-mNeonGreen pMET-CDC20 and SPC42-tdTOMATO and were SGO1-GBP (AMy28389), SGO1-GBP RTS1-nonfluorescent GFP (AMy28092), sgo1-4R-GBP (AMy28417), and sgo1-4R-GBP RTS1-nonfluorescent GFP (AMy28416). The assay was performed as described in Fig. 6 D. (E) Tethering Rts1 to wild-type Sgo1 or Sgo1-4R does not affect the initial establishment of biorientation. (F) Increased reassociation of CEN4-mNeonGreen dots was observed when Rts1 was tethered to wild-type Sgo1 or Sgo1-4R. At least 50 cells from two independent experiments were analyzed for each strain. Shown are the percentages of cells with the indicated number of switchings from two CEN4-GFP dots back to one dot. Statistics: Fisher’s exact test (*, P < 0.05; **, P < 0.01; N.S., not significant). (G) Missegregation is modestly increased when Rts1 is tethered to wild-type Sgo1 or Sgo1-4R. (H) Tethering Sgo1 to Rts1 mildly delays metaphase. Strains used carried CDC14-mRuby YFP-TUB1 and were wild type (AM29672), SGO1-GBP-3MYC RTS1-nfGFP (AM29711), and sgo1-4R-GBP-3MYC RTS1-nfGFP (AM29715). Shown are the average of three independent experiments, and error bars represent standard error. Statistics: one-tailed Student’s t test comparing to the wild-type strain (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Fig 5: Identification of phospho-degron motifs in Ame1.(A) Flag- and Myc-tagged versions of Ame1 and Okp1 were expressed from a two-micron plasmid under a bidirectional galactose-inducible promoter. Under normal growth conditions in YEP + dextrose or YEP + raffinose, no overexpression occurs, overexpression is only induced by adding galactose (GAL) to the medium. After 0, 2, and 5 hr in GAL, cell extracts were prepared and protein expression was followed using western blot analysis. (B) Western blot analysis of overexpressed Ame1-WT, -7A, and -7E variants in a wild-type strain background. (C) Quantification of protein levels of Ame1-WT and Ame1-7A after indicated times in galactose medium. Mean values and standard error of the mean are indicated, n = 7. (D) Overview of Ame1 phospho-mutants used for overexpression studies (E) or in vitro kinase assays (F). (E) Overexpression studies of individual Ame1 phospho-variants. Ame1 protein levels in this experiment are quantified below, the Ame1-WT level is set to 1. Okp1 levels are stable and used for normalization of Ame1 protein levels. (F) In vitro kinase assay of AO complexes using recombinant Cdk1-Clb2. Note reduced or lacking phosphorylation of Ame1-4A and -7A, respectively. Also Okp1 can be phosphorylated by Cdk1-Clb2. (G) Cdk1 target sites in Ame1 resemble two different types of phospho-degrons motifs that are recognized by the E3 ubiquitin ligase complex SCF-Cdc4.