Spindle Position Checkpoint Kinase Kin4 Regulates Organelle Transport in Saccharomyces cerevisiae

Figure 1. A genome-wide genetic screen to identify novel factors regulating peroxisome dynamics. (A) Schematic depiction of the genome-wide screen layout. The vps1Δdnm1Δ strain (mating type α) expressing genomically integrated peroxisomal mNG-PTS1 and cytosolic mCherry was crossed with the library of deletion mutants and the library of hypomorphic alleles of essential genes (DAmP). Both mutant libraries are mating type a. Sporulation was induced, and haploids expressing both fluorescent marker proteins and containing VPS1 and DNM1 gene deletion, as well as deletion of the library mutant, were selected. Automated imaging of the arrayed collection was performed on the growing cell cultures, and the obtained images were analysed manually. (B) Summary of the imaging analysis. The three observed main phenotypes are indicated by description, schematic depictions, and exemplified by a representative image. ‘‘Control’’ represents the phenotype obtained with most triple mutants and is identical to the phenotype observed in the control vps1Δdnm1Δ cells. The ‘‘class 1, 2, and 3′’ mutants’ phenotypes were considered hits. Arrowheads in the class 2 mutant panel indicate either bud (left) or mother…

Figure 2. SPOC kinase Kin4 is required for peroxisome transport to the bud. (A) The vps1Δdnm1Δ cells lacking INP1, INP2, or KIN4 and expressing mNG-PTS1 and cytosolic mCherry were analysed using an epifluorescence microscope. (C) Control phenotype of the vps1Δdnm1Δ cells. (B) Quantification of peroxisome distribution in strains in (A). A minimum of 108 cells were analysed per strain. (C) Quantification of peroxisome distribution in cells (at least n = 77) containing mNG-PTS1 punctate patterns from strains in (A,D). (D) Representative epifluorescence images of vps1Δdnm1Δinp1Δkin4Δ and vps1Δdnm1Δinp2Δkin4Δ cells. (E) Quantitation of peroxisome positioning in the cells (at least n = 77) from strains in (A,D). Cells expressing mNG-PTS1 and cytosolic mCherry from exponentially growing cultures were used for all epifluorescence microscopy experiments. Bright-field images were collected in one plane and processed to highlight the cell circumference in blue. Scale bar, 5 µm.

Figure 3. Frk1 is a functional paralog of Kin4. (A) Epifluorescence images were captured from the various exponentially growing S. cerevisiae mutant cells expressing mNG-PTS1. Representative images are shown as merged z-stacks. Bright-field images were collected in one plane and processed to highlight the cell circumference in blue. Scale bar, 5 µm. (B) Peroxisome quantitation of cells from the strains in (A). A minimum of 100 cells were analysed from 3 independent experiments. Statistical significance was determined using two-way ANOVA test. ns: not significant, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Error bars indicate SD: standard deviation. (C) Sequence alignment for the activation loop motifs in Elm1 substrate kinases. Elm1 phosphorylates Kin4 at T209, which is strictly conserved in Frk1. (D) Schematic representation for SPOC pathways components and their regulation. Serial dilutions of wild-type, bfa1Δ (E), and elm1Δ (F) cells overexpressing either KIN4 or FRK1 controlled by the GAL1 promoter were spotted on yeast minimal (YM) media plates containing either galactose/glucose as a sole carbon source. The plates were incubated for 3 days at 30 °C before imaging.

Figure 4. Vacuole inheritance is strongly affected in kin4Δfrk1Δ cells. The cells were grown to log phase and incubated with FM4-64 to stain the vacuoles, followed by imaging with epifluorescence microscopy. (A) Representative images for wild-type and kin4Δfrk1Δ cells are shown. Bright-field images were collected in one plane and processed to highlight the cell circumference in blue. Scale bar is 5 μm. (B) A minimum of 170 cells of each strain were scored to analyse the defect in vacuole inheritance. Categories I, II, and III represent the variable FM4-64 staining of the vacuoles in the cells.

Figure 5. Kinase activity of Kin4 is required for its function in peroxisome segregation. Kin4 and Kin4-T209A were expressed under native promoter in vps1Δdnm1Δkin4Δfrk1Δ cells. (A) Representative epifluorescence images for exponentially growing cells are shown. Bright-field images were collected in one plane and processed to highlight the cell circumference in blue. (C) vps1Δdnm1Δ. Scale bar is 5 μm. (B) Quantitation of peroxisome distribution in vps1Δdnm1Δ cells lacking either KIN4 or FRK1 or both. (C) Quantitation of peroxisome distribution in vps1Δdnm1Δkin4Δfrk1Δ cells after Kin4 and Kin4-T209A reintroduction.

Figure 6. Kin4-dependent SPOC activation and inheritance of peroxisomes are independent processes. (A) Representative epifluorescence images of vps1Δdnm1Δ cells lacking BFA1, BUB2, or RTS1 and expressing mNG-PTS1 and cytosolic mCherry. (B) Peroxisome distribution in strains in (A). A minimum of 158 cells were analysed per strain. Cells from exponentially growing cultures were used for epifluorescence microscopy experiments. C: vps1Δdnm1Δ. Scale bar, 5 µm. (C) Mitotic spindle alignment in kin4Δfrk1Δ cells is unaffected. Cells expressing GFP-Tub1 or/and mKate2-PTS1 were grown to log phase and were analysed by epifluorescence microscopy. (D) Peroxisome inheritance is not required for SPOC activation. Serial dilutions of wild-type, bfa1Δ, and inp2Δ cells overexpressing KIN4 controlled by the GAL1 promoter were spotted on yeast minimal (YM) media plates containing either galactose/glucose as a sole carbon source. The plates were incubated for 3 days at 30 °C before imaging. Epifluorescence images from exponentially grown cells expressing mNG-PTS1. Bright-field images were collected in one plane and processed to highlight the cell circumference in blue. Scale bar, 5 µm.

Figure 7. Kin4 and Frk1 are required to maintain Inp2 protein steady-state levels. (A) Inp2-ProtA was expressed in wild-type and kin4Δfrk1Δ cells. Cell lysate extracts were analysed by Western blot. (B) Intensity values for Inp2-ProtA bands were normalised against unsaturated Pgk1 bands and were plotted. Normalised ProtA signals in wild-type cells were set to 1 A. U. where A. U. is an arbitrary unit. (C) Myo2 was tagged with 3xHA in the genome. TCA extraction followed by Western blot analysis was performed on modified wild-type and kin4Δfrk1Δ strains. Error bars indicate SEM (standard error mean). N = 3. ** p < 0.01; two-tailed Student’s t-test. (D) Inp2-ProtA was expressed under native promoter in bfa1Δ and bfa1Δ+GAL-KIN4 cells. Cell lysate extracts were analysed by Western blot. (E) Representative epifluorescence images of bfa1Δ and bfa1Δ+GAL-KIN4 cells expressing HcRed-PTS1 peroxisomal marker. Cells from exponentially growing cultures were used for epifluorescence microscopy experiments. Bright-field images were collected in one plane and processed to highlight the cell circumference in blue. Arrowheads indicate mother cells lacking peroxisomal labelling. Scale bar, 5 µm. (F)…

Figure 8. Kin4 and Frk1 are required to maintain elevated Inp2ΔMIS mutant protein levels. (A) Representative epifluorescence images of inp2Δ cells expressing c-terminally mNG-tagged Inp2 wild-type and ΔMIS mutants under endogenous promoter and mRuby2-PTS1 peroxisomal marker. Cells from exponentially growing cultures were used for epifluorescence microscopy experiments. Bright-field images were collected in one plane and processed to highlight the cell circumference in blue. Scale bar, 5 µm. (B) Quantitation of peroxisome positioning in the cells from strains in (A). A minimum of 100 cells were analysed from 3 independent experiments. Statistical significance was determined using two-way ANOVA test. ns: not significant, ** p < 0.0029, **** p < 0.0001. Error bars indicate SD: standard deviation. (C) C-terminally ProtA-tagged Inp2 wild-type and ΔMIS mutants were expressed in wild-type and inp2Δ cells. Cell lysate extracts were analysed by Western blot. (D) Intensity values for Inp2-ProtA bands were normalised against unsaturated Pgk1 bands and were plotted. Normalised ProtA signals in wild-type cells were set to 1 A. U. where A. U. is an arbitrary unit. Statistical significance was determined…

Figure 9. Schematic model showing the independent roles of Kin4 and Frk1 in peroxisome transport and SPOC. (A) Kin4 and its functional paralog, Frk1, are required for peroxisome transport to the emerging bud. In addition, Kin4 is a key component of SPOC and thus negatively regulates mitotic exit in case of a misaligned spindle. Peroxisome transport is not essential for the activation of SPOC and MEN and vice versa. Though Frk1 (in grey) is a potential SPOC kinase candidate like Kin4, its direct involvement in SPOC requires further investigation. (B) Kin4 and Frk1 contribute to peroxisome transport by maintaining steady state levels of the Myo2 receptor, Inp2, on peroxisomes, thus facilitating stable/efficient Inp2-Myo2 complex formation and active and successful peroxisome transport to the bud. Dma1/2 have been implicated in Inp2 degradation in the bud to terminate peroxisome transport, ensuring directional inheritance of peroxisomes. Here, we propose that, in the absence of Kin4 and Frk1, Dma1/2 recognises Inp2 while it is in the mother cell and leads to its premature degradation, inhibiting peroxisome movement to the bud. SPOC: spindle position checkpoint; MEN: mitotic exit network.