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A complex network of interactions between mitotic kinases, phosphatases and ESCRT proteins regulates septation and membrane trafficking in S. pombe.

Bhutta MS, Roy B, Gould GW, McInerny CJ - PLoS ONE (2014)

Bottom Line: Furthermore, we observed defective endosomal sorting in mutants of plo1, ark1 and clp1, as has been reported for ESCRT mutants, consistent with a role for these kinases in the control of ESCRT function in membrane traffic.Multiple observations indicate functional interplay between polo and ESCRT components: firstly, two-hybrid in vivo interactions are reported between Plo1p and Sst4p, Vps28p, Vps25p, Vps20p and Vps32p; secondly, co-immunoprecipitation of human homologues of Vps20p, Vps32p, Vps24p and Vps2p by human Plk1; and thirdly, in vitro phosphorylation of budding yeast Vps32p and Vps20p by polo kinase.Two-hybrid analyses also identified interactions between Ark1p and Vps20p and Vps32p, and Clp1p and Vps28p.

View Article: PubMed Central - PubMed

Affiliation: Henry Wellcome Laboratory of Cell Biology, Davidson Building, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.

ABSTRACT
Cytokinesis and cell separation are critical events in the cell cycle. We show that Endosomal Sorting Complex Required for Transport (ESCRT) genes are required for cell separation in Schizosaccharomyces pombe. We identify genetic interactions between ESCRT proteins and polo and aurora kinases and Cdc14 phosphatase that manifest as impaired growth and exacerbated defects in septation, suggesting that the encoded proteins function together to control these processes. Furthermore, we observed defective endosomal sorting in mutants of plo1, ark1 and clp1, as has been reported for ESCRT mutants, consistent with a role for these kinases in the control of ESCRT function in membrane traffic. Multiple observations indicate functional interplay between polo and ESCRT components: firstly, two-hybrid in vivo interactions are reported between Plo1p and Sst4p, Vps28p, Vps25p, Vps20p and Vps32p; secondly, co-immunoprecipitation of human homologues of Vps20p, Vps32p, Vps24p and Vps2p by human Plk1; and thirdly, in vitro phosphorylation of budding yeast Vps32p and Vps20p by polo kinase. Two-hybrid analyses also identified interactions between Ark1p and Vps20p and Vps32p, and Clp1p and Vps28p. These experiments indicate a network of interactions between ESCRT proteins, plo1, ark1 and clp1 that coordinate membrane trafficking and cell separation in fission yeast.

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ESCRT proteins and Plo1p, Ark1p and Clp1p are required for vacuolar sorting in fission yeast.(a) Defective vacuolar sorting is observed in fission yeast with individual chromosomal deletions of ESCRT genes. Wild-type and ESCRT-deleted fission yeast strains, transformed with Ub-GFP-SpCPS, were cultured in liquid minimal medium at 25°C, stained with FM 4-64, and visualised using confocal microscopy. Mutants of plo1 and ark1 (b) and clp1 (c) cause defective vacuolar sorting in fission yeast. Wild-type and fission yeast plo1-ts35, ark1-T8, ark1-T11 and clp1 strains, transformed with Ub-GFP-SpCPS, were cultured in liquid minimal medium, stained with FM 4-64, and visualised using a confocal microscope. Strains were cultured at 25°C or 30°C. Scale bar, 10 µm. Experiments were performed three times with qualitatively similar results and data from a typical experiment are shown.
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pone-0111789-g006: ESCRT proteins and Plo1p, Ark1p and Clp1p are required for vacuolar sorting in fission yeast.(a) Defective vacuolar sorting is observed in fission yeast with individual chromosomal deletions of ESCRT genes. Wild-type and ESCRT-deleted fission yeast strains, transformed with Ub-GFP-SpCPS, were cultured in liquid minimal medium at 25°C, stained with FM 4-64, and visualised using confocal microscopy. Mutants of plo1 and ark1 (b) and clp1 (c) cause defective vacuolar sorting in fission yeast. Wild-type and fission yeast plo1-ts35, ark1-T8, ark1-T11 and clp1 strains, transformed with Ub-GFP-SpCPS, were cultured in liquid minimal medium, stained with FM 4-64, and visualised using a confocal microscope. Strains were cultured at 25°C or 30°C. Scale bar, 10 µm. Experiments were performed three times with qualitatively similar results and data from a typical experiment are shown.

Mentions: Various classes of ESCRT proteins function as part of the endosomal sorting machinery [24], [25], and ESCRT genes have been shown to be required for correct Ub-GFP-SpCPS localisation in fission yeast [14]. The distribution of Ub-GFP-SpCPS in cells containing mutants of plo1, ark1 and clp1 was examined. Consistent with published data, Ub-GFP-SpCPS expressed in wild-type cells exhibited a largely vacuolar pattern of sorting, identified by the fluorescent profiles of Ub-GFP-SpCPS and FM 4-64-stained vacuoles, which strongly suggest co-localisation (Fig. 6a) [14]. However, yeast strains containing ESCRT deletions, with one mutant chosen from each ESCRT class, exhibited a punctate pattern of Ub-GFP-SpCPS fluorescence associated with endosomal staining (Fig. 6a), as reported by Iwaki et al [14].


A complex network of interactions between mitotic kinases, phosphatases and ESCRT proteins regulates septation and membrane trafficking in S. pombe.

Bhutta MS, Roy B, Gould GW, McInerny CJ - PLoS ONE (2014)

ESCRT proteins and Plo1p, Ark1p and Clp1p are required for vacuolar sorting in fission yeast.(a) Defective vacuolar sorting is observed in fission yeast with individual chromosomal deletions of ESCRT genes. Wild-type and ESCRT-deleted fission yeast strains, transformed with Ub-GFP-SpCPS, were cultured in liquid minimal medium at 25°C, stained with FM 4-64, and visualised using confocal microscopy. Mutants of plo1 and ark1 (b) and clp1 (c) cause defective vacuolar sorting in fission yeast. Wild-type and fission yeast plo1-ts35, ark1-T8, ark1-T11 and clp1 strains, transformed with Ub-GFP-SpCPS, were cultured in liquid minimal medium, stained with FM 4-64, and visualised using a confocal microscope. Strains were cultured at 25°C or 30°C. Scale bar, 10 µm. Experiments were performed three times with qualitatively similar results and data from a typical experiment are shown.
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pone-0111789-g006: ESCRT proteins and Plo1p, Ark1p and Clp1p are required for vacuolar sorting in fission yeast.(a) Defective vacuolar sorting is observed in fission yeast with individual chromosomal deletions of ESCRT genes. Wild-type and ESCRT-deleted fission yeast strains, transformed with Ub-GFP-SpCPS, were cultured in liquid minimal medium at 25°C, stained with FM 4-64, and visualised using confocal microscopy. Mutants of plo1 and ark1 (b) and clp1 (c) cause defective vacuolar sorting in fission yeast. Wild-type and fission yeast plo1-ts35, ark1-T8, ark1-T11 and clp1 strains, transformed with Ub-GFP-SpCPS, were cultured in liquid minimal medium, stained with FM 4-64, and visualised using a confocal microscope. Strains were cultured at 25°C or 30°C. Scale bar, 10 µm. Experiments were performed three times with qualitatively similar results and data from a typical experiment are shown.
Mentions: Various classes of ESCRT proteins function as part of the endosomal sorting machinery [24], [25], and ESCRT genes have been shown to be required for correct Ub-GFP-SpCPS localisation in fission yeast [14]. The distribution of Ub-GFP-SpCPS in cells containing mutants of plo1, ark1 and clp1 was examined. Consistent with published data, Ub-GFP-SpCPS expressed in wild-type cells exhibited a largely vacuolar pattern of sorting, identified by the fluorescent profiles of Ub-GFP-SpCPS and FM 4-64-stained vacuoles, which strongly suggest co-localisation (Fig. 6a) [14]. However, yeast strains containing ESCRT deletions, with one mutant chosen from each ESCRT class, exhibited a punctate pattern of Ub-GFP-SpCPS fluorescence associated with endosomal staining (Fig. 6a), as reported by Iwaki et al [14].

Bottom Line: Furthermore, we observed defective endosomal sorting in mutants of plo1, ark1 and clp1, as has been reported for ESCRT mutants, consistent with a role for these kinases in the control of ESCRT function in membrane traffic.Multiple observations indicate functional interplay between polo and ESCRT components: firstly, two-hybrid in vivo interactions are reported between Plo1p and Sst4p, Vps28p, Vps25p, Vps20p and Vps32p; secondly, co-immunoprecipitation of human homologues of Vps20p, Vps32p, Vps24p and Vps2p by human Plk1; and thirdly, in vitro phosphorylation of budding yeast Vps32p and Vps20p by polo kinase.Two-hybrid analyses also identified interactions between Ark1p and Vps20p and Vps32p, and Clp1p and Vps28p.

View Article: PubMed Central - PubMed

Affiliation: Henry Wellcome Laboratory of Cell Biology, Davidson Building, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.

ABSTRACT
Cytokinesis and cell separation are critical events in the cell cycle. We show that Endosomal Sorting Complex Required for Transport (ESCRT) genes are required for cell separation in Schizosaccharomyces pombe. We identify genetic interactions between ESCRT proteins and polo and aurora kinases and Cdc14 phosphatase that manifest as impaired growth and exacerbated defects in septation, suggesting that the encoded proteins function together to control these processes. Furthermore, we observed defective endosomal sorting in mutants of plo1, ark1 and clp1, as has been reported for ESCRT mutants, consistent with a role for these kinases in the control of ESCRT function in membrane traffic. Multiple observations indicate functional interplay between polo and ESCRT components: firstly, two-hybrid in vivo interactions are reported between Plo1p and Sst4p, Vps28p, Vps25p, Vps20p and Vps32p; secondly, co-immunoprecipitation of human homologues of Vps20p, Vps32p, Vps24p and Vps2p by human Plk1; and thirdly, in vitro phosphorylation of budding yeast Vps32p and Vps20p by polo kinase. Two-hybrid analyses also identified interactions between Ark1p and Vps20p and Vps32p, and Clp1p and Vps28p. These experiments indicate a network of interactions between ESCRT proteins, plo1, ark1 and clp1 that coordinate membrane trafficking and cell separation in fission yeast.

Show MeSH
Related in: MedlinePlus