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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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Physical interactions between ESCRT proteins and Clp1p as revealed by two-hybrid analysis.(a–e) Budding yeast strains containing GAL4-galactosidase transcriptional readout were transformed with the yeast two-hybrid bait (GAL4 DNA binding domain) and prey (GAL4 transcription activation domain) constructs indicated. Clp1p was fused to GAL4 and ESCRT proteins from each class were fused to GAL4. Mbx1p, a known Clp1p interacting protein [17], was used a positive control for the assay. Transformed strains were grown for three days on selective medium with the X-gal overlay assay then performed. Experiments were performed three times with qualitatively similar results and illustrative examples of performed two-hybrid reactions are shown. (f) Summary of physical interactions between Clp1p and ESCRT proteins identified by yeast two-hybrid analysis. The table indicates the presence or absence of yeast showing a blue colour observed when each of the ESCRT proteins with Clp1p was assayed.
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pone-0111789-g005: Physical interactions between ESCRT proteins and Clp1p as revealed by two-hybrid analysis.(a–e) Budding yeast strains containing GAL4-galactosidase transcriptional readout were transformed with the yeast two-hybrid bait (GAL4 DNA binding domain) and prey (GAL4 transcription activation domain) constructs indicated. Clp1p was fused to GAL4 and ESCRT proteins from each class were fused to GAL4. Mbx1p, a known Clp1p interacting protein [17], was used a positive control for the assay. Transformed strains were grown for three days on selective medium with the X-gal overlay assay then performed. Experiments were performed three times with qualitatively similar results and illustrative examples of performed two-hybrid reactions are shown. (f) Summary of physical interactions between Clp1p and ESCRT proteins identified by yeast two-hybrid analysis. The table indicates the presence or absence of yeast showing a blue colour observed when each of the ESCRT proteins with Clp1p was assayed.

Mentions: One explanation for the observed genetic interactions is that the encoded proteins directly interact. We therefore used two-hybrid analysis to qualitatively search for invivo interactions between ESCRT proteins and Plo1p, Ark1p and Clp1p. A panel of ESCRT proteins was screened using Plo1p (wild-type, kinase-dead and polo box mutants (Fig. 3)), Ark1p (wild-type and a kinase-dead mutant (Fig. 4)) and Clp1 (wild-type and a truncation mutant (Fig. 5)). These analyses revealed that Plo1p interacts with Sst4p, Vps28p, Vps25p, Vps20p and Vps32p, but not Sst6p, Vps36p, Vps2p or Vps4p (Fig. 3). We found Ark1p interacted only in a kinase-inactivated form with Vps20p and Vps32p (Fig. 4); this is evident on qualitatively comparing Ark1.K147R to the empty vector. Clp1p was observed to interact only with Vps28p (Fig. 5); this was noted on the basis of the lack of transcriptional readout, which we propose to occur via Vps28p-mediated sequestration of Clp1aa.1-371 from transcriptional auto-activation.


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)

Physical interactions between ESCRT proteins and Clp1p as revealed by two-hybrid analysis.(a–e) Budding yeast strains containing GAL4-galactosidase transcriptional readout were transformed with the yeast two-hybrid bait (GAL4 DNA binding domain) and prey (GAL4 transcription activation domain) constructs indicated. Clp1p was fused to GAL4 and ESCRT proteins from each class were fused to GAL4. Mbx1p, a known Clp1p interacting protein [17], was used a positive control for the assay. Transformed strains were grown for three days on selective medium with the X-gal overlay assay then performed. Experiments were performed three times with qualitatively similar results and illustrative examples of performed two-hybrid reactions are shown. (f) Summary of physical interactions between Clp1p and ESCRT proteins identified by yeast two-hybrid analysis. The table indicates the presence or absence of yeast showing a blue colour observed when each of the ESCRT proteins with Clp1p was assayed.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4214795&req=5

pone-0111789-g005: Physical interactions between ESCRT proteins and Clp1p as revealed by two-hybrid analysis.(a–e) Budding yeast strains containing GAL4-galactosidase transcriptional readout were transformed with the yeast two-hybrid bait (GAL4 DNA binding domain) and prey (GAL4 transcription activation domain) constructs indicated. Clp1p was fused to GAL4 and ESCRT proteins from each class were fused to GAL4. Mbx1p, a known Clp1p interacting protein [17], was used a positive control for the assay. Transformed strains were grown for three days on selective medium with the X-gal overlay assay then performed. Experiments were performed three times with qualitatively similar results and illustrative examples of performed two-hybrid reactions are shown. (f) Summary of physical interactions between Clp1p and ESCRT proteins identified by yeast two-hybrid analysis. The table indicates the presence or absence of yeast showing a blue colour observed when each of the ESCRT proteins with Clp1p was assayed.
Mentions: One explanation for the observed genetic interactions is that the encoded proteins directly interact. We therefore used two-hybrid analysis to qualitatively search for invivo interactions between ESCRT proteins and Plo1p, Ark1p and Clp1p. A panel of ESCRT proteins was screened using Plo1p (wild-type, kinase-dead and polo box mutants (Fig. 3)), Ark1p (wild-type and a kinase-dead mutant (Fig. 4)) and Clp1 (wild-type and a truncation mutant (Fig. 5)). These analyses revealed that Plo1p interacts with Sst4p, Vps28p, Vps25p, Vps20p and Vps32p, but not Sst6p, Vps36p, Vps2p or Vps4p (Fig. 3). We found Ark1p interacted only in a kinase-inactivated form with Vps20p and Vps32p (Fig. 4); this is evident on qualitatively comparing Ark1.K147R to the empty vector. Clp1p was observed to interact only with Vps28p (Fig. 5); this was noted on the basis of the lack of transcriptional readout, which we propose to occur via Vps28p-mediated sequestration of Clp1aa.1-371 from transcriptional auto-activation.

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