Endosomal sorting complex required for transport (ESCRT) proteins are conserved between Archaea, yeast and mammalian cells. ESCRT proteins mediate membrane scission events in the downregulation of ubiquitin-labelled receptors via the multivesicular body (MVB) pathway and HIV budding from host cells. In addition, ESCRT proteins have an established role in the final stage of cytokinesis, abscission, although the functional mechanisms by which they mediate daughter cell separation have yet to be demonstrated biochemically in vivo.The ESCRT machinery is composed of four subunits: ESCRT-0, -I, -II and -III; and the modular composition of the ESCRT machinery is reflected in its various functions. ESCRT proteins are recruited sequentially to the endosomal membrane for MVB formation: first, ESCRT-0 sequesters ubiquitylated cargo destined for degradation; second, ESCRT-I and II deform the peripheral membrane to produce a bud; and third, ESCRT-III constricts the bud neck to form an intralumenal vesicle. Thereafter, AAA-ATPase Vps4 redistributes ESCRT-III subunits back into the cytoplasm to mediate further MVB formation; it is the association of ESCRT-III and Vps4 that forms the conserved membrane scission machinery in all ESCRT functions.At a precise time during late cytokinesis, ESCRT-I protein TSG101 and ESCRT-associated protein ALIX are recruited to the midbody where they localise to both sides of the dense proteinaceous Flemming body through interactions with CEP55; TSG101 and ALIX in turn recruit ESCRT-III components. Immediately before abscission, ESCRT-III redistributes outwards from the Flemming body to the abscission site; microtubules are severed and the daughter cells separate. Thereafter, ESCRT-III appears on the opposite side of the Flemming body and the process is repeated to produce the midbody remnant. How this selective and specific redistribution of ESCRT proteins is regulated in space and time remains unsolved.To this end, polo kinase and Cdc14 phosphatase were identified as potential regulators of ESCRT function, due to their significant functions in regulating cytokinesis. Homologues in the fission yeast Schizosaccharomyces pombe, Plo1p and Clp1p, are required for either formation or stabilisation of the contractile ring that drives cytoplasmic cleavage. Furthermore, human polo-like kinase, Plk1, maintains CEP55 in a phosphorylated state to negatively regulate its localisation to the midbody; and although Plk1 proteolysis facilitates abscission complex assembly, Plk1 re-emerges at the midbody late during cytokinesis. It was hypothesised, therefore, that polo kinase and Cdc14 phosphatase regulate members of the ESCRT machinery to mediate cytokinetic abscission.To address this, fission yeast was used to study interactions between Plo1p, Clp1p and ESCRT proteins. Initially, ESCRT function in fission yeast cytokinesis was examined by characterising formation of the specialised medial cell wall, the septum, in individual ESCRT deletion strains. ESCRT genes were shown to be required for cytokinesis and cell separation in fission yeast, implying a role for the ESCRT proteins in this process. A yeast genetics approach was then employed to investigate genetic interactions between ESCRT genes and plo1+ and clp1+. Double mutants were produced from crosses between ESCRT deletion strains and mutants of plo1 and clp1. Synthetic defective growth rates were observed in double mutants, indicating genetic interactions between plo1+, clp1+ and ESCRT genes. The effect of single ESCRT deletions on vacuolar sorting in fission yeast was characterised. Single mutants of plo1 and clp1 were also shown to affect vacuolar sorting, indicating novel roles for these proteins in fission yeast. Analysis of vacuolar sorting in double mutants provided further characterisation of observed genetic interactions: plo1+ was regarded to function upstream of ESCRT genes, and clp1+ downstream.The yeast two-hybrid assay was used to further analyse interactions. Physical interactions were observed between Plo1p and Sst4p (human HRS, ESCRT-0), Vps28p (VPS28, ESCRT-I), Vps25p (EAP20, ESCRT-II), Vps20p (CHMP6, ESCRT-III) and Vps32p (CHMP4, ESCRT-III). Clp1p was also shown to interact with Vps28p. Interactions were then investigated between human homologues of these proteins in HEK293 cells. Immunoprecipitation and co-immunoprecipitation methods revealed interactions between Plk1 and CHMP6, CHMP4B, CHMP3 and CHMP2A (all ESCRT-III). Furthermore, interactions were demonstrated between CDC14A and CHMP4B and CHMP2A.These results indicate that polo kinase and Cdc14 phosphatase have conserved roles in regulating ESCRT components. Characterising the nature and functional significance of this regulation may inform future approaches in disease prevention.
【 预 览 】
附件列表
Files
Size
Format
View
Investigating the role of the ESCRT proteins in cytokinesis