Clathrin-mediated endocytosis is a process by which cells can internalise diverse molecules such as nutrients, antigens and signalling-surface receptors. The creation of clathrin-coated vesicles demands interplay between the plasma membrane lipids, cargo molecules and the proteins that build up the coat. This thesis deals with the identification and characterisation of sorting nexin 9 (SNX9) as a novel component of the endocytic machinery. SNX9 belongs to a large family of proteins based on the presence of a PX domain. In addition, SNX9 harbours an SH3 domain followed by a region with predicted low-complexity and a C-terminal BAR homology domain. Binding studies demonstrated that SNX9 interacted with the endocytic core components clathrin and AP-2 and dynamin-2, a GTPase known to be crucial for vesicle scission. The C-terminal region bound to phosphatidylinositols and targeted SNX9 to artificial liposomes and cellular membranes. Consistent with a role in endocytosis, a large portion of SNX9 co-localised with AP-2 and dynamin-2 but not with markers for early endosomes, Golgi. Over-expression of truncated variants of SNX9 in K562 and HeLa cells interfered with the uptake of transferrin. SNX9 recycles between a membrane-bound and a cytosolic pool. In cytosol, SNX9 formed a resting complex together with dynamin-2 and the metabolic enzyme aldolase. Activation for membrane binding involved ATP hydrolysis and correlated with phosphorylation of SNX9 and the release of aldolase. Aldolase bound to a tryptophan-containing acidic region near the clathrin and AP-2 motifs and blocked lipid binding of purified SNX9 derivatives. SNX9 was required for membrane targeting of dynamin2 in vitro and knockdown of SNX9 in HeLa cells by RNAi resulted in impaired membrane localisation. Together these results argue strongly for a role of SNX9 in recruiting and linking of dynamin-2 to sites of vesicle creation.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:umu-197 |
| Date | January 2004 |
| Creators | Lundmark, Richard |
| Publisher | Umeå universitet, Institutionen för medicinsk kemi och biofysik, Umeå : Medicinsk biokemi och biofysik |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Doctoral thesis, comprehensive summary, info:eu-repo/semantics/doctoralThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | Umeå University medical dissertations, 0346-6612 ; 875 |
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