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Regulated membrane recruitment of dynamin-2 mediated by sorting nexin 9.
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
2004 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, no 41, 42694-42702 p.Article in journal (Refereed) Published
Abstract [en]

The endocytic proteins sorting nexin 9 (SNX9) and dynamin-2 (Dyn2) assemble in the cytosol as a resting complex, together with a 41-kDa protein. We show here that the complex can be activated for membrane binding of SNX9 and Dyn2 by incubation of cytosol in the presence of ATP. SNX9 was essential for Dyn2 recruitment, whereas the reverse was not the case. RNA interference experiments confirmed that SNX9 functions as a mediator of Dyn2 recruitment to membranes in cells. The 41-kDa component was identified as the glycolytic enzyme aldolase. Aldolase bound with high affinity to a tryptophan-containing acidic sequence in SNX9 located close to its Phox homology domain, thereby blocking the membrane binding activity of SNX9. Phosphorylation of SNX9 released aldolase from the native cytosolic complex and rendered SNX9 competent for membrane binding. The results suggest that SNX9-dependent recruitment of Dyn2 to the membrane is regulated by an interaction between SNX9 and aldolase.

Place, publisher, year, edition, pages
2004. Vol. 279, no 41, 42694-42702 p.
Keyword [en]
Adenosine Triphosphate/metabolism, Amino Acid Sequence, Carrier Proteins/*metabolism, Cell Line, Cell Membrane/*metabolism, Cytosol/metabolism, Dose-Response Relationship; Drug, Dynamin II/*metabolism, Endocytosis, Fructose-Bisphosphate Aldolase/metabolism, Glycolysis, Hela Cells, Humans, K562 Cells, Liposomes/metabolism, Models; Biological, Molecular Sequence Data, Mutation, Phosphorylation, Protein Binding, RNA Interference, RNA; Small Interfering/metabolism, Recombinant Proteins/chemistry, Temperature, Transfection, Vesicular Transport Proteins
URN: urn:nbn:se:umu:diva-6538DOI: 10.1074/jbc.M407430200PubMedID: 15299020OAI: diva2:146207
Available from: 2008-01-10 Created: 2008-01-10 Last updated: 2010-08-06Bibliographically approved
In thesis
1. Sorting nexin 9 in clathrin-mediated endocytosis
Open this publication in new window or tab >>Sorting nexin 9 in clathrin-mediated endocytosis
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Umeå: Medicinsk biokemi och biofysik, 2004. 37 p.
Umeå University medical dissertations, ISSN 0346-6612 ; 875
Cell and molecular biology, sorting nexin, dynamin, clathrin, endocytosis, human, adaptor protein complexes, Cell- och molekylärbiologi
National Category
Biochemistry and Molecular Biology
Research subject
Medical Biochemistry
urn:nbn:se:umu:diva-197 (URN)91-7305-599-9 (ISBN)
Public defence
2004-03-05, KB3A9, KBC huset, Umeå, 09:00
Available from: 2004-02-18 Created: 2004-02-18 Last updated: 2010-08-06Bibliographically approved

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Lundmark, RichardCarlsson, Sven R
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