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Sorting nexin 9 in clathrin-mediated endocytosis
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
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. , p. 37
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 875
Keywords [en]
Cell and molecular biology, sorting nexin, dynamin, clathrin, endocytosis, human, adaptor protein complexes
Keywords [sv]
Cell- och molekylärbiologi
National Category
Biochemistry and Molecular Biology
Research subject
Medical Biochemistry
Identifiers
URN: urn:nbn:se:umu:diva-197ISBN: 91-7305-599-9 (print)OAI: oai:DiVA.org:umu-197DiVA, id: diva2:142600
Public defence
2004-03-05, KB3A9, KBC huset, Umeå, 09:00
Opponent
Supervisors
Available from: 2004-02-18 Created: 2004-02-18 Last updated: 2010-08-06Bibliographically approved
List of papers
1. The beta-appendages of the four adaptor-protein (AP) complexes: structure and binding properties, and identification of sorting nexin 9 as an accessory protein to AP-2
Open this publication in new window or tab >>The beta-appendages of the four adaptor-protein (AP) complexes: structure and binding properties, and identification of sorting nexin 9 as an accessory protein to AP-2
2002 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 362, no 3, p. 597-607Article in journal (Refereed) Published
Abstract [en]

Adaptor protein (AP) complexes are essential components for the formation of coated vesicles and the recognition of cargo proteins for intracellular transport. Each AP complex exposes two appendage domains with that function to bind regulatory accessory proteins in the cytosol. Secondary structure predictions, sequence alignments and CD spectroscopy were used to relate the beta-appendages of all human AP complexes to the previously published crystal structure of AP-2. The results suggested that the beta-appendages of AP-1, AP-2 and AP-3 have similar structures, consisting of two subdomains, whereas that of AP-4 lacks the inner subdomain. Pull-down and overlay assays showed partial overlap in the binding specificities of the beta-appendages of AP-1 and AP-2, whereas the corresponding domain of AP-3 displayed a unique binding pattern. That AP-4 may have a truncated, non-functional domain was indicated by its apparent inability to bind any proteins from cytosol. Of several novel beta-appendage-binding proteins detected, one that had affinity exclusively for AP-2 was identified as sorting nexin 9 (SNX9). SNX9, which contains a phox and an Src homology 3 domain, was found in large complexes and was at least partially associated with AP-2 in the cytosol. SNX9 may function to assist AP-2 in its role at the plasma membrane.

Keywords
adaptin, coated vesicle, endocytosis, intracellular transport
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-3750 (URN)11879186 (PubMedID)
Available from: 2004-02-18 Created: 2004-02-18 Last updated: 2018-06-09Bibliographically approved
2. Sorting nexin 9 participates in clathrin-mediated endocytosis through interactions with the core components
Open this publication in new window or tab >>Sorting nexin 9 participates in clathrin-mediated endocytosis through interactions with the core components
2003 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 47, p. 46772-46781Article in journal (Refereed) Published
Abstract [en]

Sorting nexin 9 (SNX9) belongs to a family of proteins, the sorting nexins, that are characterized by the presence of a subclass of the phosphoinositide-binding phox domain. SNX9 has in its amino terminus a Src homology 3 domain and a region with predicted low complexity followed by a carboxyl-terminal part containing the phox domain. We previously found that SNX9 is one of the major proteins in hematopoietic cells that binds to the alpha and beta2-appendages of adaptor protein complex 2 (AP-2), a protein with a critical role in the formation of clathrin-coated vesicles at the plasma membrane. In the present study we show that clathrin and dynamin-2, two other essential molecules in the endocytic process, also interact with SNX9. We found that both AP-2 and clathrin bind to the low complexity region in SNX9 in a cooperative manner, whereas dynamin-2 binds to the Src homology 3 domain. In the cytosol, SNX9 is present in a 14.5 S complex containing dynamin-2 and an unidentified 41-kDa protein. In HeLa cells, SNX9 co-localized with both AP-2 and dynamin-2 at the plasma membrane or on vesicular structures derived from it but not with the early endosomal marker EEA1 or with AP-1. The results suggest that SNX9 may be recruited together with dynamin-2 and become co-assembled with AP-2 and clathrin at the plasma membrane. Overexpression in both K562 and HeLa cells of truncated forms of SNX9 interfered with the uptake of transferrin, consistent with a role of SNX9 in endocytosis.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-3751 (URN)10.1074/jbc.M307334200 (DOI)12952949 (PubMedID)
Available from: 2004-02-18 Created: 2004-02-18 Last updated: 2018-06-09Bibliographically approved
3. Regulated membrane recruitment of dynamin-2 mediated by sorting nexin 9.
Open this publication in new window or tab >>Regulated membrane recruitment of dynamin-2 mediated by sorting nexin 9.
2004 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, no 41, p. 42694-42702Article 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.

Keywords
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
Identifiers
urn:nbn:se:umu:diva-6538 (URN)10.1074/jbc.M407430200 (DOI)15299020 (PubMedID)
Available from: 2008-01-10 Created: 2008-01-10 Last updated: 2018-06-09Bibliographically approved

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