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A cytosolic factor mediating membrane recruitment of AP-1 clathrin adaptors

Suri, Gregor. A cytosolic factor mediating membrane recruitment of AP-1 clathrin adaptors. 2008, Doctoral Thesis, University of Basel, Faculty of Science.

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Abstract

Transport of cargo within the endocytic and secretory pathway is generally mediated by coated vesicles. These vesicles are formed through the recruitment of cytosolic coat proteins to the donor membrane that act as a scaffold to form coated buds and vesicles. At the same time they selectively concentrate cargo proteins by interacting with cytosolic signals. Clathrin, in combination with different adaptor proteins (APs), is the major coat protein for vesicle formation at the plasma membrane, endosomes and the trans-Golgi network. Best characterized is clathrin mediated endocytosis at the plasma membrane which involves AP-2 and a network of associated proteins. Much less is known about AP-1 mediated clathrin coated vesicle formation at the TGN/endosomes. In vitro studies demonstrated that the minimal requirements to recruit AP-1 to liposome membranes are activated Arf1, phosphoinositides, and either sorting signals or an unknown cytosolic factor. In order to identify this factor, we fractionated calf brain cytosol by several chromatographic methods. Fractions were tested for factor dependent AP-1 recruitment activity using an in vitro assay. Purification via ammonium sulfate precipitation, hydrophobic interaction chromatography, anion/cation exchange chromatography or hydroxyapatite chromatography produced a final fraction containing three major proteins: amphiphysin 1, amphiphysinand endophilin A1. All three proteins are known accessory factors in clathrin coated vesicle formation at the plasma membrane. Co-immunodepletion of amphiphysinandresulted in a strong reduction of AP-1 recruitment activity. Therefore we conclude that a heterodimer of amphiphysinandis the long searched for cytosolic factor, required to recruit AP-1 in the absence of sorting signals in vitro. Our results strongly suggest that amphiphysin 1, amphiphysinand endophilin A1 are also involved in AP-1 mediated clathrin coated vesicle formation at the TGN and endosomes in vivo.
Advisors:Spiess, Martin
Committee Members:Hauri, Hans-Peter
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Growth & Development > Biochemistry (Spiess)
UniBasel Contributors:Spiess, Martin and Hauri, Hans-Peter
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:8514
Thesis status:Complete
Number of Pages:133
Language:English
Identification Number:
edoc DOI:
Last Modified:05 Apr 2018 17:32
Deposited On:13 Feb 2009 16:50

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