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"In vitro" reconstitution of "trans"-Golgi exit and the effect of GAG attachment on protein sorting

Kobialka, Szymon. "In vitro" reconstitution of "trans"-Golgi exit and the effect of GAG attachment on protein sorting. 2007, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_8398

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Abstract

The trans-Golgi network is one of the main sorting stations for proteins and lipids in the eukaryotic endomembrane system. Yet, the mechanisms for sorting and transport out of the trans-Golgi network remain poorly understood. In this work, an in vitro procedure to reconstitute carrier formation at the trans-Golgi was established. Using this assay, it could be shown that formation of carriers containing the asialoglycoprotein receptor H1 and the cation dependent mannose 6-phosphate receptor is independent of the clathrin machinery, but dependent on GTP hydrolysis. This is in contrast to the common notion that the mannose 6phosphate receptor exits the Golgi in clathrin coated vesicles, but is consistent with recent evidence for tubular export carriers or maturation. The assay is based on sulfation, which can only take place in the trans-Golgi and thus defines the starting compartment. To improve the radioactive sulfation signal in the assay, a novel tag was developed that contains an attachment site for glycosaminoglycans (long, linear and heavily sulfated carbohydrate polymers). To investigate the influence of glycosaminoglycan attachment on protein sorting, biosynthetic exocytosis, endocytosis, endocytic recycling, and overall stability of H1 with or without glycan was examined. While stability and recycling were unaffected, exocytosis was accelerated and endocytosis was slowed down by carbohydrate attachment. Endocytosis was impaired due to interactions of glycosaminoglycan with the extracellular matrix, but the alteration of exocytosis rate requires further investigation, as it suggests that the glycan diverts the protein to a different sorting route.
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:8398
Thesis status:Complete
Bibsysno:Link to catalogue
Number of Pages:90
Language:English
Identification Number:
Last Modified:05 Apr 2018 17:32
Deposited On:13 Feb 2009 16:36

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