Clathrin-mediated endocytosis is a selective pathway for the entry of transmembrane proteins into the cell through the generation of a short-lived vesicular intermediate. Cells and tissues depend on this process for obtaining nutrients, modulation of signaling and cell migration. The clathrin-coated structure intermediate is assembled on the plasma membrane from a cohort of 20-30 distinct proteins that aid in cargo selection, scaffolding, membrane bending and scission of the vesicle. Exactly how these complex assemblies are nucleated at the plasma membrane remains unclear although the lipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) plays an important role by anchoring many of the endocytic components. The work in this thesis helps to clarify the nucleation phase by describing the molecular details of the interaction between a PtdIns(4,5)P2-generating lipid kinase PIPKIgamma and the heterotetrameric clathrin adaptor AP-2. By engaging a subdomain on the AP-2 beta2 subunit appendage, the kinase is strategically positioned at assembly sites to generate PtdIns(4,5)P2 and drive coat assembly forward. Clathrin binds to the same subdomain on the beta2 appendage but with a higher apparent affinity. I therefore invoke a model in which PtdIns(4,5)P2 production for nucleation is negatively regulated by PIPKIgamma displacement from AP-2 by clathrin at later stages of assembly. I also demonstrate that a cargo-sorting alternate adaptor that binds to the other subsite on the AP-2 beta2 appendage is not subject to displacement by clathrin during clathrin-coated vesicle budding, ensuring non-competitive cargo incorporation into the vesicle. Finally, the PtdIns(4,5)P2-binding EFC domain proteins FCHO1 and FCHO2 have been proposed to act as dedicated nucleators of clathrin-coated structures on the plasma membrane. I demonstrate in multiple cell lines that these proteins are not invariantly required for placement of clathrin-coated assemblies on the plasma membrane despite being early arriving components themselves. FCHO1/2 are involved in the regulation of the size and number of these assemblies in some cellular contexts. My data support the model of PtdIns(4,5)P2 regulated, not protein regulated, nucleation of clathrin-coated structures; however multiple parallel pathways may contribute to initiation of endocytic buds.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-07182011-221052 |
| Date | 20 July 2011 |
| Creators | Thieman, James Robert |
| Contributors | Linton Traub, Meir Aridor, Alessandro Bisello, Simon C. Watkins, Gerard L. Apodaca |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-07182011-221052/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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