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Regulation of clathrin-coated vesicle nucleation

Thieman, James Robert (2011) Regulation of clathrin-coated vesicle nucleation. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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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.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Thieman, James Robertjat33@pitt.eduJAT33
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairAridor, Meiraridor@pitt.eduARIDOR
Committee MemberBisello, Alessandroalb138@pitt.eduALB138
Committee MemberApodaca, Gerard Lgla6@pitt.eduGLA6
Committee MemberTraub, Lintontraub@pitt.eduTRAUB
Committee MemberWatkins, Simon Cswatkins@pitt.eduSWATKINS
Date: 20 July 2011
Date Type: Completion
Defense Date: 9 June 2011
Approval Date: 20 July 2011
Submission Date: 18 July 2011
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Cell Biology and Molecular Physiology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: clathrin lattice; initiation; plasma membrane; siRNA
Other ID:, etd-07182011-221052
Date Deposited: 10 Nov 2011 19:52
Last Modified: 15 Nov 2016 13:46


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