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Protein Interaction Domains in Clathrin-Mediated Endocytosis

Hawryluk, Matthew John (2006) Protein Interaction Domains in Clathrin-Mediated Endocytosis. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Clathrin-mediated endocytosis is the major process by which cells internalize nutrients, extracellular macromolecules, and membrane constituents to regulate such diverse processes as cell polarity, development, and motility. Over twenty proteins comprise a large protein interaction web that is pertinent to this process. This work investigates proteins that act as clathrin-associated sorting proteins (CLASPs), and their interactions with other endocytic components. Epsin 1 is shown to be a CLASP that engages components of the endocytic clathrin coat and selects for polyubiquitinated cargo. The interaction with polyubiquitin is enabled through epsin 1's UIM domains. I show that polyubiquitin is an efficient endocytic signal, which is relevant for physiological mammalian substrates such as the epithelial sodium channel (ENaC). Stonin 2 behaves as an unconventional CLASP, as it doesn't directly engage clathrin or the plasma membrane. My work uses this protein to biochemically characterize the WXXF motif and identify a privileged binding site located on the sandwich subdomain of the AP-2 ? appendage. This work supports a model in which arrays of binding motifs and multiple engagement sites on the ? appendage allow for an increase in binding affinity, which affects the temporal ordering of endocytic accessory protein interactions during clathrin-mediated endocytosis. These studies have defined important protein interactions that have improved our understanding of the molecular mechanism of clathrin-mediated endocytosis.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Hawryluk, Matthew
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairApodaca, Gerard Lgla6@pitt.eduGLA6
Committee MemberLevitan, Edwin Slevitan@server.pharm.pitt.eduELEVITAN
Committee MemberJohnson, John Pjohnson@msx.dept-med.pitt.eduJOHNSONJ
Committee MemberAridor, Meiraridor@pitt.eduARIDOR
Date: 30 August 2006
Date Type: Completion
Defense Date: 9 August 2006
Approval Date: 30 August 2006
Submission Date: 27 August 2006
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: AP-2; CLASP; Clathrin; Clathrin-Mediated Endocytosis; Endocytosis; Eps15; Epsin; Membrane Traffic; Protein Sorting Signals; Stonin; Ubiquitin
Other ID:, etd-08272006-230458
Date Deposited: 10 Nov 2011 20:01
Last Modified: 15 Nov 2016 13:49


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