All eukaryotic cells depend on extracellular nutrients and environmental cues for long-term survival. This constant flow of information frequently leads to compensatory remodeling of the plasma membrane. Clathrin- coated transport vesicles can rapidly and selectively convey nutrient receptors, signaling molecules, ion channels, transporters, and structural proteins off the surface membrane, delivering them to an endosomal sorting station for further processing. The cargo selection process is managed by adaptor proteins, traditionally thought primarily to be the heterotetrameric AP-2 complex. In the previous funding cycle, we provided firm experimental evidence that cargo recognition is expanded by a set of highly-selective clathrin- associated sorting proteins (CLASPs). We also made substantial progress in delineating peptide-based interaction motifs within CLASPs that facilitate direct clathrin and AP-2 binding, and have defined structurally some of the cognate interaction surfaces upon the core coat components. In this application, our goals are to extend this characterization of the molecular principles of clathrin coat operation. On the basis of both Saccharomyces cerevisiae and mammalian live-cell imaging studies, it appears that the time of entry and exit of endocytic factors at the bud site reflects a specific role in the assembly/sorting process. Yet, how this is dictated by the protein-protein interaction elements contained within individual endocytic components is not fully apparent. Using a combination of structural studies, biochemical assays for clathrin assembly, RNA interference and functional uptake assays, ultrastructural analysis, and live-cell microscopy, we will dissect individual protein-protein interactions to unravel their contribution and importance in the coat assembly and sorting process. In particular, we will focus on the clathin-AP-2 beta2 subunit interaction hub, and on Disabled-2 and the autosomal recessive hypercholesterolemia (ARM) protein, two CLASPs involved in low density lipoprotein internalization. Additionally, the concept of compositionally heterogeneous, differentially cargo-selective clathrin coats at the cell surface will be tested experimentally. It is anticipated that fundamental new information regarding the molecular mechanisms that link clathrin coat polymerization to cargo capture at the cell surface will be obtained through these investigations.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK053249-15
Application #
8111187
Study Section
Special Emphasis Panel (ZRG1-CB-G (02))
Program Officer
Haft, Carol R
Project Start
1998-03-15
Project End
2013-07-31
Budget Start
2011-08-01
Budget End
2013-07-31
Support Year
15
Fiscal Year
2011
Total Cost
$285,909
Indirect Cost
Name
University of Pittsburgh
Department
Physiology
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Traub, Linton M; Bonifacino, Juan S (2013) Cargo recognition in clathrin-mediated endocytosis. Cold Spring Harb Perspect Biol 5:a016790
Umasankar, P K; Sanker, Subramaniam; Thieman, James R et al. (2012) Distinct and separable activities of the endocytic clathrin-coat components Fcho1/2 and AP-2 in developmental patterning. Nat Cell Biol 14:488-501
Jha, Anupma; Watkins, Simon C; Traub, Linton M (2012) The apoptotic engulfment protein Ced-6 participates in clathrin-mediated yolk uptake in Drosophila egg chambers. Mol Biol Cell 23:1742-64
Traub, Linton M (2012) An MBoC favorite: regulation of the vitellogenin receptor during Drosophila melanogaster oogenesis. Mol Biol Cell 23:3277
Mitra, Shalini; Lukianov, Stefan; Ruiz, Wily G et al. (2012) Requirement for a uroplakin 3a-like protein in the development of zebrafish pronephric tubule epithelial cell function, morphogenesis, and polarity. PLoS One 7:e41816
Lemmon, Sandra K; Traub, Linton M (2012) Getting in touch with the clathrin terminal domain. Traffic 13:511-9
Traub, Linton M (2011) Regarding the amazing choreography of clathrin coats. PLoS Biol 9:e1001037
Pedersen, Gitte Albinus; Chakraborty, Souvik; Steinhauser, Amie L et al. (2010) AMN directs endocytosis of the intrinsic factor-vitamin B(12) receptor cubam by engaging ARH or Dab2. Traffic 11:706-20
Traub, Linton M; Wendland, Beverly (2010) Cell biology: How to don a coat. Nature 465:556-7
Thieman, James R; Mishra, Sanjay K; Ling, Kun et al. (2009) Clathrin regulates the association of PIPKIgamma661 with the AP-2 adaptor beta2 appendage. J Biol Chem 284:13924-39

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