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Identification of a novel clathrin-binding motif in yeast auxilin Leslie Kim Spring 2007

Identification of a novel clathrin-binding motif in yeast auxilin Leslie Kim Spring 2007 Research Advisor: Dr. Todd Graham BSCI 296. Vesicle-Mediated Protein Transport. Juan S. Bonifacino and Benjamin S. Glick, Cell 2004. Clathrin. Barbara Pearse. The Clathrin Cycle.

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Identification of a novel clathrin-binding motif in yeast auxilin Leslie Kim Spring 2007

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  1. Identification of a novel clathrin-binding motif in yeast auxilin Leslie Kim Spring 2007 Research Advisor: Dr. Todd Graham BSCI 296

  2. Vesicle-Mediated Protein Transport Juan S. Bonifacino and Benjamin S. Glick, Cell 2004

  3. Clathrin BarbaraPearse

  4. The ClathrinCycle Cremona O, Dev Cell. 2001

  5. Interaction of Auxilin with Clathrin is Mediated by Short Peptide Motifs in an Unstructured Region J Auxilin Scheele, U. et al. J. Biol. Chem. 2003;278:25357-25368

  6. Interaction of GST-Swa2 fusion proteins with clathrin 1 2 3 4 5 6 7 8 9 10 11 12

  7. Test role of UBA domain • Solution Structure of UBA domain • Purpose of UBA domain? Nicholas Chim, Walter E. Gall, Jing Xiao, Mark P. Harris, Todd R. Graham, and Andrzej M. Krezel* (2004)

  8. Two Forms of Clathrin Ub Swa2p Hsc70

  9. Testing the Role of the UBA domain GST-(101-237) GST-(1-237) GST-(1-100) GST-(1-181) GST-(1-140) GST U B U B U B U B U B U B αCHC1 GST-(1-100) GST-(1-140) GST-(1-181) GST-(101-237) GST-(1-237)

  10. Conclusions Part A • UBA domain does not contribute to increased interaction of Swa2 with clathrin • Amino acid region 101-140 likely contains a novel clathrin binding motif

  11. Construction of New GST-Swa2 proteins 1 2 3 4 5 6 7 • GST Fusion Proteins • GST • 1-100 • 1-110 • 1-120 • 1-130 • 1-140 • Marker

  12. Clathrin Pulldown Trial 3 GST-(1-140) GST GST-(1-100) GST-(1-110) GST-(1-120) GST-(1-130) U BU BU BU BU BU B αCHC1

  13. Results from Pulldown experiments GST 1-100 1-120 1-130 1-140 1-110 GST – fusion proteins

  14. Conclusions Part B • Novel CB element appears – (101-110) • How do we identify it? • Peptide Inhibition Assay with synthetic 10-mer peptide • Functional Assay

  15. Peptide Inhibition Assay peptide inhibitor : GST-Swa2p-1-140 1 : 1 0 : 1 10 : 1 U B U B U B αCHC1 • 101-110 amino acid sequence F N Q L F G M G T V

  16. Novel Clathrin binding element • 101-110 amino acid sequence F N Q L F G M G T V 110

  17. Acidic phenylalanine motif • ENTH domain containing proteins– (amino-terminal homology) Ent3p, Ent5p (Duncan, et al., 2004) • γ ear domains of Gga2 and AP1 (Apl4) • Acidic phenylalanine motifs • Ent3a DEEEDDDDEFSEFQ • Swa2 DDDFNQLF 98101 105 • Create Construct GST-Swa2 (1-90) (91-140) (101-140)

  18. Functional Assay Xiao et al., 2006

  19. Functional visual assay to test CB motifs F ATG TPR J R 363 373 pRS315 Δ2-362 pRS315 Δ2-362 R2 F1 RevPCRSwa2F F – 5’ ACG CGTGAT CTT TTA AGA TCT ... 3’ Mlu IDLLBgl II CB domain 2 R – 5’AGA TCT TAA AAG ATC ACG CGT ... 3’ Bgl IILLDMlu I

  20. PCR Products 1 2 3 4 5 6 2500 2000 2000 1500 • Lane_ • 1. 3. 5 DNA Marker • 2. PCR Product 1 (320 bp) • 4. PCR Product 2 (1780 bp) • 6. Full length PCR product (2079 bp)

  21. Conclusions Part C • Peptide 101-110 does not inhibit Swa2-1-140 interaction with clathrin • 101-110 peptide inhibitor does not contain complete motif • Novel motif likely surrounds 101 residue • Novel clathrin interaction element likely to involve acidic phenylalanine motif

  22. Future Directions • Define acidic phenylalanine motif • Interaction with clathrin, Gga2, AP1 • Perform functional assay to test contribution of CB motif in vivo

  23. Acknowledgements • Dr. Graham • Jing Xiao • The Graham Lab

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