Identification of a novel clathrin-binding motif in yeast auxilin

Leslie KimSpring 2007

Research Advisor: Dr. Todd GrahamBSCI 296

Juan S. Bonifacino and Benjamin S. Glick, Cell 2004

Vesicle-Mediated Protein Transport

Clathrin

Barbara Pearse

Cremona O, Dev Cell. 2001

The Clathrin Cycle

Scheele, U. et al. J. Biol. Chem. 2003;278:25357-25368

Interaction of Auxilin with Clathrin is Mediated by Short Peptide Motifs in an Unstructured Region

J

Auxilin

Interaction of GST-Swa2 fusion proteins with clathrin

1 2 3 4 5 6 7 8 9 10 11 12

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)

Two Forms of Clathrin

Ub

Swa2p

Hsc70

Testing the Role of the UBA domain

GST-(1-140)

GST

U B U B U B U B U B U B

GST

-(1-1

00)

GST

-(1-1

40)

GST

-(1-1

81)

GST

-(101

-237

)

GST

-(1-2

37)

GST-(1-181)

GST-(1-100)

GST-(101-237)

GST-(1-237)

αCHC1

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

Construction of New GST-Swa2 proteins

GST Fusion Proteins

1. GST

2. 1-100

3. 1-110

4. 1-120

5. 1-130

6. 1-140

7. Marker

1 2 3 4 5 6 7

Clathrin Pulldown Trial 3

U B U B U B U B U B U B

GST

-(1-1

40)

GST

-(1-1

30)

GST

-(1-1

20)

GST

-(1-1

10)

GST

-(1-1

00)

GST

αCHC1

Results from Pulldown experiments

GST 1-100 1-110 1-120 1-130 1-140

GST – fusion proteins

Conclusions Part B

Novel CB element appears – (101-110) How do we identify it?

Peptide Inhibition Assay with synthetic 10-mer peptide

Functional Assay

Peptide Inhibition Assay

U B U B U B

αCHC1

0 : 1

1 : 1

10 :

1

peptide inhibitor : GST-Swa2p-1-140

101-110 amino acid sequence

F N Q L F G M G T V

Novel Clathrin binding element

110

101-110 amino acid sequence

F N Q L F G M G T V

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 98 101 105

Create Construct GST-Swa2 (1-90) (91-140) (101-140)

Functional Assay

Xiao et al., 2006

Functional visual assay to test CB motifs

pRS315 Δ2-362

F

R

pRS315 Δ2-362

RevPCRSwa2FF – 5’ ACG CGT GAT CTT TTA AGA TCT ... 3’ Mlu I D L L Bgl II CB domain 2

R – 5’ AGA TCT TAA AAG ATC ACG CGT ... 3’ Bgl II L L D Mlu I

AT

G

363 373

TPR J

R2

F1

PCR Products 1 2 3 4 5 6

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)

2000

1500

2500

2000

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

Future Directions

Define acidic phenylalanine motif Interaction with clathrin, Gga2, AP1

Perform functional assay to test contribution of CB motif in vivo

Acknowledgements

Dr. Graham Jing Xiao The Graham Lab