identification of a novel clathrin-binding motif in yeast auxilin leslie kim spring 2007 research...
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Identification of a novel clathrin-binding motif in yeast auxilin
Leslie KimSpring 2007
Research Advisor: Dr. Todd GrahamBSCI 296
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
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)
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
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
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 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