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The subcellular localization of SF-1 Fu Huang. Lab Introduction Lab 429, Institute of Molecular Biology, Academia Sinica PI: Bon-chu Chung, Ph.D. Research.

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Presentation on theme: "The subcellular localization of SF-1 Fu Huang. Lab Introduction Lab 429, Institute of Molecular Biology, Academia Sinica PI: Bon-chu Chung, Ph.D. Research."— Presentation transcript:

1 The subcellular localization of SF-1 Fu Huang

2 Lab Introduction Lab 429, Institute of Molecular Biology, Academia Sinica PI: Bon-chu Chung, Ph.D. Research interests I. Steroid Function and Regulation II. Zebrafish Development

3 zSumoylation regulates nuclear distribution of SF-1 zCytoplasmic distribution of SF-1.

4 Schematic overview of the biosynthetic pathways for steroid hormones SF1 regulates the expression of P450 steroid hydroxylases.

5 Steroidogenic factor 1 (SF-1) zThe Ftz-F1 box of SF-1 contains NLS sequence. (Li et al., 1999) zSF-1 is an orphan nuclear receptor. zSF-1 was always found in nucleus in previous studies. SF-1

6 SUMO-1 zSUMO-1 is a 101 amino acid polypeptide and only 1-97 a.a. can be attached to target proteins. zSF-1 has two sumoylation sites, K-119 and K-194. zSUMO-1 can trigger the formation of PML nuclear bodies. SF-1

7 Cloning of FLAG-RFP-SUMO-1 fusion protein SUMO-1 WT: 94 95 96 97 CAA ACG GGG GGT Gly Gly SUMO-1 AA (mutant): 94 95 96 97 CAA ACG GCG GCT Ala Ala

8 Western blot of FLAG-RFP-SUMO-1 FLAG-RFP-SUMO-1 Sumoylated proteins WTAA

9 Sumoylation regulates nuclear distribution of SF-1 RFP-SUMO-1 WTSF-1-GFPMerge RFP-SUMO-1 AASF-1-GFPMerge WT AA

10 zSumoylation regulates nuclear distribution of SF-1 zCytoplasmic distribution of SF-1.

11 Cytoplasmic distribution of SF-1 SF-1 AbSF-1-GFP PhaseMerge

12 Cytoplasmic distribution of SF-1 SF-1-GFP RFP Merge 3D view

13 Cytoplasmic localization of SF-1 Actin AbSF-1-GFP Merge3D view

14 Cytoplasmic localization of SF-1 Tublin Ab SF-1-GFP Merge3D view

15 Cytoplasmic localization of SF-1 SCC AbSF-1-GFP Merge

16 LMB does not affect cytoplasmic distribution of SF-1 15min45min1h15min1h45min 2h15min2h45min3h15min3h45min 4h15min4h45min5h15min5h45min 6h15min6h45min (After LMB treatment)

17 SF-1 contains LMB- insensitive NES homologue SF-1 FF1b AR SF-1 FF1b AR (743-817) (306-381) (359-436) AR MR ER MR ER (743-817) (809-883) (386-455) (809-883) (386-455) (Saporita et al., 2003)

18 The LBD domain of SF-1 contains LMB-independent NES homologue SF-1

19 Future work zExamine the interaction of SF-1 and SUMO-1 in living cell under different stimulus. zExamine the candidate NES fragment of SF-1. zUse electro-microscopy to find out the exact location of cytoplasmic SF-1.

20 Acknowledgments zDr. Bon-chu Chung zMr. Wei-yi Chen zMr. Wen-chih Lee zMs. Lee zAll the lab members

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