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 interests

I. Steroid Function and Regulation

II. Zebrafish Development

Sumoylation regulates nuclear distribution of SF-1

Cytoplasmic distribution of SF-1.

Schematic overview of the biosynthetic pathways for steroid hormones

SF1 regulates the expression of P450 steroid hydroxylases.

Steroidogenic factor 1 (SF-1)

The Ftz-F1 box of SF-1 contains NLS sequence. (Li et al., 1999)

SF-1 is an orphan nuclear receptor.SF-1 was always found in nucleus in previous stu

dies.

SF-1

SUMO-1

SUMO-1 is a 101 amino acid polypeptide and only 1-97 a.a. can be attached to target proteins.

SF-1 has two sumoylation sites, K-119 and K-194.SUMO-1 can trigger the formation of PML nuclear

bodies.

SF-1

Cloning of FLAG-RFP-SUMO-1 fusion protein

SUMO-1 WT: 94 95 96 97CAA ACG GGG GGT Gly Gly

SUMO-1 AA (mutant): 94 95 96 97CAA ACG GCG GCT Ala Ala

Western blot of FLAG-RFP-SUMO-1

FLAG-RFP-SUMO-1

Sumoylated proteins

WT AA

Sumoylation regulates nuclear distribution of SF-1

RFP-SUMO-1 WT SF-1-GFP Merge

RFP-SUMO-1 AA SF-1-GFP Merge

WT

AA

Sumoylation regulates nuclear distribution of SF-1

Cytoplasmic distribution of SF-1.

Cytoplasmic distribution of SF-1

SF-1 Ab SF-1-GFP

Phase Merge

Cytoplasmic distribution of SF-1

SF-1-GFP RFP

Merge 3D view

Cytoplasmic localization of SF-1

Actin Ab SF-1-GFP

Merge 3D view

Cytoplasmic localization of SF-1

Tublin Ab SF-1-GFP

Merge 3D view

Cytoplasmic localization of SF-1

SCC Ab SF-1-GFP

Merge

LMB does not affect cytoplasmic distribution of SF-1

15min 45min 1h15min 1h45min

2h15min 2h45min 3h15min 3h45min

4h15min 4h45min 5h15min 5h45min

6h15min 6h45min (After LMB treatment)

SF-1 contains LMB- insensitive NES homologue SF-1

FF1bAR

SF-1

FF1b

AR

(743-817)

(743-817)

(306-381)

(306-381)

(359-436)

(359-436)

AR

AR

MRER

MRER

(743-817)

(743-817)(809-883)(386-455)

(809-883)(386-455)

(Saporita et al., 2003)

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

SF-1

Future work

Examine the interaction of SF-1 and SUMO-1 in living cell under different stimulus.

Examine the candidate NES fragment of SF-1.

Use electro-microscopy to find out the exact location of cytoplasmic SF-1.

Acknowledgments

Dr. Bon-chu ChungMr. Wei-yi ChenMr. Wen-chih LeeMs. LeeAll the lab members