Expression and characterization of SUMO-conjugated metal-responsive transcription factor 1: SIM-dependent cross-interaction and distinct DNA binding activity.
Metal-responsive transcription factor 1 (MTF-1) regulates a variety of genes involving in metal homeostasis and oxidative stress. We have shown that MTF-1 can be conjugated by small ubiquitin-like modifier (SUMO) and forms complexes with cellular factor(s) in a SUMO-interacting motif (SIM)-dependent manner. To investigate whether the interaction of MTF-1 and its SUMO conjugate occurs, we expressed and isolated MTF-1 and sumoylated MTF-1 (S-MTF-1) for functional studies. Various conditions were examined to optimize the expressions of MTF-1 and S-MTF-1. Results from affinity column chromatography demonstrated that the unmodified MTF-1 consistently co-eluted with the S-MTF-1. Mutations at the SIM did not reduce the level of MTF-1 sumoylation but the sumoylated product can then be purified to homogeneity. The presence of MTF-1 cross-interaction was further supported by in vitro pull-down assays. The ability of the purified proteins in binding metal-responsive element (MRE) was assessed with electrophoretic mobility shift assay. Noticeably, MTF-1 required the presence of cell extracts to render the binding activity. However, S-MTF-1 binds MRE in void of other cellular factors. The same characteristic was found for MTF-1 with SUMO fusion at the carboxyl terminus. These results indicate that the presence of SUMO moiety allows the protein to interact directly with MRE.
Institute of Molecular and Cellular Biology and Department of Life Science, National Tsing Hua University, No. 101, Section 2, Kuang-Fu Road, Hsinchu 30013, Taiwan.
Chang-Yi
CY
Lin
Ya-Chuan
YC
Liu
Meng-Chieh
MC
Lin
Thao
T
Thi Nguyen
Ming F
MF
Tam
Chih-Yuan
CY
Chein
Meng-Ting
MT
Lin
Lih-Yuan
LY
Lin
D020816:Amino Acid Motifs; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D051379:Mice; D009154:Mutation; D060066:Protein Interaction Maps; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020218:Response Elements; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism; D058207:Sumoylation; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism*; D000097809:Transcription Factor MTF-1
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D014157:Transcription Factors; D000097809:Transcription Factor MTF-1