In vivo fluorescent detection of Fe-S clusters coordinated by human GRX2.
A major challenge to studying Fe-S cluster biosynthesis in higher eukaryotes is the lack of simple tools for imaging metallocluster binding to proteins. We describe the first fluorescent approach for in vivo detection of 2Fe2S clusters that is based upon the complementation of Venus fluorescent protein fragments via human glutaredoxin 2 (GRX2) coordination of a 2Fe2S cluster. We show that Escherichia coli and mammalian cells expressing Venus fragments fused to GRX2 exhibit greater fluorescence than cells expressing fragments fused to a C37A mutant that cannot coordinate a metallocluster. In addition, we find that maximal fluorescence in the cytosol of mammalian cells requires the iron-sulfur cluster assembly proteins ISCU and NFS1. These findings provide evidence that glutaredoxins can dimerize within mammalian cells through coordination of a 2Fe2S cluster as observed with purified recombinant proteins.
Hoff
Kevin G
KG
Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 E California Boulevard, Mail Code 210-41, Pasadena, CA 91125, USA. khoff@caltech.edu
Culler
Stephanie J
SJ
Nguyen
Peter Q
PQ
McGuire
Ryan M
RM
Silberg
Jonathan J
JJ
Smolke
Christina D
CD
D001665:Binding Sites; D002460:Cell Line; D002942:Circular Dichroism; D019281:Dimerization; D054477:Glutaredoxins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007501:Iron / Q000737:chemistry*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009154:Mutation; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013455:Sulfur / Q000737:chemistry*
C516008:GLRX2 protein, human; D054477:Glutaredoxins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D013455:Sulfur; D007501:Iron