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Published December 24, 2009 | Supplemental Material
Journal Article Open

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.

Additional Information

© 2009 Elsevier Ltd. Received 9 September 2009; revised 28 October 2009; accepted 2 November 2009. Published: December 23, 2009. Available online 24 December 2009. This work was supported by the American Heart Association and the Friedreich's Ataxia Research Alliance #09BGIA2220299 (to J.J.S.), Arnold and Mabel Beckman Foundation, and the Caltech Joseph Jacobs Institute for Molecular Engineering for Medicine (to C.D.S.), the Robert A. Welch Foundation C 1614 (to J.J.S.), and the National Institutes of Health fellowship 5F32GM078901 (to K.G.H) and training grant 2T32 GM008362 (to P.Q.N.).

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