Abstract
The COP1 gene of Arabidopsis thaliana encodes a protein mediating the switch between the two developmental pathways utilized in light and darkness. A cysteine-rich motif identified the COP1 protein as a member of a group of regulatory proteins which share the amino acid motif Cys-X-X-Cys-loop I-Cys-X-His-X-X-Cys-X-X-Cys-loop II-Cys-X-X-Cys (ring finger). Although this new class of cysteine-rich motifs has been proposed to bind metal ions, no direct evidence supporting this has been presented. By analyzing the COP1 protein expressed in Escherichia coli, we demonstrate here that each COP1 molecule can bind up to two zinc atoms. The two zinc ions are bound with different affinities. One is tightly bound and resistant to urea and EDTA, whereas the other one is labile under those conditions. It is further shown that deletion of the ring finger motif abolishes the metal-binding capacity of COP1. We conclude that the ring finger motif constitutes a zinc-coordinating element distinct from previously characterized zinc-binding domains.
Highlights
The COPl gene of Arabidopsis thaliana encodes a fingers featuring the pattern Cysz-Hisz(9)
The two zinc ions are bound with sis thalianu
It is further shown that dele- suggests that COPlacts as a mastesruppressor of photomortion of the ring finger motif abolishes the metal-bind- phogenesis, presumably through repression of gene expression ing capacity of COP1.We conclude that the ring finger (13)
Summary
Osborn Memorial Laboratory, Yale University, P. 0. COPlAZ was filled in as described above and the plasmid religated concentrations of buffers and mock protein preparations in the abto give pM-COP1 NAZ. Of zinc content by atomic absorption spectroscopy was according to pMal-c itself will produce a fusion protein consisting of the maltose- a described procedure (33), and the buffer alone contained less than binding protein and a short peptide derived from @-galactosidaseF. This was confirmed by Western blot analysis with a COP1-specific antiserum and analysis of the digestion pattern of M-COP1 Nwith factor
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