Abstract
The constitutive photomorphogenic 1 (COP1) protein of Arabidopsis functions as a molecular switch for the seedling developmental fates: photomorphogenesis under light conditions and skotomorphogenesis in darkness. The COP1 protein contains a cysteine-rich zinc-binding RING finger motif found in diverse groups of regulatory proteins. To understand the role of the COP1 RING finger in mediating protein-protein interaction, we have performed a yeast two-hybrid screen and isolated a novel protein with a RING-H2 motif, a variant type of the RING finger. This protein, designated COP1 Interacting Protein 8 (CIP8), is encoded by a single copy gene and localized to cytosol in a transient assay. In addition to the RING-H2 motif, the predicted protein has a C4 zinc finger, an acidic region, a glycine-rich cluster, and a serine-rich cluster. The COP1 RING finger and the CIP8 RING-H2 domains are sufficient for their interaction with each other both in vitro and in yeast, whereas neither motif displayed significant self-association. Moreover, site-directed mutagenesis studies demonstrated that the expected zinc-binding ligands of the RING finger and RING-H2 fingers are essential for their interaction. Our findings indicate that the RING finger motif, in this case, serves as autonomous protein-protein interaction domain. The allele specific effect of cop1 mutations on the CIP8 protein accumulation in seedlings indicates that its stability in vivo is dependent on the COP1 protein.
Highlights
Zinc ions provide structural integrity to many regulatory proteins, most often through cysteine and histidine ligands that form a tetrahedral geometry
Interaction of RING Finger and RING-H2 Motif fused with the LexA DNA binding domain (LexA-N-terminal 282 amino acid (N282)) was used as a bait to screen a light-grown Arabidopsis seedling cDNA library fused to a synthetic activation domain in yeast (Fig. 1A)
The RING Finger Motif as Protein-Protein Interaction Domain—This study clearly demonstrated a role of the constitutive photomorphogenic 1 (COP1) RING finger as an autonomous protein-protein interaction module
Summary
Zinc ions provide structural integrity to many regulatory proteins, most often through cysteine and histidine ligands that form a tetrahedral geometry. In an effort to understand the function of the COP1 RING finger domain, we have performed a yeast two-hybrid screen to identify interacting proteins.
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