The RING Finger Motif of Photomorphogenic Repressor COP1 Specifically Interacts with the RING-H2 Motif of a NovelArabidopsis Protein

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 zincbinding 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.Zinc ions provide structural integrity to many regulatory proteins, most often through cysteine and histidine ligands that form a tetrahedral geometry. The RING finger motif is a cysteine-rich zinc-binding domain defined by the consensus sequence: CX 2 CX (9 -39) CX (1-3) HX (2-3) CX 2 CX (4 -48) CX 2 C (C 3 HC 4 ). A unique feature of this motif is that the consensus residues coordinate two zinc ions in a "cross-braced" fashion (1, 2). Thus, the RING finger forms one integrated structural unit, rather than forming two tandem zinc finger modules. Proteins containing RING finger domains are found in viruses and all eukaryotes, including yeast, plants, and animals. They have diverse cellular functions, including oncogenesis, viral gene expression, signal transduction, peroxisome biogenesis, DNA repair and recombination, and membrane vesicle sorting (2). The RING finger motif has been shown in many cases to play a role in mediating protein-protein interactions. However, the precise role of the RING finger, as well as specific interactive target motifs of the RING finger, have yet to be clearly defined.The Arabidopsis COP1 1 protein serves as a repressor of photomorphogenesis. In the dark, the COP1 protein localizes to the nucleus and represses photomorphogenesis by inhibiting transcription factors that promote light-inducible gene expression (3-5). Light stimuli abrogate the nuclear localization of COP1 and allows seedlings to pursue photomorphogenic development (6). COP1 contains an N-terminal RING finger motif (7,8), which has been demonstrated to bind tw...

Loss-of-Function Mutations in Chitin Responsive Genes Show Increased Susceptibility to the Powdery Mildew Pathogen Erysiphe cichoracearum

“…Several important Arabidopsis genes with RING domains have been identified including COP1, a photomorphogenesis regulator (Torii et al, 1999), A-RZF, a gene involved in seed development (Zou and Taylor, 1997), and ATL2, which is known to be strongly induced by exposure to chitin and inactivated cellulase preparations and has been characterized as an early-response gene (Salinas-Mondragó n et al, 1999). Members of the ATL gene family are postulated to be involved in the early stages of the defense response (Martinez-Garcia et al, 1996), suggesting that At2g35000 may be involved in the initial signaling responses to chitin leading to plant defense.…”

Dimerization of cotton fiber cellulose synthase catalytic subunits occurs via oxidation of the zinc-binding domains

“…In Arabidopsis, for example, the constitutive photomorphogenic 1 (COP1) interacts via its RING-finger domain type C 3 HC 4 with the COP1 interacting protein 8 (CIP8) RING-H2 finger (32). As a result of the COP1-CIP8 interaction, the stability of the CIP8 in seedlings is increased.…”

Comparison of the genetic determinism of two key phenological traits, flowering and maturity dates, in three Prunus species: peach, apricot and sweet cherry

“…The other five proteins could be more directly involved in the control of flowering date. CIP8 is a protein that binds to COP1 (Torii et al, 1999) similar to SPA proteins, and possess ubiquitin ligase activity (Hardtke et al, 2002). PhyA is a light-labile cytoplasmic red/far-red light photoreceptor involved in the regulation of photomorphogenesis.…”