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

doi:10.1074/jbc.274.39.27674

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

From the ^‡Department of Molecular, Cellular, and Developmental Biology and the ^‖Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8104

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 bothin 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.

Footnotes

↵* This work was supported by National Institutes of Health Grants GM47850 (to X. W. D.), DK09070 (to J. E. C.), and Human Frontier Science Program Grant RG0043/97 (to M. M. and X. W. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) .
↵§ These authors contributed equally to this work.
↵¶ Recipient of the Postdoctoral Fellowship for Research Abroad from the Japan Society for the Promotion of Science. Current address: Dept. of Biology, University of Michigan, Ann Arbor, MI 48109-1048.
↵** Current address: Laboratory for Photoperception and Signal Transduction, Frontier Research Program, RIKEN, Saitama 351-01, Japan.
↵‡ Recipient of the National Science Foundation Presidential Faculty Fellow Award. To whom correspondence should be addressed: Dept. of Molecular, Cellular, and Developmental Biology, Yale University, P.O. Box 208104, 165 Prospect St., OML 301, New Haven, CT 06520-8104. Tel.: 203-432-8908; Fax: 203-432-3854; E-mail: xingwang.deng@ yale.edu.
Abbreviations:

COP1

constitutive photomorphogenic 1 protein

CIP8

COP1 interacting protein 8

PAGE

polyacrylamide gel electrophoresis

PCR

polymerase chain reaction

MBP

maltose-binding protein

GUS

β-glucuronidase
- Received April 27, 1999.
- Revision received June 28, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.