Arabidopsis COP 1 protein specifically interacts in vitro with a cytoskeleton - associated protein , CIP 1 ( signal transduction / light regulation / coiled coil / plant development / subcellular trafficking )

Arabidopsis COP1 acts inside the nucleus to suppress photomorphogenic cellular development, and light inactivation of COP1 may involve a specific control of its nuclear activity in hypocotyls and cotyledons, but not in roots, of developing seedlings. To understand the molecular mechanisms ofCOP1 action during light-mediated development, we initiated a screen for Arabidopsis cDNAs encoding proteins which interact directly with COP1 in vitro as a step to identify the cellular components involved. We report here the isolation and characterization of a cDNA clone encoding a protein designated CIP1 (COPl-interactive protein 1). CIP1 is predominantly a-helical and most likely involved in coiled-coil formation. It interacts specifically with the putative coiledcoil region of COP1 in vitro. Further, CIP1 is encoded by a single gene inArabidopsis, and its mRNA and protein levels are not regulated by light. Immunofluorescent labeling of CIP1 in Arabidopsis seedling protoplasts demonstrated that CIP1 is part of, or associated with, a cytoskeletal structure in hypocotyl and cotyledon cells, but not in roots. Our results are consistent with a possible role of CIP1 in mediating light control of COP1 nuclear activity by regulating its nucleocytoplasmic partitioning. Arabidopsis seedlings are able to follow two distinct morphogenetic pathways dependent on the ambient light conditions (for review, see ref. 1). Light-grownArabidopsis seedlings have a short hypocotyl and open and enlarged cotyledons with chloroplast development. In contrast, dark-grown seedlings have a long hypocotyl, an apical hook, and closed and undeveloped cotyledons with etioplasts. To identify genes involved in the light control of plant development, Arabidopsis mutants with altered seedling developmental patterns in response to light have been isolated (for review, see refs. 2 and 3). Among them, mutations in six genetic loci-DETI, COP1, and COP8COP11-result in dark-grown seedlings with pleiotropic photomorphogenic characteristics, including short hypocotyls, open and enlarged cotyledons with differentiated cell types, and expression of light-inducible genes (2-4). Four of the pleiotropic photomorphogenic genes have been cloned. While COP11/FUS6 (5), COP9 (6), and DET1 (7) encode novel proteins without significant homology to any reported sequence in the gene bank, COP1 encodes a protein with three recognizable domains (8, 9). From the N terminus, these are a ring-finger-type zinc-binding motif (10), a predicted coiled-coil domain, and a domain with multiple WD-40 repeats, homologous to the 3 subunit of the heterotrimeric guanine nucleotide-binding proteins (G proteins). Significant amino acid sequence homology between COP1, excluding the zinc-binding domain, and the entire TAFII80 subunit of the Drosophila TFIID transcription factor complex has been reported (11). The COP1 structure suggests not only a potential to interact with nucleic acids through its zinc-finger domain but The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 4239 also contacts with other proteins through its coiled-coil and GP homologous domains. The recessive nature of the mutations in the COP1, DET1, and COP8-COP11 loci implies that their wild-type gene products act as negative regulators of photomorphogenic seedling development in darkness and that light acts to abrogate their repressive function. Recently we demonstrated that overexpression of COP1 protein alone is sufficient to suppress many aspects of photomorphogenic seedling development, such as hypocotyl elongation and cotyledon enlargement (12). This result provides direct evidence for COP1 being a lightinactivatable molecular repressor for photomorphogenic seedling development. Furthermore, cytological studies with P-glucuronidase-COP1 fusion protein have indicated that COP1 acts in the nucleus to achieve the suppression of photomorphogenic development, and light inactivation may involve a cell-type-specific depletion of nuclear COP1 activity (13). To understand the molecular mechanisms of COP1 in repressing photomorphogenic development and its light inactivation, it will be necessary to identify other cellular components that directly interact with COP1. Toward this goal, we initiated a screen for Arabidopsis cDNAs encoding proteins which interact with COP1 in vitro. We report here the isolation and characterization of a cDNA clone encoding a protein, designated CIP1, which interacts with the putative coiled-coil domain of COP1 and is part of, or associated with, the cytoskeleton.t MATERIALS AND METHODS COP1 Probe, cDNA Library Screening, and ProteinInteraction Assay. The COP1 open reading frame was cloned as a BamHI-Bgl II fragment into the BamHI site of the vector pET3b (Novagen). Expression inEscherichia coli BL21(DE3)/ pLysS (Novagen) resulted in a COP1 protein in which 11 vector-derived amino acids replaced the 6 residues at the N terminus of COP1. COP1 was purified from inclusion bodies of isopropyl 3-D-thiogalactopyranoside (IPTG)-induced bacteria according to a published procedure (10), except that 0.1 mM ZnCl2 was present in the final dialysis buffer. The purified COP1 protein was biotinylated (14) with 25 tug of COP1 protein and 2 ,tg of biotin N-hydroxysuccinimide ester (BRL). Biotinylated COP1 was dialyzed for 16 hr at 4°C and used as a protein probe to screenArabidopsis Agtll cDNA libraries made from poly(A)+ mRNAs of either dark-grown seedlings or leaves of adult Arabidopsis thaliana (Columbia) plants (8). The cDNA libraries were plated with E. coli Y1090 (r-) and screened with biotinylated COP1 essentially accordAbbreviations: BSA, bovine serum albumin; GST, glutathione Stransferase; MBP, maltose-binding protein. *Present address: Molecular Biology Laboratory, Nippon Medical School, 1-396 Kosugicho, Nakahara, Kawasaki 211, Japan. tThe sequence reported in this paper has been deposited in the GenBank database (accession no. U20810). 4240 Plant Biology: Matsui et al ing to a published protocol (14), except that biotinylated COP1 protein was used at 0.5 ,tg/ml. For analysis of the interaction of COP1 with the protein encoded by the selected cDNA clone, CIP1, the 1.5-kb cDNA insert was subcloned to the EcoRI site of the pUR291 expression vector (24), which resulted in a P-galactosidase (LacZ)CIP1 fusion protein. Total proteins from E. coli DHSaF' carrying this plasmid, either uninduced or IPTG-induced, were separated by SDS/10% PAGE and blotted onto nitrocellulose filter for far-Western analysis according to the same procedure as for the cDNA screen. Various deletion versions (or fusions) of COP1 were biotinylated and used as probes for far-Western assay. The full-length COPl fusion to the E. coli maltose-binding protein (MBP) and its derivatives, a deletion of the WD40 repeats, and a deletion of the zinc-binding motif have been described (10). The glutathione S-transferase (GST)COP1 fusion was generated by expressing the COP1 cDNA in the pGEX vector (Pharmacia) and purified by glutathione affinity chromatography. A COP1 fragment encompassing aa 105-205 was expressed from a cDNA fragment adapted for cloning into the Nco I and BamHI sites of pET15b (Novagen) by polymerase chain reaction. In this protein, COP1 residue 104 (leucine) is replaced by a methionine-valine dipeptide. RNA and Protein Gel Blot Analysis. Six-day-old seedlings were used for RNA and protein analysis. Plant growth, RNA extraction, and Northern analysis (15) and protein extraction and analysis (9) were according to published procedures. Rabbit polyclonal antibodies produced against a purified E. coli-expressed GST-CIP1 fusion were affinity-purified against a MBP-CIP1 fusion and used in Western and cytological studies. Immunpcytological Localization of CIP1. Protoplasts were released from dissected organs of Arabidopsis seedlings by incubation in enzyme solution [10 mM Mes, pH 5.7/0.4 M mannitol/1% cellulase R10/0.25% macerozyme R10 (Yakult Pharmaceutical, Tokyo)/0.1% bovine serum albumin (BSA)/ 30 mM CaCl2/5 mM 2-mercaptoethanol] at 22°C under dark or light conditions for 1-3 hr. The protoplasts were filtered through 65-ugm nylon mesh and pelleted by a brief centrifugation at 150 x g. They were washed once with 4 mM Mes, pH 5.7/20 mM KCl/0.5 M mannitol and transferred to polylysinecoated slides to settle for about 3 hr at room temperature under the required light conditions. Bound protoplasts on the slides were fixed for 10 min with 2% formaldehyde in PHEM solution (60 mM Pipes/25 mM Hepes/10 mM EGTA, 2 mM MgCl2, pH 6,9) and then permeabilized for 5 min with 0.5% Nonidet P-40 in PHEM. The slides were soaked twice for 15 min each in methanol/acetone (1:1, vol/vol) at -20°C and air dried. They were rehydrated in phosphate-buffered saline (PBS) and blocked with 1% BSA in PHEM. After 2 hr of reaction with the CIP1 antibody at 6 ,tg/ml in PHEM with 0.5% BSA, they were washed first with PBS, then with 0.1% Tween 20 in PBS, and lastly with PBS again. A fluoresceinlabeled polyclonal antibody against rabbit IgG (Sigma) at a dilution of 1:100 was applied for 2 hr of incubation at room temperature. Slides were washed as above and mounted with 90% glycerol/1% p-phenylenediamine at pH 9.0 and 4',6diamidino-2-phenylindole (1 tLg/ml).