Modular domain structure of Arabidopsis COP1. Reconstitution of activity by fragment complementation and mutational analysis of a nuclear localization signal in planta.
The Arabidopsis COP1 protein functions as a developmental regulator, in part by repressing photomorphogenesis in darkness. Using complementation of a cop1 loss-of-function allele with transgenes expressing fusions of cop1 mutant proteins and beta-glucuronidase, it was confirmed that COP1 consists of two modules, an amino terminal module conferring a basal function during development and a carboxyl terminal module conferring repression of photomorphogenesis. The amino-terminal zinc-binding domain of COP1 was indispensable for COP1 function. In contrast, the debilitating effects of site-directed mutations in the single nuclear localization signal of COP1 were partially compensated by high-level transgene expression. The carboxyl-terminal module of COP1, though unable to substantially ameliorate a cop1 loss-of-function allele on its own, was sufficient for conferring a light-quality-dependent hyperetiolation phenotype in the presence of wild-type COP1. Moreover, partial COP1 activity could be reconstituted in vivo from two non-covalently linked, complementary polypeptides that represent the two functional modules of COP1. Evidence is presented for efficient association of the two sub-fragments of the split COP1 protein in Arabidopsis and in a yeast two-hybrid assay.
Stacey
M G
MG
Department of Botany, The University of Tennessee, Knoxville, Tennessee 37996-1100, USA.
Kopp
O R
OR
Kim
T H
TH
von Arnim
A G
AG
D017360:Arabidopsis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005816:Genetic Complementation Test; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D008027:Light; D016297:Mutagenesis, Site-Directed; D019913:Nuclear Localization Signals / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010641:Phenotype; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018514:Plant Structures / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D030821:Plants, Genetically Modified; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D044767:Ubiquitin-Protein Ligases
D029681:Arabidopsis Proteins; D002352:Carrier Proteins; D019913:Nuclear Localization Signals; D010446:Peptide Fragments; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C471983:AT2G32950 protein, Arabidopsis; D044767:Ubiquitin-Protein Ligases; D005966:Glucuronidase