Modular domain structure of Arabidopsis COP1. Reconstitution of activity by fragment complementation and mutational analysis of a nuclear localization signal in planta

Abstract

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.

Figure 1

Structure of COP1 mutants. The capacity of the respective mutants to complement the cop1-5 mutation is indicated on the right. All COP1 mutants were expressed as GUS-fusions. Yes, Full complementation, i.e. a wild-type phenotype; Partial, plants that retained a mild cop1 phenotype, but produced seeds (e.g. Fig. 2, COP1[1-282]); Weak, a slight amelioration of the cop1-5 seedling phenotype (Fig. 2, COP1[293-675]), which was insufficient to overcome lethality of cop1-5. The structural domains of COP1 are indicated by patterned boxes. CLS and NLS denote the cytoplasmic and nuclear localizaton signal of COP1, respectively.

Figure 2

Complementation phenotypes of cop1-5 homozygous plants expressing COP1 mutant proteins. A cop1-5 mutant seedling and COP1 wild-type plants (WT) are shown for comparison. The cop1-5 mutant looks identical in light and darkness. All COP1 mutant proteins were expressed as GUS fusions. Seedlings were germinated on agar medium for 5 d in darkness (top row) or for 12 d in the light (bottom row). Bars = 1 mm.

Figure 3

Complementation phenotypes of cop1-5 homozygous plants expressing the indicated NLS mutants of COP1 or wild-type COP1 (wt). All proteins were expressed as GUS fusions. The relative transgene expression levels are indicated by the GUS activities (picomoles of methylumbelliferone per minute per microgram protein) given below the panels. Seedlings were germinated on agar medium for 5 d in darkness (top row) or for 12 d in the light (bottom row). Bars = 1 mm. Note that the unmutated GUS-COP1 transgene complements the cop1-5 allele to the level of wild-type Arabidopsis.

Figure 4

Western-blot data and co-immunoprecipitation between COP1-4 and GUS-COP1(293-675) (GUS-COP1C). A, Equal amounts of total protein from wild-type non-transgenic seedlings (−) or from seedlings expressing GUS-COP1C or GUS-COP1 were separated by SDS-PAGE, blotted, and probed with a polyclonal COP1 antiserum (pc), a COP1 monoclonal antibody (mc), or a GUS antibody (GUS). The monoclonal antibody recognizes an N-terminal epitope absent from GUS-COP1C. Migration positions of the COP1 proteins are indicated. Less than full-length bands are due to protein degradation. For the GUS-COP1 sample, a 1:10 dilution was also loaded to compare the expression levels of GUS-COP1 and wild-type COP1. The GUS-COP1 extracts were from cop1-5 mutant seedlings and therefore lack the wild-type COP1 signal. The affinity of the monoclonal antibody is insufficient for western-blot detection of wild-type COP1. B, Immunoprecipitates from cop1-4 mutant or COP1 wild-type seedlings transgenic for GUS-COP1C were prepared using the polyclonal serum (pc) or the monoclonal antibody (mc), which recognizes COP1-4, but not COP1C. Antibody was omitted from controls (−). The immunoprecipitates were separated, blotted, and probed with an anti-GUS antiserum. Note that GUS-COP1C was coprecipitated with each anti-COP1 antibody from cop1-4 mutant extracts. One control immunoprecipitation was conducted with an anti-GUS antiserum (GUS).

Figure 5

Complementation of the cop1-4 allele by GUS-COP1(293-675). Transgenic seedlings or non-transgenic controls were germinated in darkness for 5 d (top) or grown under light conditions for 5 weeks (bottom). The two transgenic seedlings shown in B and C represent the range of variation seen among complemented seedlings. A and D, cop 1-4; B, C, and E, cop 1-4, GUS-COP1(293-675); F, wild type.

Figure 6

Complementation of cop1-4, but not of cop1-6, by GUS-COP1(293-675). Rosette leaves were stained for GUS activity (top). Complementation of the cop1-4 mutant was abolished by silencing of the transgene. Bottom, Unstained plants are shown to demonstrate the lack of complementation of the cop1-6 phenotype.

Figure 7

Hypocotyl lengths of cop1-4 mutant or wild-type COP1 seedlings with or without the GUS-COP1(293-675) transgene after germination for 5 d under the indicated constant light conditions. Error bars represent standard deviations.

Figure 8

Cotransformation of onion epidermal cells with GFP-COP1-4 and GUS-COP1(293-675). The representative nuclei shown demonstrate that GFP-COP1-4 localizes to nuclear foci when co-expressed with GUS-COP1(293-675), but not when expressed alone. Left, GFP fluorescence; right, brightfield images of the same cells. Broken lines demarcate the nuclei. The cytoplasmic GFP-COP1-4 protein is concentrated in cytoplasmic inclusion bodies, which are outside the field of view shown here. A and B, GFP-COP1-4 and GUS-COP1(293-675); C and D, GFP-COP1-4 alone.