Arabidopsis COP10 is a ubiquitin-conjugating enzyme variant that acts together with COP1 and the COP9 signalosome in repressing photomorphogenesis

Abstract

A group of evolutionarily conserved pleiotropic COP/DET/FUS proteins was initially defined by their ability to repress photomorphogenesis in Arabidopsis. It was proposed that this regulation be mediated by targeting degradation of key cellular regulators that promote photomorphogenesis. Among them, COP1 and the COP9 signalosome have been hypothesized to fulfill the roles as an ubiquitin ligase (E3) and an essential E3 modulator. Here we report that COP10 encodes a protein similar to ubiquitin-conjugating enzyme (E2) variant proteins (UEV). COP10 is part of a nuclear protein complex and capable of directly interacting with both COP1 and the COP9 signalosome. Our data indicates that COP10 defines a possible E2 activity, thus validating the working hypothesis that the pleiotropic COP/DET/FUS group of proteins defined a protein ubiquitination pathway.

Figure 1

Phenotype characteristics of cop10 mutants and the map-base cloning of COP10. (a) Morphology of the light- or dark-grown cop10 mutant seedlings. (Panels 1–5) Six-day-old light-grown wild- type, cop10-4, cop10-1, cop10-2, and cop10-3 seedlings, respectively. (Panels 6–10) Six-day-old dark-grown wild-type, cop10-4, cop10-1, cop10-2, and cop10-3 seedlings, respectively. (Panels 11,12) Eight-day-old dark-grown wild-type and cop10-4 seedlings, respectively. Bars, 1 mm. (b) A 5-week-old cop10-2 mutant plant. Bar, 1 mm. (c) Complementation of the cop10-4 mutation by a genomic fragment of the COP10 gene. From left to right, dark-grown 8-day-old wild-type seedling (left), cop10-4 (center), and cop10-4 transformed with COP10 genomic fragment (right). Bar, 1 mm. (d) Summary for the positional cloning of the COP10 gene. Partial genetic map of Arabidopsis chromosome III between the markers ATHCHIB and nga162 with several P1, TAC, and BAC clones are shown. (Gray lines) Open reading frames (ORFs) predicted by GENSCAN analysis. (e) Structure of COP10 and molecular nature of the mutations found in the four cop10 alleles. Boxes with Roman numerals indicate exons, whereas the introns are shown as lines between the exons. Shadowed area of exons indicates ORF coding region. The two splicing junctions where all four cop10 mutations are located are shown as inserts. The intron and exon sequences are shown as small and capital letters.

Figure 2

The COP10 mRNA and protein expression profiles. (a) RNA blot analysis. RNAs were prepared from 5-day-old wild-type and cop10-4, cop10-1, cop10-2 and cop10-3 mutant seedlings grown under continuous white light. Total RNA from equal numbers of seedlings was used. The band in the wild-type lane corresponds to a size of 0.9 kb. As a loading control, the blot was reprobed with the 18S ribosomal RNA gene. (b) Immunoblot of light-grown wild-type (WT), cop10-4, cop10-1, cop10-2, and cop10-3 seedling extracts using anti-COP10 antibodies. (c) COP10 immunoblot from light- and dark-grown wild-type, light-grown cop10-1, cop9-1, fus6-1 and cop1-5 seedlings. (d) COP10 immunoblot of protein extracts from different tissue types. Flower, root, stem, or leaf protein extracts were obtained from 4-week-old light-grown mature plants. The seedlings were 5-day-old under continuous white light. A tubulin immunoblot was used as control for b and c and a Rpt5 immunoblot was used as a loading control in d.

Figure 3

Comparison of COP10 and ubiquitin conjugation enzyme E2 related proteins. (a) Sequence alignment of COP10 from Arabidopsis (AtCOP10), soybean (GmCOP10, GenBank AI460798), and tomato (LeCOP10, GenBank AW223526), and E2 enzymes from Saccharomyces (ScUBC4, GenBank S22857) and Arabidopsis UBC9 (AtUBC9, GenBank AAG40371). (Black boxes) Identical amino acids. Asterisk denotes active-site cysteine of E2 enzymes. (b) Sequence comparison between consensus catalytic center of UBC (PROSITE: PDOC00163) and Arabidopsis COP10. The catalytic cysteine and its corresponding serine in COP10 were highlighted. (c) Phylogenetic relationships of selected UBCs, COP10s, Ubc domain-like regions of TSG101, and UEVs. The gene families lacking catalytic cysteine are clustered in three gray boxes. The tree was constructed by phylogenetic analysis using ClustalW (Thompson et al. 1994). The accession numbers for the proteins are given below in parentheses: Homo sapiens (Hs)UbcM2 (AAD40197), HsUEV1 (AAB72016), HsMMS2 (CAA66717), HsTSG101 (AAC52083); Arabidopsis thaliana (At)UBC1 (P25865), AtUBC3 (P42746), AtUBC5 (P42749), AtUBC8 (P35131), AtUBC15 (AAC39324), AtMMS2 (AAK68786), AtTSG101-h1 (AAG51025), AtTSG101-h2 (BAB11114); Drosophila melanogaster (Dm)TSG101 (AAG29564); Saccharomyces cerevisiae (Sc)UBC9 (S52414), ScUBC13 (NP010377), and ScMMS2 (AAC24241). Note that Arabidopsis contains members in all three groups of E2V protein families.

Figure 4

The conformation, protein interaction, and nuclear localization of COP10. (a) Gel filtration analysis of the COP10 complex. Protein extracts were prepared from 5-day-old seedlings of continuous light or dark-grown wild-type, and cop10-1, cop9-1, fus6-1, and cop1-5 mutants in continuous light condition. Numbers below the panel indicate the approximate molecular mass. T represents total soluble protein extracts before gel filtration. The fraction numbers are indicated on the top of the panel. An anti-CSN1 immunoblot of light-grown wild type was performed for revealing the different peak position of the COP9 signalosome. (b) Interaction of COP10 with truncated COP1 proteins (Ang et al. 1998) and individual subunits (Serino et al. 1999) of the COP9 signalosome by a yeast two-hybrid assay (Ang et al. 1998). (c) COP10 immunoblot of protein extracts from nuclear or cytosolic fractions of cauliflower tissues. Anti-tubulin or anti-histone immunoblots were used as a cytosolic or nuclear protein controls, respectively. (d) Subcellular localization of the GUS–COP10 fusion protein in transiently transfected onion epidermal cells (von Arnim and Deng 1994). The entire COP10 coding region was fused to COOH end of GUS (von Arnim and Deng 1994). Cells stained for GUS and DAPI are shown next to each other. Results of GUS–NIa fusion protein and GUS alone were used as controls.

Figure 5

A working model depicting possible functional relationships of COP1, COP9 signalosome, and COP10 complex in protein ubiquitination and proteasome-mediated degradation. The COP9 signalosome directly interacts with COP10 complex and regulates its accumulation and assembly, whereas COP10 complex acts as E2 activity for the COP1-mediated substrate ubiquitination. In repressing photomorphogenesis, those three components work together to target photomorphogenesis-promoting transcription factors (such as HY5) degradation via 26S proteasome. It is assumed that COP10 complex contains at least one more subunit that provides the E2 catalytic activity. Stacked parallel lines indicated the direct protein–protein interactions. The other abbreviations are: Ub, ubiquitin; RING, RING-finger motif; Coil, Coil–coil domain; WD40, WD-40 repeat domain.