Arabidopsis has two redundant Cullin3 proteins that are essential for embryo development and that interact with RBX1 and BTB proteins to form multisubunit E3 ubiquitin ligase complexes in vivo.

Cullin-based E3 ubiquitin ligases play important roles in the regulation of diverse developmental processes and environmental responses in eukaryotic organisms. Recently, it was shown in Schizosaccharomyces pombe, Caenorhabditis elegans, and mammals that Cullin3 (CUL3) directly associates with RBX1 and BTB domain proteins in vivo to form a new family of E3 ligases, with the BTB protein subunit functioning in substrate recognition. Here, we demonstrate that Arabidopsis thaliana has two redundant CUL3 (AtCUL3) genes that are essential for embryo development. Besides supporting anticipated specific AtCUL3 interactions with the RING protein AtRBX1 and representative Arabidopsis proteins containing a BTB domain in vitro, we show that AtCUL3 cofractionates and specifically associates with AtRBX1 and a representative BTB protein in vivo. Similar to the AtCUL1 subunit of the SKP1-CUL1-F-box protein-type E3 ligases, the AtCUL3 subunit of the BTB-containing E3 ligase complexes is subjected to modification and possible regulation by the ubiquitin-like protein Related to Ubiquitin in vivo. Together with the presence of large numbers of BTB proteins with diverse structural features and expression patterns, our data suggest that Arabidopsis has conserved AtCUL3-RBX1-BTB protein E3 ubiquitin ligases to target diverse protein substrates for degradation by the ubiquitin/proteasome pathway.

Cytomegalovirus stimulated mRNA accumulation and cell surface expression of the oxidized LDL scavenger receptor, CD36.

OBJECTIVE: Cytomegalovirus (CMV) has been epidemiologically associated with multiple disease processes including coronary, carotid and cardiac graft atherosclerosis. An early initiating event in atherogenesis is the uptake by macrophages of oxidized low-density lipoproteins (OxLDL) via the scavenger receptor, CD36. Because CMV can activate host-cell gene transcription, we hypothesized that CMV may upregulate CD36 expression. METHODS AND RESULTS: THP-1 monocyte/macrophage cells were treated with Davis strain CMV and cell surface CD36 expression measured by flow cytometry. Virus challenge increased the percentage of cells expressing CD36 from 21.8 +/- 1.7 to 48.2 +/- 4.0% (mean +/- S.D. for three experiments, P=0.0005); CD36 mRNA accumulation was increased by CMV treatment as determined by reverse transcription-polymerase chain reaction. Viral challenge also upregulated the mitogen-activated protein kinase p38; further, the specific p38 inhibitor, SB203580, reversed the CMV-induced CD36 cell surface expression from 57.2% of cells to baseline levels (29.0 and 30.1% for SB203580 treated and control cells, respectively; P=0.001). Treatment with virus also stimulated uptake of OxLDL: microscopically, virus-treated cells had a mean of 32 +/- 4.0 lipid vacuoles compared with 20 +/- 1.3 for control cells (P=0.01). CONCLUSIONS: These findings suggest that CMV-induced CD36 expression is one mechanism through which CMV may promote atherosclerosis. Other CMV-associated atherogenic mechanisms may exist; additional investigation is necessary.

Analysis of far-red light-regulated genome expression profiles of phytochrome A pathway mutants in Arabidopsis.

Phytochrome A (phyA) is the primary photoreceptor responsible for various far-red (FR) light-mediated responses. Previous studies have identified multiple phyA signaling mutants, including both positive and negative regulators of the phyA-mediated responses. How these defined intermediates act to mediate FR light responses is largely unknown. Here a cDNA microarray was used to examine effects of those mutations on the far-red light control of genome expression. Clustering analysis of the genome expression profiles supports the notion that phyA signaling may entail a network with multiple paths, controlling overlapping yet distinct sets of gene expression. FHY1, FAR1 and FHY3 most likely act upstream in the phyA signaling network, close to the phyA photoreceptor itself. FIN219, SPA1 and REP1 most likely act somewhere more downstream in the network and control the expression of smaller sets of genes. Further, this study also provides genomics evidence for the partial functional redundancy between FAR1 and FHY3. These two homologous proteins control the expression of a largely overlapping set of genes, and likely act closely together in the phyA-mediated FR light responses.

Characterization of a strong dominant phytochrome A mutation unique to phytochrome A signal propagation.

Here, we report the isolation and characterization of a strong dominant-negative phytochrome A (phyA) mutation (phyA-300D) in Arabidopsis. This mutation carries a single amino acid substitution at residue 631, from valine to methionine (V631M), in the core region within the C-terminal half of PHYA. This PHYA core region contains two protein-interactive motifs, PAS1 and PAS2. Val-631 is located within the PAS1 motif. The phyA-V631M mutant protein is photochemically active and accumulates to a level similar to wild type in dark-grown seedlings. Overexpression of PHYA-V631M in a wild-type background results in a dominant-negative interference with endogenous wild-type phyA, whereas PHYA-V631M in a phyA null mutant background is inactive. To investigate the specificity of this mutation within the phytochrome family, the corresponding amino acid substitution (V664M) was created in the PHYTOCHROME B (PHYB) polypeptide. We found that the phyB-V664M mutant protein is physiologically active in phyB mutant and causes no interfering effect in a wild-type background. Together, our results reveal a unique feature in phyA signal propagation through the C-terminal core region.