The defective proteasome but not substrate recognition function is responsible for the null phenotypes of the Arabidopsis proteasome subunit RPN10.

Ubiquitylated substrate recognition during ubiquitin/proteasome-mediated proteolysis (UPP) is mediated directly by the proteasome subunits RPN10 and RPN13 and indirectly by ubiquitin-like (UBL) and ubiquitin-associated (UBA) domain-containing factors. To dissect the complexity and functional roles of UPP substrate recognition in Arabidopsis thaliana, potential UPP substrate receptors were characterized. RPN10 and members of the UBL-UBA-containing RAD23 and DSK2 families displayed strong affinities for Lys-48-linked ubiquitin chains (the major UPP signals), indicating that they are involved in ubiquitylated substrate recognition. Additionally, RPN10 uses distinct interfaces as primary proteasomal docking sites for RAD23s and DSK2s. Analyses of T-DNA insertion knockout or RNA interference knockdown mutants of potential UPP ubiquitin receptors, including RPN10, RPN13, RAD23a-d, DSK2a-b, DDI1, and NUB1, demonstrated that only the RPN10 mutant gave clear phenotypes. The null rpn10-2 showed decreased double-capped proteasomes, increased 20S core complexes, and pleiotropic vegetative and reproductive growth phenotypes. Surprisingly, the observed rpn10-2 phenotypes were rescued by a RPN10 variant defective in substrate recognition, indicating that the defectiveness of RPN10 in proteasome but not substrate recognition function is responsible for the null phenotypes. Our results suggest that redundant recognition pathways likely are used in Arabidopsis to target ubiquitylated substrates for proteasomal degradation and that their specific roles in vivo require further examination.

Expression and characterization of a constitutively active STAT6 from Tetraodon.

In this paper, we report the cloning and characterization of the STAT6 gene from the pufferfish, Tetraodon nigroviridis. The TnSTAT6 gene is composed of 20 exons and 19 introns. The exon-intron organization of this gene is similar to that of HsSTAT6 except for the exons encoding the C-terminal transactivation domain. The full-length complementary (c)DNA of TnSTAT6 encodes a 794-amino acid protein that is 31% identical to human STAT6. We generated a constitutively active TnSTAT6-JH1 by fusing the kinase domain of carp JAK1 to the C-terminal end of TnSTAT6 and demonstrated that the fusion protein has specific DNA-binding ability and can activate a reporter construct carrying multiple copies of mammalian IL-4-response elements. Interestingly, TnSTAT6-JH1 associated with and phosphorylated TnSTAT6 on Tyr661. Mutation of this residue, Y661W, in TnSTAT6 abolished its association with TnSTAT6-JH1. This is consistent with the importance of the corresponding Tyr641 of HsSTAT6 in tyrosine phosphorylation and dimer formation. On the other hand, treatment of mammalian IL-4 did not induce tyrosine phosphorylation of wild-type TnSTAT6, suggesting that both the divergent N-terminal domain and coiled-coiled domain of TnSTAT6 may affect the interaction of TnSTAT6 with mammalian IL-4 receptor complexes.

The 8ab protein of SARS-CoV is a luminal ER membrane-associated protein and induces the activation of ATF6.

The 8ab protein of SARS-CoV is a group-specific accessory protein, which is lost when the virus was transmitted from animals to humans due to a 29-nucleotide deletion in the ORF8ab region. Here we found that 8ab protein is associated with ER membrane at luminal surface. 8ab protein was found to up-regulate the synthesis of endogenous ER-resident chaperons involved in protein folding through the activation of the transcription factor ATF6, while it showed no effect on the CHOP induction and XBP1 splicing associated with the unfolded protein response (UPR). When ectopically expressed in mammalian cells, 8ab induced the proteolysis of ATF6 and the translocation of its cleaved DNA-binding and transcription-activation domains from the ER to the nucleus. Finally, we showed that 8ab binds to the luminal domain of ATF6. These findings suggest that 8ab could modulate the UPR by activating ATF6 to facilitate protein folding and processing. Thus, the loss of 8ab in SARS-CoV through viral evolution in animals may play a role in its pathogenicity.

Genomic structure, expression and characterization of a STAT5 homologue from pufferfish (Tetraodon fluviatilis).

The STAT5 (signal transducer and activator of transcription 5) gene was isolated and characterized from a round-spotted pufferfish genomic library. This gene is composed of 19 exons spanning 11 kb. The full-length cDNA of Tetraodon fluviatilis STAT5 (TfSTAT5) contains 2461 bp and encodes a protein of 785 amino acid residues. From the amino acid sequence comparison, TfSTAT5 is most similar to mouse STAT5a and STAT5b with an overall identity of 76% and 78%, respectively, and has < 35% identity with other mammalian STATs. The exon/intron junctions of the TfSTAT5 gene were almost identical to those of mouse STAT5a and STAT5b genes, indicating that these genes are highly conserved at the levels of amino acid sequence and genomic structure. To understand better the biochemical properties of TfSTAT5, a chimeric STAT5 was generated by fusion of the kinase-catalytic domain of carp Janus kinase 1 (JAK1) to the C-terminal end of TfSTAT5. The fusion protein was expressed and tyrosine-phosphorylated by its kinase domain. The fusion protein exhibits specific DNA-binding and transactivation potential toward an artificial fish promoter as well as authentic mammalian promoters such as the beta-casein promoter and cytokine inducible SH2 containing protein (CIS) promoter when expressed in both fish and mammalian cells. However, TfSTAT5 could not induce the transcription of beta-casein promoter via rat prolactin and Nb2 prolactin receptor. To our knowledge, this is the first report describing detailed biochemical characterization of a STAT protein from fish.