Structure and Biochemical Function of a Prototypical Arabidopsis U-box Domain

Abstract

U-box proteins, as well as other proteins involved in regulated protein degradation, are apparently over-represented in Arabidopsis compared with other model eukaryotes. The Arabidopsis protein AtPUB14 contains a typical U-box domain followed by an Armadillo repeat region, a domain organization that is frequently found in plant U-box proteins. In vitro ubiquitination assays demonstrated that AtPUB14 functions as an E3 ubiquitin ligase with specific E2 ubiquitin-conjugating enzymes. The structure of the AtPUB14 U-box domain was determined by NMR spectroscopy. It adopts the ␤␤␣␤ fold of the Prp19p U-box and RING finger domains. In these proteins, conserved hydrophobic residues form a putative E2-binding cleft. By contrast, they contain no common polar E2 binding site motif. Two hydrophobic cores stabilize the AtPUB14 U-box fold, and hydrogen bonds and salt bridges interconnect the residues corresponding to zinc ion-coordinating residues in RING domains. Residues from a C-terminal ␣-helix interact with the core domain and contribute to stabilization. The Prp19p U-box lacks a corresponding C-terminal ␣-helix. Chemical shift analysis suggested that aromatic residues exposed at the N terminus and the C-terminal ␣-helix of the AtPUB14 U-box participate in dimerization. Thus, AtPUB14 may form a biologically relevant dimer. This is the first plant U-box structure to be determined, and it provides a model for studies of the many plant U-box proteins and their interactions. Structural insight into these interactions is important, because ubiquitindependent protein degradation is a prevalent regulatory mechanism in plants.The ubiquitin proteolytic pathway plays an important role in regulated protein degradation (1). Proteins designated for degradation are covalently modified by attachment of a ubiquitin polymer and degraded by the 26 S proteasome. A ubiquitinactivating enzyme (E1) 1 catalyzes ATP-dependent formation of a thioester bond between ubiquitin and itself and transfers the activated ubiquitin to a ubiquitin-conjugating enzyme (E2). Formation of an isopeptide bond between ubiquitin and a substrate is facilitated by a ubiquitin-protein ligase (E3) that can bind both the E2-ubiquitin complex and the substrate. Members of the HECT and RING protein families are the best characterized E3 ligases, but recently U-box proteins have also been shown to function as E3s (2, 3). This may be the general function of U-box proteins, although they were initially suggested to function as ubiquitin chain assembly factors (E4s) (4). Recently, research on U-box proteins, especially the carboxyl terminus of Hsc70-interacting proteins (CHIP), has focused on their ability to interact with molecular chaperones and selectively ubiquitinate unfolded proteins. Thus ubiquitination can also function in protein quality control (5).The U-box motif is a peptide chain that contains ϳ70 amino acid residues, with characteristics suggesting that it is a structural variant of the RING fold but lacks the signature zincbinding amino acids of the RING doma...

Isolation and characterization of a copalyl diphosphate synthase gene promoter from Salvia miltiorrhiza

“…3) [44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59][60]. Comparison of the obtained data with trans-factors identified by the in silico searches of S. miltiorrhiza CPS promoter showed no common protein (Tab.…”

“…The apoptosis was regulated in A. thaliana by the putative ubiquitin-ligase and F-box/RNI-like/FBD-like domains-containing protein, both co-expressed with the CPS gene (Tab. 3) [56,57]. Also in the S. miltiorrhiza, CPS promoter was observed a cis-active motif participating in the response to protein unfolding (Tab.…”

Interactions between theS-Domain Receptor Kinases and AtPUB-ARM E3 Ubiquitin Ligases Suggest a Conserved Signaling Pathway in Arabidopsis

“…Finally, AtPUB44 was chosen to represent the dual ARM-repeat clade (U-box:ARM:ARM) and is the most distantly related AtPUB relative to ARC1 (Mudgil et al, 2004;Samuel et al, 2006). All six of these AtPUB-ARM proteins have been shown to have in vitro E3 ubiquitin ligase activity (Andersen et al, 2004;Mudgil et al, 2004;J. Salt, M.A.…”

“…The larger known Arabidopsis families include the RING family with approximately 469 predicted proteins and the F-box family with approximately 700 predicted proteins (Gagne et al, 2004;Stone et al, 2005). The Arabidopsis U-box family is a smaller predicted family with 62 members (Azevedo et al, 2001;Andersen et al, 2004).…”

“…The PUB-ARM family comprises the largest group with 41 predicted members in the Arabidopsis genome and 43 members in the rice (Oryza sativa) genome (Mudgil et al, 2004;Samuel et al, 2006). Despite the limited knowledge about the biological functions for these predicted PUB-ARM proteins, they have been shown to function as E3 ubiquitin ligases (Andersen et al, 2004;Mudgil et al, 2004). In various plant species, diverse biological functions have emerged for related PUB-ARM proteins.…”