The Ubiquitin-Proteasome Pathway and Plant Development

Abstract

The importance of the ubiquitin-proteasome pathway to cellular regulation in eukaryotes has become increasingly apparent during the last several years. This fact was formally acknowledged recently by the awarding of the 2004 Nobel Prize in Chemistry to Aaron Ciechanover, Avram Hershko, and Irwin Rose for the discovery of ubiquitin-mediated protein degradation. In plants, regulated protein degradation by the ubiquitin/26S proteosome contributes significantly to development by affecting a wide range of processes, including embryogenesis, hormone signaling, and senescence. In Arabidopsis thaliana more than 1400 genes (∼5% of the proteome) encode components of the ubiquitin/26S proteasome (Ub/26S) pathway (Smalle and Vierstra, 2004). Approximately 90% of these genes encode subunits of the E3 ubiquitin ligases, which confer substrate specificity to the pathway. Two of the primary objectives in the field are to identify the substrates of the Ub/26S proteasome pathway and to characterize the E3 ubiquitin ligases. This review will discuss how the Ub/26S proteasome affects plant development by focusing primarily on the action of the E3 ubiquitin ligases. Classes of E3 Ubiquitin Ligases in Plants. Cullin and cullin-like proteins are green, RING proteins are red, and substrate specificity factors are purple. The 26S proteasome is a multisubunit complex that consists of a cylindrical 20S core protease capped on each end by a 19S regulatory particle (Groll and Huber, 2003). The 19S regulatory particle can be further divided into lid and base components. The lid contains nine subunits and is responsible for recognizing ubiquitinated substrates and for removing the Ub chains. The base contains several subunits that work to unfold the substrate. As a whole, the regulatory particle serves as the gate into the interior of the core protease. The core protease consists of a stack of proteolytic α and β subunits surrounding a narrow chamber. It is here that the substrates are finally degraded into short peptides, after which the constituent amino acids can be recycled (Voges et al., 1999; Yang et al., 2004). Subunits of the regulatory particle are encoded by gene families (Yang et al., 2004). The loss of some of these subunits can lead to stabilization of specific targets. For example, Arabidopsis plants deficient in the rpn10 gene, a base component, show defects in ABA signaling. This defect can be explained, at least in part, by stabilization of the transcription factor ABI5 (Smalle et al., 2003). Furthermore, a mutation in rpn12a, a lid component, results in the upregulation of two cytokinin-induced genes, CYCD3 and NIA1 (Smalle et al., 2002). It is unclear whether these regulatory particle subunits act in a specific manner or whether the effects on these signaling pathways are because of a general defect in proteasome activity. However, in both cases, other proteins that are degraded by the Ub/26S proteasome pathway remain unaffected. The E3 ubiquitin ligases comprise a large and diverse family of proteins or protein complexes containing either a HECT domain or a RING/U-box domain (Figure 1). The RING domain E3s can be further divided into single subunit RING/U-box E3s, such as Constitutive Photomorphogenesis1 (COP1), SEVEN IN ABSENTIA IN ARABIDOPSIS THALIANA 5 (SINAT5), and Arm Repeat-Containing1 (ARC1), and multisubunit RING E3s, which include the SCF, CUL3-BTB, and APC complexes. Whereas the Arabidopsis RING domain–containing family includes several hundreds of proteins, the HECT family is relatively small. HECT E3s are large proteins, typically 100 400 kD. The HECT domain is a 350–amino acid motif and contains both a ubiquitin binding site and a Ub E2 binding site (Pickart, 2001). In addition to the HECT domain, these E3s have a protein–protein interaction domain, such as an SH3 domain, RING-finger, or coiled-coil domain. This part of the protein is probably involved in target recognition and/or localization. Exceptional among ubiquitin ligases, HECT E3s form a covalent bond with ubiquitin before transferring it to the substrate protein (Pickart, 2001). Although many HECT E3s have been identified in animals (up to 50 putative HECT E3s in humans, for example), only seven HECT E3s are encoded by the Arabidopsis genome (Schwarz et al., 1998; Downes et al., 2003). The first examples of plant HECT E3s, UPL1 (for Ubiquitin Protein Ligase1)and UPL2, were reported in 1999 (Bates and Vierstra, 1999). Since then, an examination of the Arabidopsis genomic sequence identified five more HECT E3 genes, called UPL3 through UPL7 (Downes et al., 2003). RT-PCR data show that each of these genes is expressed in seedlings. T-DNA insertions have been isolated in each gene, the best characterized of which is the upl3 mutant. The most striking feature of the upl3 mutant is the highly branched trichomes (up to seven branches versus two to three in the wild type). The gibberellic acid pathway regulates trichome development, and the constitutive