Review
Protein degradation in signaling

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Abstract

Recent studies have linked proteolysis by the ubiquitin/proteasome pathway to a variety of signaling pathways in higher plants. These links were uncovered by characterization of mutants altered in signaling or by targeted disruption of components of the proteolytic pathway. Significant advances have recently revealed connections between proteolysis and hormone responses, light perception, environmental adaptation, and floral development.

Introduction

Accumulating evidence indicates that the degradation of many cytosolic and nuclear proteins is controlled by the ubiquitin/proteasome pathway in eukaryotes (reviewed recently in [1] and see Figure 1). In this pathway, ubiquitin becomes covalently attached to cellular proteins by an ATP-dependent reaction cascade. E1 (or ubiquitin-activating enzyme [UBA]), catalyzes the formation of an ubiquitin adenylate that is then transferred to an E1 cysteinyl sulfhydryl group. E1 transfers this activated ubiquitin to a cysteinyl sulfhydryl group on a second protein called E2 (i.e. ubiquitin-conjugating enzyme [UBC]). E2s are a large family of mostly 25–35 kDa proteins sharing a conserved core domain that contains the catalytic cysteine. In Arabidopsis, there are at least 36 E2 isoforms that can be clustered into 12 groups (RD Vierstra et al., unpublished data).

The transfer of activated ubiquitin to the ϵ-lysyl residue of target proteins typically requires a third protein or protein complex called an E3 (i.e. ubiquitin ligase). Work in yeast and mammalian systems clearly indicates that the specificity of the ubiquitin pathway derives from the activity of specific E3s or E2/E3 combinations. No single conserved domain identifies E3 proteins in toto; therefore database searching can not identify all E3s. Various studies in plants have recently implicated specific E3 activities in signaling pathways (see below).

So far, four E3 types have been identified in animals and yeast with related sequences that are also present in plants (Figure 2). The first group is the HECT (homology to E6-AP carboxy terminus) domain proteins that share a 350-amino-acid region with mammalian E6-AP, the first member of this type to be discovered. So far, we have identified seven HECT E3s in Arabidopsis, two of which are single 405 kDa polypeptides [2]. The second E3 type, represented by yeast ubiquitin amino-end recognizing protein 1 (Ubr1p), degrades substrates with specific amino-terminal amino acids as well as other proteins by unknown signals [3]. Arabidopsis PROTEOLYSIS (PRT1) appears to be a relative of Ubr1p, and has been shown to be important for degradation of amino-end-rule substrates [4]. Ubr1 and PRT1 both contain a RING-H2 motif, a C-X2-C-Xn-C-X1-3-H-X2-3-C/H-X2-C-Xn-C-X2-C linear sequence (using the single-letter code for amino acids) that is thought to coordinate two Zn atoms, and may be important for E2 activation. The APC/C (anaphase-promoting complex/cyclosome) E3 is responsible for the degradation of anaphase inhibitors, mitotic cyclins, and components of the mitotic spindle (reviewed in [5]). It is a complex of 8–12 subunits in yeast and Xenopus, many of which have orthologs in Arabidopsis.

The Skp1-cullin-F-box (SCF)/RING-H2 E3 ligase complex (reviewed in [6]) is named after the three original yeast protein subunits: suppressor of kinetochore protein 1 (Skp1p); cell-division cycle 53 (Cdc53p) (called cullins in other species); and F-box proteins (which were named after the first member to be discovered, human cyclin F). More recently, a fourth subunit called Roc1/Rbx1/Hrt1 (for regulator of cullins 1/RING-box protein 1/high-level expression reduces Ty3 transposition 1) that contains a RING-H2 motif was identified. Cdc53p and Hrt1p bind the cognate E2, and Skp1p appears to link Cdc53p/Hrt1p/E2 to the F-box protein though interaction with the latter’s approximately 45-amino-acid F-box. F-box proteins appear to be responsible for target specificity by binding substrate through additional protein–protein interaction motifs, such as leucine-rich, WD-40, or other motifs. Database searches have identified many putative F-box-containing sequences in Arabidopsis, suggesting how important these recognition factors may be.

Additional components of the ubiquitin pathway include the family of ubiquitin-specific hydrolases (UBPs) that hydrolyze a variety of ubiquitin linkages, either before or after proteolysis. Individual UBPs function in mammalian cytokine signaling and cell-to-cell communication 7, 8. Their role in plants is unclear. Arabidopsis contains at least 28 UBPs; genetic disruption shows that several UBPs are essential (J Doelling, RD Vierstra, unpublished observations).

Ubiquitinated proteins are most often targeted for degradation by a large multi-catalytic protease, the 26S proteasome. The 26S proteasome is comprised of two large subcomplexes, the 20S proteasome and the 19S regulatory complex. The plant counterpart appears to be similar in organization and structure to the fungal and animal proteasomes, and probably functions in an analogous manner 9, 10, 11. Proteolysis occurs in the lumen of the 20S central aqueous compartment, releasing small peptides of 7–10 residues that are then degraded to free amino acids by cellular peptidases. The 19S regulatory complex can be divided further into the base, a ring of six ATPase subunits associated with diverse cellular activities (AAA) ATPase subunits and two non-ATPase subunits, and a lid of approximately 12 polypeptides. The 19S regulatory complex appears to be responsible for recognizing the ubiquitylated protein, unfolding the tertiary and secondary structures of the protein, and directing the unfolding polypeptide into the lumen of the 20S complex.

The discovery of other protein modification systems that operate in a manner analogous to the ubiquitin pathway is a new and exciting area of research. Our current knowledge of these systems in plants has been reviewed recently [12], and only those aspects that relate to signaling and proteolysis will be discussed here. This review highlights new information on proteolysis and its linkage to signaling pathways in higher plants.

Section snippets

Auxin signaling pathways are linked to the ubiquitin/proteasome pathway

Genetic approaches to elucidating the molecular mechanisms of auxin action have revealed links between hormone responses and the ubiquitin pathway. The cloning of transport inhibitor response 1 (tir1), an auxin response mutant, was a significant advance. Sequence analysis of the TIR1 protein revealed its relation to F-box proteins, the specificity factors of the SCF class of ubiquitin E3 ligases (Figure 2) [13]. It was subsequently shown, by yeast two-hybrid screening and

Regulated proteolysis is important for photomorphogenesis

One of the first studies that linked proteolysis to light perception was that of Butler and coworkers (reviewed in [31]) that showed rapid phytochrome A (phyA) degradation upon phototransformation to the far-red (Pfr) form. It has since been shown that this degradation probably involves the ubiquitin/proteasome pathway. Modulation of the intracellular levels of phyA by rapidly removing the activated receptor appears to be critical for the continuous sensing of and adaptation to the ambient

Other signaling pathways linked to the ubiquitin pathway

The responses of plants to hormones other than auxin also appear to require specific components of the ubiquitin/proteasome pathway. Disruptions of the proteasome subunit RPN10 in Physomitrella and RPN12a in Arabidopsis appear to alter cytokinin signaling. Loss of the HECT-E3 UBIQUITIN PROTEIN LIGASE (UPL2) causes Arabidopsis plants to become hypersensitive to abscissic acid (PW Bates, RD Vierstra, unpublished results).

SCF complexes in other organisms regulate multiple signaling pathways. In

Proteasome inhibitors link proteolysis and signaling

Inhibitors that are highly specific for the proteasome have become useful in studies of the role of the ubiquitin/proteasome pathway in higher plants. Cell permeable inhibitors such as the tripeptide aldehyde MG132 and clasto-lactacystin lactone have implicated the proteasome in trachiary-element differentiation [44], pathogen response [45], and wound signaling [46]. Although care must be taken in the interpretation of results obtained using such inhibitors because of their possible indirect

Conclusions

It is clear that the ubiquitin/proteasome pathway plays important roles in various signaling pathways in yeast and animals. These processes include intercellular communication, regulation of the abundance of receptors, and degradation of cytosolic transcriptional repressors and nuclear transcriptional activators. The ultimate consequence may be either activation or repression of entire signaling pathways and/or genetic programs. The few examples in plant systems described here are just the tip

Acknowledgements

Research in the Callis laboratory is supported by grants from the National Science Foundation (IBN 98-08791) and the Department of Energy (DOE). JC thanks Dr Charles Gasser for helpful discussions. The Vierstra laboratory was supported by grants from the US Department of Agriculture National Research Initiative Competitive Grants Program (USDA-NRICGP), the US Department of Energy (DOE), and the Consortium for Plant Biotechnology Research.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • ••of outstanding interest

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