Arabidopsis membrane-anchored ubiquitin-fold (MUB) proteins localize a specific subset of ubiquitin-conjugating (E2) enzymes to the plasma membrane

The covalent attachment of ubiquitin (Ub) to various intracellular proteins plays important roles in altering the function, localization, processing, and degradation of the modified target. A minimal ubiquitylation pathway uses a three-enzyme cascade (E1, E2, and E3) to activate Ub and select target proteins for modification. Although diverse E3 families provide much of the target specificity, several factors have emerged recently that coordinate the subcellular localization of the ubiquitylation machinery. Here, we show that the family of membrane-anchored ubiquitin-fold (MUB) proteins recruits and docks specific E2s to the plasma membrane. Protein interaction screens with Arabidopsis MUBs revealed that interacting E2s are limited to a well defined subgroup that is phylogenetically related to human UbcH5 and yeast Ubc4/5 families. MUBs appear to interact noncovalently with an E2 surface opposite the active site that forms a covalent linkage with Ub. Bimolecular fluorescence complementation demonstrated that MUBs bind simultaneously to the plasma membrane via a prenyl tail and to the E2 in planta. These findings suggest that MUBs contribute subcellular specificity to ubiquitylation by docking the conjugation machinery to the plasma membrane.     

Autophagy: a multifaceted intracellular system for bulk and selective recycling

Plants have evolved sophisticated mechanisms to recycle intracellular constituents. One gaining in appreciation is autophagy, which involves specialized vesicles engulfing and delivering unwanted cytoplasmic material to the vacuole for breakdown. Central to this process is the ubiquitin-fold protein autophagy (ATG)-8, which becomes tethered to the developing autophagic membranes by lipidation. Here, we review data showing that the ATG8 moiety provides a docking site not only for proteins that help shape the enclosing vesicles and promote their fusion with the tonoplast, but also for a host of receptors that recruit appropriate autophagic cargo. The identity of these receptors has dramatically altered the view of autophagy as being a relatively nonspecific mechanism to one that may selectively sequester aggregated proteins, protein complexes, organelles, and even invading pathogens.     

Genetic analyses of the Arabidopsis 26S proteasome regulatory particle reveal its importance during light stress and a specific role for the N-Terminus of RPT2 in development.

The 26S proteasome subunit RPT2 is a component of the hexameric ring of AAA-ATPases that forms the base of the 19S regulatory particle (RP). This subunit has specific roles in the yeast and mammalian proteasomes by helping promote assembly of the RP with the 20S core protease (CP) and gate the CP to prevent indiscriminate degradation of cytosolic and nuclear proteins. In plants, this subunit plays an important role in diverse processes that include shoot and root apical meristem maintenance, cell size regulation, trichome branching, and stress responses. Recently, we reported that mutants in RPT2 and several other RP subunits have reduced histone levels, suggesting that at least some of the pleiotropic phenotypes observed in these plants result from aberrant nucleosome assembly. Here, we expand our genetic analysis of RPT2 in Arabidopsis to shed additional light on the roles of the N- and C-terminal ends. We also present data showing that plants bearing mutations in RP subunit genes have their seedling phenotypes exacerbated by prolonged light exposure.     

The ubiquitin-specific protease UBP14 is essential for early embryo development in Arabidopsis thaliana

The ubiquitin/26S proteasome pathway is a major route for selectively degrading cytoplasmic and nuclear proteins in eukaryotes. In this pathway, chains of ubiquitins become attached to short-lived proteins, signalling recognition and breakdown of the modified protein by the 26S proteasome. During or following target degradation, the attached multi-ubiquitin chains are released and subsequently disassembled by ubiquitin-specific proteases (UBPs) to regenerate free ubiquitin monomers for re-use. Here, we describe Arabidopsis thaliana UBP14 that may participate in this recycling process. Its amino acid sequence is most similar to yeast UBP14 and its orthologues, human IsoT1-3 and Dictyostelium UbpA, and it can functionally replace yeast UBP14 in a ubp14Delta mutant. Like its orthologues, AtUBP14 can disassemble multi-ubiquitin chains linked internally via epsilon-amino isopeptide bonds using Lys48 and can process some, but not all, translational fusions of ubiquitin linked via alpha-amino peptide bonds. However, unlike its yeast and Dictyostelium orthologues, AtUBP14 is essential in Arabidopsis. T-DNA insertion mutations in the single gene that encodes AtUBP14 cause an embryonic lethal phenotype, with the homozygous embryos arresting at the globular stage. The arrested seeds have substantially increased levels of multi-ubiquitin chains, indicative of a defect in ubiquitin recycling. Taken together, the data demonstrate an essential role for the ubiquitin/26S proteasome pathway in general and for AtUBP14 in particular during early plant development.     

Sequences within both the N- and C-terminal domains of phytochrome A are required for PFR ubiquitination and degradation

Photoconversion of the plant photoreceptor phytochrome A (phyA) from its inactive Pr form to its biologically active Pfr from initiates its rapid proteolysis. Previous kinetic and biochemical studies implicated a role for the ubiquitin/26S proteasome pathway in this breakdown and suggested that multiple domains within the chromoprotein are involved. To further resolve the essential residues, we constructed a series of mutant PHY genes in vitro and analyzed the Pfr-specific degradation of the resulting photoreceptors expressed in transgenic tobacco. One important site is within the C-terminal half of the polypeptide as its removal stabilizes oat phyA as Pfr. Within this half is a set of conserved lysines that are potentially required for ubiquitin attachment. Substitution of these lysines did not prevent ubiquitination or breakdown of Pfr, suggesting either that they are not the attachment sites or that other lysines can be used in their absence. A small domain just proximal to the C-terminus is essential for the form-dependent breakdown of the holoprotein. Removal of just six amino acids in this domain generated a chromoprotein that was not rapidly degraded as Pfr. Using chimeric photoreceptors generated from potato PHYA and PHYB, we found that the N-terminal half of phyA is also required for Pfr-specific breakdown. Only those chimeras containing the N-terminal sequences from phyA were ubiquitinated and rapidly degraded as Pfr. Taken together, our data demonstrate that, whereas an intact C-terminal domain is essential for phyA degradation, the N-terminal domain is responsible for the selective recognition and ubiquitination of Pfr.     

Polypeptide tags,ubiquitous modifiers for plant protein regulation

Evidence has emerged over the past few years that plants, like animals and fungi, employ a variety of polypeptides as tags to reversibly or irreversibly affect the function, structure, location, and/or turnover of numerous intracellular proteins. In plants, known polypeptide tags include ubiquitin, SUMO, RUB, and APG12, with the possibility of others. These modifiers are typically added post-translationally using individual sets of conjugase pathways that attach the polypeptides via an isopeptide bond to -lysyl amino group(s) in the targets. Often the tags can be removed subsequently by unique proteases that specifically cleave only the isopeptide bond. Examples also exist where the tag is added during translation upon fusion of the coding sequence of the tag with that of the target. Based on the number and diversity of targets, ubiquitin is the most influential modifier which mainly serves as a reusable signal for selective protein degradation by the 26S proteasome. In contrast, SUMO, RUB and APG12 become attached to a more limited number of targets and appear to have specialized functions, including roles in nuclear pore assembly/function, cell-cycle regulation, and lysosomal/vacuole trafficking, respectively. Based on their widespread occurrence in plants and their pervasive role in various biological processes, polypeptide tags likely play a prominent role in plant cell regulation.     

The serine-rich N-terminal domain of oat phytochrome a helps regulate light responses and subnuclear localization of the photoreceptor

Phytochrome (phy) A mediates two distinct photobiological responses in plants: the very-low-fluence response (VLFR), which can be saturated by short pulses of very-low-fluence light, and the high-irradiance response (HIR), which requires prolonged irradiation with higher fluences of far-red light (FR). To investigate whether the VLFR and HIR involve different domains within the phyA molecule, transgenic tobacco (Nicotiana tabacum cv Xanthi) and Arabidopsis seedlings expressing full-length (FL) and various deletion mutants of oat (Avena sativa) phyA were examined for their light sensitivity. Although most mutants were either partially active or inactive, a strong differential effect was observed for the Delta6-12 phyA mutant missing the serine-rich domain between amino acids 6 and 12. Delta6-12 phyA was as active as FL phyA for the VLFR of hypocotyl growth and cotyledon unfolding in Arabidopsis, and was hyperactive in the VLFR of hypocotyl growth and cotyledon unfolding in tobacco, and the VLFR blocking subsequent greening under white light in Arabidopsis. In contrast, Delta6-12 phyA showed a dominant-negative suppression of HIR in both species. In hypocotyl cells of Arabidopsis irradiated with FR phyA:green fluorescent protein (GFP) and Delta6-12 phyA:GFP fusions localized to the nucleus and coalesced into foci. The proportion of nuclei with abundant foci was enhanced by continuous compared with hourly FR provided at equal total fluence in FL phyA:GFP, and by Delta6-12 phyA mutation under hourly FR. We propose that the N-terminal serine-rich domain of phyA is involved in channeling downstream signaling via the VLFR or HIR pathways in different cellular contexts.