Richard D. Vierstra

The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53

The ubiquitin-dependent proteolytic pathway plays a major role in selective protein degradation. Ubiquitination of proteins requires the sequential action of the ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzymes (E2), and in some cases ubiquitin-protein ligases (E3s). The oncogenic human papillomavirus (HPV) types 16 and 18 utilize this cellular proteolytic system to target the tumor suppressor protein p53. The HPV E6 oncoprotein binds to a cellular protein of 100 kd, termed E6-associated protein (E6-AP). The E6-E6-AP complex specifically interacts with p53, resulting in the rapid ubiquitin-dependent degradation of p53. Here we report the purification and identification of the factors necessary for the E6-E6-AP-mediated ubiquitination of p53. The ubiquitination of p53 requires the E1 enzyme and a novel E2 in mammalian cells, while E3 activity is conferred by the E6-E6-AP complex. Furthermore, E6-AP appears to have ubiquitin-protein ligase activity in the absence of E6.

The ubiquitin–26S proteasome system at the nexus of plant biology

Plants, like other eukaryotes, rely on proteolysis to control the abundance of key regulatory proteins and enzymes. Strikingly, genome-wide studies have revealed that the ubiquitin-26S proteasome system (UPS) in particular is an exceedingly large and complex route for protein removal, occupying nearly 6% of the Arabidopsis thaliana proteome. But why is the UPS so pervasive in plants? Data accumulated over the past few years now show that it targets numerous intracellular regulators that have central roles in hormone signalling, the regulation of chromatin structure and transcription, tailoring morphogenesis, responses to environmental challenges, self recognition and battling pathogens.

The F-box subunit of the SCF E3 complex is encoded by a diverse superfamily of genes in Arabidopsis

The covalent attachment of ubiquitin is an important determinant for selective protein degradation by the 26S proteasome in plants and animals. The specificity of ubiquitination is often controlled by ubiquitin-protein ligases (or E3s), which facilitate the transfer of ubiquitin to appropriate targets. One ligase type, the SCF E3s are composed of four proteins, cullin1/Cdc53, Rbx1/Roc1/Hrt1, Skp1, and an F-box protein. The F-box protein, which identifies the targets, binds to the Skp1 component of the complex through a degenerate N-terminal ≈60-aa motif called the F-box. Using published F-boxes as queries, we have identified 694 potential F-box genes in Arabidopsis, making this gene superfamily one of the largest currently known in plants. Most of the encoded proteins contain interaction domains C-terminal to the F-box that presumably participate in substrate recognition. The F-box proteins can be classified via a phylogenetic approach into five major families, which can be further organized into multiple subfamilies. Sequence diversity within the subfamilies suggests that many F-box proteins have distinct functions and/or substrates. Representatives of all of the major families interact in yeast two-hybrid experiments with members of the Arabidopsis Skp family supporting their classification as F-box proteins. For some, a limited preference for Skps was observed, suggesting that a hierarchical organization of SCF complexes exists defined by distinct Skp/F-box protein pairs. Collectively, the data shows that Arabidopsis has exploited the SCF complex and the ubiquitin/26S proteasome pathway as a major route for cellular regulation and that a diverse array of SCF targets is likely present in plants.

The ubiquitin/26S proteasome pathway, the complex last chapter in the life of many plant proteins

Plants use a repertoire of methods to control the level and activity of their constituent proteins. One method, whose prominence is only now being appreciated, is selective protein breakdown by the ubiquitin/26S proteasome pathway. Remarkably, recent analyses of the near-complete Arabidopsis thaliana genome identified >1300 genes, or approximately 5% of the proteome, involved in the ubiquitin/26S proteasome pathway, making it one of the most elaborate regulatory mechanisms in plants. Molecular genetic analyses have also connected individual components to almost all aspects of plant biology, including the cell-cycle, embryogenesis, photomorphogenesis, circadian rhythms, hormone signaling, homeosis, disease resistance and senescence. Consequently, it appears that the ubiquitin/26S proteasome pathway rivals transcription complexes and protein kinase cascades as the main player in plant cell regulation.

The APG8/12-activating Enzyme APG7 Is Required for Proper Nutrient Recycling and Senescence in Arabidopsis thaliana

The vacuole/lysosome serves an important recycling function during starvation and senescence in eukaryotes via a process called autophagy. Here bulk cytosolic constituents and organelles become sequestered in specialized autophagic vesicles, which then deliver their cargo to the vacuole for degradation. In yeasts, genetic screens have identified two novel post-translational modification pathways remarkably similar to ubiquitination that are required for autophagy. From searches of the Arabidopsisgenome, we have identified gene families encoding proteins related to both the APG8 and −12 polypeptide tags and orthologs for all components required for their attachment. A single APG7gene encodes the ATP-dependent activating enzyme that initiates both conjugation pathways. Phenotypic analysis of anAPG7 disruption indicates that it is not essential for normal growth and development in Arabidopsis. However, theapg7-1 mutant is hypersensitive to nutrient limiting conditions and displays premature leaf senescence. mRNAs for both APG7 and APG8 preferentially accumulate as leaves senesce, suggesting that both conjugation pathways are up-regulated during the senescence syndrome. These findings show that the APG8/12 conjugation pathways have been conserved in plants and may have important roles in autophagic recycling, especially during situations that require substantial nitrogen and carbon mobilization.

Soluble, highly fluorescent variants of green fluorescent protein (GFP) for use in higher plants

Green fluorescent protein (GFP) from Aequorea victoria has rapidly become a standard reporter in many biological systems. However, the use of GFP in higher plants has been limited by aberrant splicing of the corresponding mRNA and by protein insolubility. It has been shown that GFP can be expressed in Arabidopsis thaliana after altering the codon usage in the region that is incorrectly spliced, but the fluorescence signal is weak, possibly due to aggregation of the encoded protein. Through site-directed mutagenesis, we have generated a more soluble version of the codon-modified GFP called soluble-modified GFP (smGFP). The excitation and emission spectra for this protein are nearly identical to wild-type GFP. When introduced into A. thaliana, greater fluorescence was observed compared to the codon-modified GFP, implying that smGFP is ‘brighter’ because more of it is present in a soluble and functional form. Using the smGFP template, two spectral variants were created, a soluble-modified red-shifted GFP (smRS-GFP) and a soluble-modified blue-fluorescent protein (smBFP). The increased fluorescence output of smGFP will further the use of this reporter in higher plants. In addition, the distinct spectral characters of smRS-GFP and smBFP should allow for dual monitoring of gene expression, protein localization, and detection of in vivo protein-protein interactions.

A light-sensing knot revealed by the structure of the chromophore-binding domain of phytochrome

Phytochromes are red/far-red light photoreceptors that direct photosensory responses across the bacterial, fungal and plant kingdoms. These include photosynthetic potential and pigmentation in bacteria as well as chloroplast development and photomorphogenesis in plants. Phytochromes consist of an amino-terminal region that covalently binds a single bilin chromophore, followed by a carboxy-terminal dimerization domain that often transmits the light signal through a histidine kinase relay. Here we describe the three-dimensional structure of the chromophore-binding domain of Deinococcus radiodurans phytochrome assembled with its chromophore biliverdin in the Pr ground state. Our model, refined to 2.5 Å resolution, reaffirms Cys 24 as the chromophore attachment site, locates key amino acids that form a solvent-shielded bilin-binding pocket, and reveals an unusually formed deep trefoil knot that stabilizes this region. The structure provides the first three-dimensional glimpse into the photochromic behaviour of these photoreceptors and helps to explain the evolution of higher plant phytochromes from prokaryotic precursors.

The multiubiquitin-chain-binding protein Mcb1 is a component of the 26S proteasome in Saccharomyces cerevisiae and plays a nonessential, substrate- specific role in protein turnover

The 26S proteasome is an essential proteolytic complex that is responsible for degrading proteins conjugated with ubiquitin. It has been proposed that the recognition of substrates by the 26S proteasome is mediated by a multiubiquitin-chain-binding protein that has previously been characterized in both plants and animals. In this study, we identified a Saccharomyces cerevisiae homolog of this protein, designated Mcb1. Mcb1 copurified with the 26S proteasome in both conventional and nickel chelate chromatography. In addition, a significant fraction of Mcb1 in cell extracts was present in a low-molecular-mass form free of the 26S complex. Recombinant Mcb1 protein bound multiubiquitin chains in vitro and, like its plant and animal counterparts, exhibited a binding preference for longer chains. Surprisingly, (delta)mcb1 deletion mutants were viable, grew at near-wild-type rates, degraded the bulk of short-lived proteins normally, and were not sensitive to UV radiation or heat stress. These data indicate that Mcb1 is not an essential component of the ubiquitin-proteasome pathway in S.cerevisiae. However, the (delta)mcb1 mutant exhibited a modest sensitivity to amino acid analogs and had increased steady-state levels of ubiquitin-protein conjugates. Whereas the N-end rule substrate, Arg-beta-galactosidase, was degraded at the wild-type rate in the (delta)mcb1 strain, the ubiquitin fusion degradation pathway substrate, ubiquitin-Pro-beta-galactosidase, was markedly stabilized. Collectively, these data suggest that Mcb1 is not the sole factor involved in ubiquitin recognition by the 26S proteasome and that Mcb1 may interact with only a subset of ubiquitinated substrates.

Proteolysis in plants: mechanisms and functions

Proteolysis is essential for many aspects of plant physiology and development. It is responsible for cellular housekeeping and the stress response by removing abnormal/misfolded proteins, for supplying amino acids needed to make new proteins, for assisting in the maturation of zymogens and peptide hormones by limited cleavages, for controlling metabolism, homeosis, and development by reducing the abundance of key enzymes and regulatory proteins, and for the programmed cell death of specific plant organs or cells. It also has potential biotechnological ramification in attempts to improve crop plants by modifying protein levels. Accumulating evidence indicates that protein degradation in plants is a complex process involving a multitude of proteolytic pathways with each cellular compartment likely to have one or more. Many of these have homologous pathways in bacteria and animals. Examples include the chloroplast ClpAP protease, vacuolar cathepsins, the KEX2-like proteases of the secretory system, and the ubiquitin/26S proteasome system in the nucleus and cytoplasm. The ubiquitin-dependent pathway requires that proteins targeted for degradation become conjugated with chains of multiple ubiquitins; these chains then serve as recognition signals for selective degradation by the 26S proteasome, a 1.5 MDa multisubunit protease complex. The ubiquitin pathway is particularly important for developmental regulation by selectively removing various cell-cycle effectors, transcription factors, and cell receptors such as phytochrome A. From insights into this and other proteolytic pathways, the use of phosphorylation/dephosphorylation and/or the addition of amino acid tags to selectively mark proteins for degradation have become recurring themes.

Autophagic Nutrient Recycling in Arabidopsis Directed by the ATG8 and ATG12 Conjugation Pathways

Autophagy is an important mechanism for nonselective intracellular breakdown whereby cytosol and organelles are encapsulated in vesicles, which are then engulfed and digested by lytic vacuoles/lysosomes. In yeast, this encapsulation employs a set of autophagy (ATG) proteins that direct the conjugation of two ubiquitin-like protein tags, ATG8 and ATG12, to phosphatidylethanolamine and the ATG5 protein, respectively. Using an Arabidopsis (Arabidopsis thaliana) atg7 mutant unable to ligate either tag, we previously showed that the ATG8/12 conjugation system is important for survival under nitrogen-limiting growth conditions. By reverse-genetic analyses of the single Arabidopsis gene encoding ATG5, we show here that the subpathway that forms the ATG12-ATG5 conjugate also has an essential role in plant nutrient recycling. Similar to plants missing ATG7, those missing ATG5 display early senescence and are hypersensitive to either nitrogen or carbon starvation, which is accompanied by a more rapid loss of organellar and cytoplasmic proteins. Multiple ATG8 isoforms could be detected immunologically in seedling extracts. Their abundance was substantially elevated in both the atg5 and atg7 mutants, caused in part by an increase in abundance of several ATG8 mRNAs. Using a green fluorescent protein-ATG8a fusion in combination with concanamycin A, we also detected the accumulation of autophagic bodies inside the vacuole. This accumulation was substantially enhanced by starvation but blocked in the atg7 background. The use of this fusion in conjunction with atg mutants now provides an important marker to track autophagic vesicles in planta.