Konstantin Tomanov

Lysine63-linked ubiquitylation of PIN2 auxin carrier protein governs hormonally controlled adaptation of Arabidopsis root growth

Cross-talk between plant cells and their surroundings requires tight regulation of information exchange at the plasma membrane (PM), which involves dynamic adjustments of PM protein localization and turnover to modulate signal perception and solute transport at the interface between cells and their surroundings. In animals and fungi, turnover of PM proteins is controlled by reversible ubiquitylation, which signals endocytosis and delivery to the cell’s lytic compartment, and there is emerging evidence for related mechanisms in plants. Here, we describe the fate of Arabidopsis PIN2 protein, required for directional cellular efflux of the phytohormone auxin, and identify cis- and trans-acting mediators of PIN2 ubiquitylation. We demonstrate that ubiquitin acts as a principal signal for PM protein endocytosis in plants and reveal dynamic adjustments in PIN2 ubiquitylation coinciding with variations in vacuolar targeting and proteolytic turnover. We show that control of PIN2 proteolytic turnover via its ubiquitylation status is of significant importance for auxin distribution in root meristems and for environmentally controlled adaptations of root growth. Moreover, we provide experimental evidence indicating that PIN2 vacuolar sorting depends on modification specifically by lysine63-linked ubiquitin chains. Collectively, our results establish lysine63-linked PM cargo ubiquitylation as a regulator of polar auxin transport and adaptive growth responses in higher plants.

Arabidopsis PIAL1 and 2 Promote SUMO Chain Formation as E4-Type SUMO Ligases and Are Involved in Stress Responses and Sulfur Metabolism

Substrates modified by covalent attachment of the small ubiquitin-related modifier (SUMO) usually receive a single SUMO moiety. Existence of enzymes that promote formation of SUMO chains indicates that such chains have distinct roles in plants, and mutant analysis indicates involvement in regulation of sulfur metabolism and stress responses. The Arabidopsis thaliana genes PROTEIN INHIBITOR OF ACTIVATED STAT LIKE1 (PIAL1) and PIAL2 encode proteins with SP-RING domains, which occur in many ligases of the small ubiquitin-related modifier (SUMO) conjugation pathway. We show that PIAL1 and PIAL2 function as SUMO ligases capable of SUMO chain formation and require the SUMO-modified SUMO-conjugating enzyme SCE1 for optimal activity. Mutant analysis indicates a role for PIAL1 and 2 in salt stress and osmotic stress responses, whereas under standard conditions, the mutants show close to normal growth. Mutations in PIAL1 and 2 also lead to altered sulfur metabolism. We propose that, together with SUMO chain binding ubiquitin ligases, these enzymes establish a pathway for proteolytic removal of sumoylation substrates.

SUMOylation represses SnRK1 signaling in Arabidopsis

The SnRK1 protein kinase balances cellular energy levels in accordance with extracellular conditions and is thereby key for plant stress tolerance. In addition, SnRK1 has been implicated in numerous growth and developmental processes from seed filling and maturation to flowering and senescence. Despite its importance, the mechanisms that regulate SnRK1 activity are poorly understood. Here, we demonstrate that the SnRK1 complex is SUMOylated on multiple subunits and identify SIZ1 as the E3 Small Ubiquitin-like Modifier (SUMO) ligase responsible for this modification. We further show that SnRK1 is ubiquitinated in a SIZ1-dependent manner, causing its degradation through the proteasome. In consequence, SnRK1 degradation is deficient in siz1-2 mutants, leading to its accumulation and hyperactivation of SnRK1 signaling. Finally, SnRK1 degradation is strictly dependent on its activity, as inactive SnRK1 variants are aberrantly stable but recover normal degradation when expressed as SUMO mimetics. Altogether, our data suggest that active SnRK1 triggers its own SUMOylation and degradation, establishing a negative feedback loop that attenuates SnRK1 signaling and prevents detrimental hyperactivation of stress responses.

Ubiquitin Lys 63 chains - second-most abundant, but poorly understood in plants

Covalent attachment of the small modifier ubiquitin to Lys ε-amino groups of proteins is surprisingly diverse. Once attached to a substrate, ubiquitin is itself frequently modified by ubiquitin, to form chains. All seven Lys residues of ubiquitin, as well as its N-terminal Met, can be ubiquitylated, implying cellular occurrence of different ubiquitin chain types. The available data suggest that the synthesis, recognition, and hydrolysis of different chain types are precisely regulated. This remarkable extent of control underlies a versatile cellular response to substrate ubiquitylation. In this review, we focus on roles of Lys63-linked ubiquitin chains in plants. Despite limited available knowledge, several recent findings illustrate the importance of these chains as signaling components in plants.

Small Ubiquitin-Like Modifier Conjugating Enzyme with Active Site Mutation Acts as Dominant Negative Inhibitor of SUMO Conjugation in Arabidopsis

Small ubiquitin-like modifier (SUMO) conjugation affects a broad range of processes in plants, including growth, flower initiation, pathogen defense, and responses to abiotic stress. Here, we investigate in vivo and in vitro a SUMO conjugating enzyme with a Cys to Ser change in the active site, and show that it has a dominant negative effect. In planta expression significantly perturbs normal development, leading to growth retardation, early flowering and gene expression changes. We suggest that the mutant protein can serve as a probe to investigate sumoylation, also in plants for which poor genetic infrastructure precludes analysis via loss-of-function mutants.

Protein sumoylation and phosphorylation intersect in Arabidopsis signaling

Summary Conjugation of the small ubiquitin‐related modifier (SUMO) to protein substrates has an impact on stress responses and on development. We analyzed the proteome and phosphoproteome of mutants in this pathway. The mutants chosen had defects in SUMO ligase SIZ1, which catalyzes attachment of single SUMO moieties onto substrates, and in ligases PIAL1 and PIAL2, which are known to form SUMO chains. A total of 2657 proteins and 550 phosphopeptides were identified and quantified. Approximately 40% of the proteins and 20% of the phosphopeptides showed differences in abundance in at least one of the analyzed genotypes, demonstrating the influence of SUMO conjugation on protein abundance and phosphorylation. The data show that PIAL1 and PIAL2 are integral parts of the SUMO conjugation system with an impact on stress response, and confirm the involvement of SIZ1 in plant defense. We find a high abundance of predicted SUMO attachment sites in phosphoproteins (70% versus 40% in the total proteome), suggesting convergence of phosphorylation and sumoylation signals onto a set of common targets.

SUMO Chain Formation by Plant Enzymes.

SUMO conjugation is a conserved process of eukaryotes, and essential in metazoa. Different isoforms of SUMO are present in eukaryotic genomes. Saccharomyces cerevisiae has only one SUMO protein, humans have four and Arabidopsis thaliana has eight, the main isoforms being SUMO1 and SUMO2 with about 95 % identity. Functionally similar to human SUMO2 and SUMO3, Arabidopsis SUMO1 and 2 can form chains, even though they do not possess a consensus SUMOylation motif. The surprising finding that plants have dedicated enzymes for chain synthesis implies a specific role for SUMO chains in plants. By the cooperative action with SUMO chain recognizing ubiquitin ligases, chains might channel substrates into the ubiquitin-dependent degradation pathway.A method is described to generate SUMO chains, using plant enzymes produced in E. coli. In vitro SUMO chain formation may serve for further analysis of SUMO chain functions. It can also provide an easy-to-synthesize substrate for SUMO-specific proteases.

Sumoylation and phosphorylation: hidden and overt links

Post-translational modifications are essential mediators between stimuli from development or the environment and adaptive transcriptional patterns. Recent data allow a first glimpse at how two modifications, phosphorylation and sumoylation, act interdependently to modulate stress responses. In particular, many components of the SUMO conjugation system are phosphoproteins, and some regulators and enzymes of protein phosphorylation can be sumoylated. Equally important, however, a number of proteins can be subject to both modifications. These substrates also have the capacity to connect stimuli transmitted via sumoylation with those transmitted via phosphorylation. As a prime example, we review data suggesting that nitrate reductase is a hub that integrates cues from these two modifications. Powerful proteomics approaches allowed the identification of additional common substrates, paving the way for studies to understand, on a broader basis, the cross-talk of phosphorylation with sumoylation and how it contributes to plant growth.

SUMO chain formation relies on the amino-terminal region of SUMO conjugating enzyme and has dedicated substrates in plants

The small ubiquitin-related modifier (SUMO) conjugation apparatus usually attaches single SUMO moieties to its substrates, but SUMO chains have also been identified. To better define the biochemical requirements and characteristics of SUMO chain formation, mutations in surface-exposed Lys residues of Arabidopsis SUMO-conjugating enzyme (SCE) were tested for in vitro activity. Lys-to-Arg changes in the amino-terminal region of SCE allowed SUMO acceptance from SUMO-activating enzyme and supported substrate mono-sumoylation, but these mutations had significant effects on SUMO chain assembly. We found no indication that SUMO modification of SCE promotes chain formation. A substrate was identified that is modified by SUMO chain addition, showing that SCE can distinguish substrates for either mono-sumoylation or SUMO chain attachment. It is also shown that SCE with active site Cys mutated to Ser can accept SUMO to form an oxyester, but cannot transfer this SUMO moiety onto substrates, explaining a previously known dominant negative effect of this mutation.

Expression and Purification of the Arabidopsis E4 SUMO Ligases PIAL1 and PIAL2

The proteins PIAL1 (At1g08910) and PIAL2 (At5g41580) are members of the recently discovered group of plant E4 SUMO ligases. This protocol allows quick and simple expression of the recombinant proteins in Escherichia coli (E. coli) and subsequent affinity purification using a maltose binding protein (MBP) tag. The proteins can be used in SUMOylation reactions, where the MBP part of the protein can be detected with a commercially available antibody, or additional purification steps can be applied.