SUMO Conjugation and SUMO Chain Formation by Plant Enzymes.

SUMO conjugation is a conserved process of eukaryotes, and essential in metazoa. Similar to ubiquitylation, a SUMO-activating enzyme links to the SUMO carboxyl-terminal Gly in a thioester bond, and a SUMO-conjugating enzyme accepts activated SUMO and can transfer it to substrates. Unlike ubiquitylation, this transfer can also occur, in an unspecified number of cases, in the absence of ligase-like enzymes.

ATM controls meiotic DNA double-strand break formation and recombination and affects synaptonemal complex organization in plants.

Meiosis is a specialized cell division that gives rise to genetically distinct gametic cells. Meiosis relies on the tightly controlled formation of DNA double-strand breaks (DSBs) and their repair via homologous recombination for correct chromosome segregation. Like all forms of DNA damage, meiotic DSBs are potentially harmful and their formation activates an elaborate response to inhibit excessive DNA break formation and ensure successful repair.

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.

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 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.

Protein sumoylation and phosphorylation intersect in Arabidopsis signaling.

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.

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.

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.

Expression and Purification of the 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 () 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.

Arabidopsis PIAL1 and 2 promote SUMO chain formation as E4-type SUMO ligases and are involved in stress responses and sulfur metabolism.

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.

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.

Small ubiquitin-like modifier conjugating enzyme with active site mutation acts as dominant negative inhibitor of SUMO conjugation in arabidopsis(F).

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.

Update on sumoylation: defining core components of the plant SUMO conjugation system by phylogenetic comparison.

The conjugation of the small ubiquitin-related modifier, SUMO, to substrate proteins is a reversible and dynamic process, and an important response of plants to environmental challenges. Nevertheless, reliable data have so far been restricted largely to the model plant Arabidopsis thaliana. The increasing availability of genome information for other plant species offers the possibility to identify a core set of indispensable components, and to discover species-specific features of the sumoylation pathway.

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.