Self-incompatibility in flowering plants prevents inbreeding and promotes outcrossing to generate genetic diversity. In Solanaceae, a multiallelic gene, S-locus F-box (SLF), was previously shown to encode the pollen determinant in self-incompatibility. It was postulated that an SLF allelic product specifically detoxifies its non-self S-ribonucleases (S-RNases), allelic products of the pistil determinant, inside pollen tubes via the ubiquitin-26S-proteasome system, thereby allowing compatible pollinations. However, it remained puzzling how SLF, with much lower allelic sequence diversity than S-RNase, might have the capacity to recognize a large repertoire of non-self S-RNases. We used in vivo functional assays and protein interaction assays to show that in Petunia, at least three types of divergent SLF proteins function as the pollen determinant, each recognizing a subset of non-self S-RNases. Our findings reveal a collaborative non-self recognition system in plants.

Post-translational modification by ubiquitin (Ub) regulates diverse cellular processes. Ubiquitin ligases (E3s) catalyze the final step of Ub transfer from E2 ubiquitin-conjugating enzyme thioesterified with Ub (E2~Ub) to a lysine side chain of substrate. RING-type E3s are the largest family of E3s with approximately 600 members in humans. RING E3s function by recruiting E2~Ub via the RING domain and promote direct transfer of Ub from E2 to the substrate lysine. The extent of substrate ubiquitination depends on the processivity of the RING E3-E2~Ub complex. RING domain activates E2~Ub by stabilizing E2~Ub in the closed conformation such that the thioester bond is optimally oriented for nucleophilic attack by the substrate lysine. Moreover, we showed that non-covalent Ub binding to the "backside" of E2 UbcH5B stimulates the catalytic efficiency of the RING E3-E2~Ub complex by enhancing RING E3's affinity for E2~Ub complex. Recently we have developed small protein molecules that modulate the activity of RING E3s. I will present these results in this meeting

The covalent attachment of SUMO (small ubiquitin-like modifier) to other intracellular proteins affects a broad range of nuclear processes in yeast and animals, including chromatin maintenance, transcription, and transport across the nuclear envelope, as well as protects proteins from ubiquitin addition. Substantial increases in SUMOylated proteins upon various stresses have also implicated this modification in the general stress response. To help understand the role(s) of SUMOylation in plants, we developed a stringent method to isolate SUMO-protein conjugates from Arabidopsis thaliana that exploits a tagged SUMO1 variant that faithfully replaces the wild-type protein. Following purification under denaturing conditions, SUMOylated proteins were identified by tandem mass spectrometry from both nonstressed plants and those exposed to heat and oxidative stress. The list of targets is enriched for factors that direct SUMOylation and for nuclear proteins involved in chromatin remodeling/repair, transcription, RNA metabolism, and protein trafficking. Targets of particular interest include histone H2B, components in the LEUNIG/TOPLESS corepressor complexes, and proteins that control histone acetylation and DNA methylation, which affect genome-wide transcription. SUMO attachment site(s) were identified in a subset of targets, including SUMO1 itself to confirm the assembly of poly-SUMO chains. SUMO1 also becomes conjugated with ubiquitin during heat stress, thus connecting these two posttranslational modifications in plants. Taken together, we propose that SUMOylation represents a rapid and global mechanism for reversibly manipulating plant chromosomal functions, especially during environmental stress. mass spectrometry | SUMO | TOPLESS | chromatin remodeling | stress P osttranslational modification of proteins has emerged as a central regulatory mechanism that underpins a wide range of cellular processes. One essential modification in eukaryotes involves the reversible attachment of the ∼100-amino-acid protein small ubiquitin-like modifier (SUMO) to other intracellular proteins (1, 2). SUMO becomes covalently linked by an isopeptide bond between its C-terminal glycine and the ε-amino group of lysines within the target via an ATP-dependent reaction cascade involving the sequential action of single E1-activating and E2-conjugating enzymes, and a diverse collection of E3-ligase enzymes. SUMO addition can then be reversed by a family of de-SUMOylating enzymes which cleave the isopeptide linkage. Most often a single SUMO moiety is attached to the target but in some cases polymeric SUMO chains are assembled (1, 2). Many attachment sites conform to a consensus ΨKXE sequence, where Ψ is a large hydrophobic amino acid and K represents the lysine that binds SUMO (3).Over a decade of work, primarily in yeast and mammalian cell cultures, shows that SUMOylation controls a broad spectrum of cellular activities through the modification of predominantly, but not exclusively, nuclear proteins. These include roles in gene expression, ma...

Highlights d A new binding site for autophagy adaptors and receptors was discovered on ATG8 d This site engages UIM-like sequences rather than the canonical ATG8-interacting motif d UIM-type autophagy adaptors and receptors can be found in plants, yeast, and humans d UIM-containing UBX domain proteins mediate autophagic clearance of CDC48/p97

Petunia inflata S-locus F-box (Pi SLF) is thought to function as a typical F-box protein in ubiquitin-mediated protein degradation and, along with Skp1, Cullin-1, and Rbx1, could compose an SCF complex mediating the degradation of nonself S-RNase but not self S-RNase. We isolated three P. inflata Skp1s (Pi SK1, -2, and -3), two Cullin-1s (Pi CUL1-C and -G), and an Rbx1 (Pi RBX1) cDNAs and found that Pi CUL1-G did not interact with Pi RBX1 and that none of the three Pi SKs interacted with Pi SLF 2 . We also isolated a RING-HC protein, S-RNase Binding Protein1 (Pi SBP1), almost identical to Petunia hybrida SBP1, which interacts with Pi SLFs, S-RNases, Pi CUL1-G, and an E2 ubiquitin-conjugating enzyme, suggesting that Pi CUL1-G, SBP1, and SLF may be components of a novel E3 ligase complex, with Pi SBP1 playing the roles of Skp1 and Rbx1. S-RNases interact more with nonself Pi SLFs than with self Pi SLFs, and Pi SLFs also interact more with nonself S-RNases than with self S-RNases. Bacterially expressed S 1 -, S 2 -, and S 3 -RNases are degraded by the 26S proteasomal pathway in a cell-free system, albeit not in an S-allele-specific manner. Native glycosylated S 3 -RNase is not degraded to any significant extent; however, deglycosylated S 3 -RNase is degraded as efficiently as the bacterially expressed S-RNases. Finally, S-RNases are ubiquitinated in pollen tube extracts, but whether this is mediated by the Pi SLFcontaining E3 complex is unknown.