Covalent posttranslational modification of target proteins with ubiquitin and ubiquitin-like proteins regulates many important cellular processes. However, the molecular mechanisms by which these proteins are activated and conjugated to substrates has yet to be fully understood. NMR studies have shown that the ubiquitin-like proteins SUMO-1, -2, and -3 interact with the same N-terminal region of the E2 conjugating enzyme Ubc9 with similar affinities. This is correlated to their almost identical utilization by Ubc9 in the SUMO conjugation pathway. To investigate the functional significance of this interaction, site-directed mutagenesis was used to alter residues in the SUMO binding surface of Ubc9, and the effect of the amino acid substitutions on binding and conjugation to SUMO-1 and target protein RanGAP1 was investigated by isothermal titration calorimetry and biochemical analysis. R13A/K14A and R17A/K18A mutations in Ubc9 disrupted the interaction with SUMO-1 but did not completely abolish the interaction with E1. While these Ubc9 mutants displayed a significantly reduced efficiency in the transfer of SUMO-1 from E1 to E2, their ability to recognize substrate and transfer SUMO-1 from E2 to the target protein was unaffected. These results suggest that the noncovalent binding site of SUMO-1 on Ubc9, although distant from the active site, is important for the transfer of SUMO-1 from the E1 to the E2. The conservation of E2 enzymes across the ubiquitin and ubiquitin-like protein pathways indicates that analogous N-terminal sites of E2 enzymes are likely to have similar roles in general.
To control gene expression by directly responding to hormone concentrations, both animal and plant cells have exploited comparable mechanisms to sense small-molecule hormones in nucleus. Whether nuclear entry of these hormones is actively transported or passively diffused, as conventionally postulated, through the nuclear pore complex, remains enigmatic. Here, we identified and characterized a jasmonate transporter in Arabidopsis thaliana, AtJAT1/AtABCG16, which exhibits an unexpected dual localization at the nuclear envelope and plasma membrane. We show that AtJAT1/AtABCG16 controls the cytoplasmic and nuclear partition of jasmonate phytohormones by mediating both cellular efflux of jasmonic acid (JA) and nuclear influx of jasmonoyl-isoleucine (JA-Ile), and is essential for maintaining a critical nuclear JA-Ile concentration to activate JA signaling. These results illustrate that transporter-mediated nuclear entry of small hormone molecules is a new mechanism to regulate nuclear hormone signaling. Our findings provide an avenue to develop pharmaceutical agents targeting the nuclear entry of small molecules.
Summary• Seed dormancy controls germination and plays a crucial role in the life cycle of plants. Chromatin modifications are involved in the regulation of seed dormancy; however, little is known about the underlying mechanism.• KYP ⁄ SUVH4 is required for histone H3 lysine 9 dimethylation. Mutations in this gene cause increased seed dormancy. KYP ⁄ SUVH4-overexpressing Arabidopsis plants show decreased dormancy. KYP ⁄ SUVH4 expression is regulated by abscisic acid (ABA) and gibberellins (GA). The sensitivity of seed germination to ABA and paclobutrazol (PAC) is enhanced slightly in kryptonite-2 (kyp-2) and suvh4-2 ⁄ suvh5 mutants, but weakened in KYP ⁄ SUVH4-overexpressing plants.• In the kyp-2 mutant, several dormancy-related genes, including DOG1 and ABI3, show increased expression levels, in agreement with a negative role for KYP ⁄ SUVH4 in gene transcription.• Genetic analysis showed that DOG1 and HUB1 are epistatic to KYP ⁄ SUVH4, suggesting that these genes regulate seed dormancy in the same genetic pathway.
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