Elucidation of ubiquitin-conjugating enzymes that interact with RBR-type ubiquitin ligases using a liquid-liquid phase separation-based method.

RING-between RING (RBR)-type ubiquitin (Ub) ligases (E3s) such as Parkin receive Ub from Ub-conjugating enzymes (E2s) in response to ligase activation. However, the specific E2s that transfer Ub to each RBR-type ligase are largely unknown because of insufficient methods for monitoring their interaction. To address this problem, we have developed a method that detects intracellular interactions between E2s and activated Parkin. Fluorescent homotetramer Azami-Green fused with E2 and oligomeric Ash (Assembly helper) fused with Parkin form a liquid-liquid phase separation (LLPS) in cells only when E2 and Parkin interact. Using this method, we identified multiple E2s interacting with activated Parkin on damaged mitochondria during mitophagy. Combined with in vitro ubiquitination assays and bioinformatics, these findings revealed an underlying consensus sequence for E2 interactions with activated Parkin. Application of this method to other RBR-type E3s including HOIP, HHARI, and TRIAD1 revealed that HOIP forms an LLPS with its substrate NEMO in response to a proinflammatory cytokine and that HHARI and TRIAD1 form a cytosolic LLPS independent of Ub-like protein NEDD8. Since an E2-E3 interaction is a prerequisite for RBR-type E3 activation and subsequent substrate ubiquitination, the method we have established here can be an in-cell tool to elucidate the potentially novel mechanisms involved in RBR-type E3s.

Critical role of mitochondrial ubiquitination and the OPTN-ATG9A axis in mitophagy.

Damaged mitochondria are selectively eliminated in a process called mitophagy. Parkin and PINK1, proteins mutated in Parkinson's disease, amplify ubiquitin signals on damaged mitochondria with the subsequent activation of autophagic machinery. Autophagy adaptors are thought to link ubiquitinated mitochondria and autophagy through ATG8 protein binding. Here, we establish methods for inducing mitophagy by mitochondria-targeted ubiquitin chains and chemical-induced mitochondrial ubiquitination. Using these tools, we reveal that the ubiquitin signal is sufficient for mitophagy and that PINK1 and Parkin are unnecessary for autophagy activation per se. Furthermore, using phase-separated fluorescent foci, we show that the critical autophagy adaptor OPTN forms a complex with ATG9A vesicles. Disruption of OPTN-ATG9A interactions does not induce mitophagy. Therefore, in addition to binding ATG8 proteins, the critical autophagy adaptors also bind the autophagy core units that contribute to the formation of multivalent interactions in the de novo synthesis of autophagosomal membranes near ubiquitinated mitochondria.

Relaxed Chromatin Formation and Weak Suppression of Homologous Pairing by the Testis-Specific Linker Histone H1T.

Linker histones bind to nucleosomes and compact polynucleosomes into a higher-order chromatin configuration. Somatic and germ cell-specific linker histone subtypes have been identified and may have distinct functions. In this study, we reconstituted polynucleosomes containing human histones H1.2 and H1T, as representative somatic and germ cell-specific linker histones, respectively, and found that H1T forms less compacted chromatin, as compared to H1.2. An in vitro homologous pairing assay revealed that H1T weakly inhibited RAD51/RAD54-mediated homologous pairing in chromatin, although the somatic H1 subtypes, H1.0, H1.1, H1.2, H1.3, H1.4, and H1.5, substantially suppressed it. An in vivo recombination assay revealed that H1T overproduction minimally affected the recombination frequency, but significant suppression was observed when H1.2 was overproduced in human cells. These results suggested that the testis-specific linker histone, H1T, possesses a specific function to produce the chromatin architecture required for proper chromosome regulation, such as homologous recombination.