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Background: Diazaborine is the only known inhibitor of eukaryotic ribosome biogenesis, but its target is unknown. Results: Diazaborine binds the AAA-ATPase Drg1 and inhibits its ATP hydrolysis, thereby blocking release of Rlp24 from pre-60S particles. Conclusion: Diazaborine blocks ribosome biogenesis by inhibiting the physiological activity of Drg1. Significance: Our results highlight the potential of the ribosome biogenesis pathway as target for novel inhibitors.

During endoplasmic-reticulum-associated protein degradation (ERAD), misfolded proteins are polyubiquitinated, extracted from the ER membrane and degraded by the proteasome 1-4. In a process called retrotranslocation, misfolded luminal proteins first need to traverse the ER membrane before ubiquitination can occur in the cytosol. It was suggested that the membrane-embedded ubiquitin ligase Hrd1 forms a retrotranslocation pore regulated by cycles of autoand deubiquitination 5-8. However, the mechanism by which auto-ubiquitination affects Hrd1 and allows polypeptides to cross the membrane and whether Hrd1 forms a membranespanning pore remained unknown. Here, using purified Hrd1 incorporated into different model membranes, we show that Hrd1 auto-ubiquitination leads to the opening of a pore. Substrate binding increases the pore size and its activity, whereas deubiquitination closes the pore and renders it unresponsive to substrate. We identify two binding sites for misfolded proteins in Hrd1, a low-affinity luminal site and a high-affinity cytoplasmic site formed following auto-ubiquitination of specific lysine residues in Hrd1's RING domain. We propose that the affinity difference between the luminal and cytoplasmic binding sites provides the initial driving force for substrate movement through Hrd1. In Saccharomyces cerevisiae, ERAD of luminal proteins (ERAD-L) requires the Hrd1 complex, which is composed of the ubiquitin ligase Hrd1, three other membrane proteins (Hrd3, Usa1 and Der1) and the lumen-soluble Yos9 (refs. 9-16). Ubiquitination depends on the ubiquitin-conjugating enzyme Ubc7 and its cofactor Cue1 (refs. 17-19) and recruits the Cdc48 complex, which is composed of the ATPase Cdc48 and its cofactors Npl4, Ufd1 and Ubx2. This complex catalyses the extraction of substrates from the ER membrane in an ATP-dependent manner 6,20-25. Overexpression of Hrd1 obviates the need for the other components of the Hrd1 complex 5,26. In further support of a crucial role of Hrd1 in retrotranslocation, reconstitution experiments with purified Hrd1 showed that a membrane-anchored version of a misfolded mutant of carboxypeptidase Y (CPY*) 27 is translocated across the membrane when specific lysine residues of Hrd1 are available for auto-ubiquitination 7. A cryo-electron microscopy structure of Hrd1 in complex with Hrd3 revealed a hydrophilic cavity on the cytoplasmic side of Hrd1 and it was speculated that it represents a closed state of the retrotranslocon 28. We hypothesized that retrotranslocation-competent Hrd1 forms a water-filled channel under appropriate conditions and is thus suitable for electrophysiological characterization.

At physiological pH‐values CO2 and HCO−3are the dominant inorganic carbon species and the interconversion between both is catalyzed by carbonic anhydrase (EC 4.2.1.1). This enzyme is widely distributed among photosynthetic organisms. In the first part of the review, the similarities and the differences of carbonic anhydrases from plants and animals are briefly described. In the second part recent advances in molecular biology to understand the structure of carbonic anhydrase from higher terrestrial plants as well as its involvement in photosynthetic CO2 fixation are summarized. Lastly, the review deals with the presence of carbonic anhydrase in aquatic organisms including cyanobacteria, microalgae, macroalgae and angiosperms. Evidence for the presence of extracellular and intracellular isozymes in these organisms are discussed. The properties and function(s) of carbonic anhydrase during the operation of the inorganic carbon concentrating mechanism are also described.