The endoplasmic reticulum (ER)-resident intramembrane rhomboid protease RHBDL4 generates metastable protein fragments and together with the ER-associated degradation (ERAD) machinery provides a clearance mechanism for aberrant and surplus proteins. However, the endogenous substrate spectrum and with that the role of RHBDL4 in physiological ERAD is mainly unknown. Here, we use a substrate trapping approach in combination with quantitative proteomics to identify physiological RHBDL4 substrates. This revealed oligosaccharyltransferase (OST) complex subunits such as the catalytic active subunit STT3A as substrates for the RHBDL4dependent ERAD pathway. RHBDL4-catalysed cleavage inactivates OST subunits by triggering dislocation into the cytoplasm and subsequent proteasomal degradation. RHBDL4 thereby controls the abundance and activity of OST, suggesting a novel link between the ERAD machinery and glycosylation tuning.

In this communication, we report the adaptation of the CRISPR-Cas9 technology in Ustilago trichophora prototrophic wild-type isolate obtained from its natural host Echinochloa crus-galli. The established CRISPR vector and method enable a rapid and marker-free introduction of Cas9-induced non-homologous end-joining (NHEJ) dependent mutation at the targeted gene. Moreover, the method allows a specific modification of the chromosomal DNA sequence by Cas9-induced homologous recombination using short DNA repair templates. The results demonstrate the applicability of the CRISPR-Cas9 technology in U. trichophora for both gene knock-out by the NHEJ pathway and specific gene modification by templated genome editing, paving the way for rapid metabolic engineering of this Ustilago species for industrial applications.

Schüler:innen-Mentoring-Programme sollten einen festen Bestandteil im Mobbingpräventionskonzept von Schulen bilden, da sie der hohen Dunkelziffer von Mobbing entgegenwirken können, indem zusätzliche Anlaufstellen geschaffen werden. Außerdem werden die sozialen Kompetenzen und das konstruktive Konfliktverhalten der Schüler:innen gefördert. Aufgrund verschiedener Hürden bei der Umsetzung bedarf es einer überlegten und konsequenten Implementierung durch schulinterne und externe Expert:innen.

The Endoplasmic Reticulum (ER)-resident intramembrane rhomboid protease RHBDL4 generates metastable protein fragments and together with the ER-associated degradation (ERAD) machinery provides a clearance mechanism for aberrant and surplus proteins.However, the endogenous substrate spectrum and with that the role of RHBDL4 in physiological ERAD is mainly unknown. Here, we use quantitative proteomics to identify physiological RHBDL4 substrates. This revealed oligosacharyltransferase (OST) complex subunits such as the catalytic active subunit STT3A as substrates for the RHBDL4dependent ERAD pathway. RHBDL4-catalyzed cleavage inactivates OST subunits by triggering dislocation into the cytoplasm and subsequent proteasomal degradation. RHBDL4catalyzed cleavage is enhanced by positive charged transmembrane residues, which are recognized by a putative negative-charged docking site at the rhomboid active site gate.RHBDL4 controls the abundance and activity of OST, suggesting a novel link between the ERAD machinery and glycosylation tuning in the ER. AbbreviationsERAD, ER-associated degradation; OST, oligosacharyltransferase; TM, transmembrane; UIM, ubiquitin-interacting motif.