Our first main aim was to understand the translocation processes that are involved in ERAD on a molecular level. We studied two ubiquitin ligase complexes involved in this process, which are named the Hrd1 complex and the Doa10 complex. Because ERAD is conserved in all nucleated cell, we use the simple model organism Saccharomyces cerevisiae (Baker’s yeast) for our experiments. We have devised assays with purified proteins and model membranes with which we can quantitatively measure translocation across a membrane or extraction of membrane proteins from a membrane. To avoid non-native interactions of membrane proteins in detergent during the reconstitution procedure we developed a system that employs SNARE proteins to fuse two liposome populations containing either the membrane protein substrate or Doa10. This system overcomes the only seemingly trivial problem of how to mix membrane proteins (Figure 1).
Using this reconstitution system, we found that Doa10 facilitates the extraction of a ubiquitinated substrate from a membrane. In this process, a folded domain residing on the luminal face of the membrane is unfolded by the cytoplasmic ATPase Cdc48. How Doa10 mediates this process precisely is a matter of ongoing research. Answering this question require structural insight into Doa10. The ultimate aim is to capture Doa10 in the process of translocating a membrane protein substrate. This work has been published in the journal elife in 2020 (Schmidt et al., 2020, life) and has been presented to international audiences at the EMBO workshops Ubiquitin (Dubrovnik, Croatia, 2019), and "The endoplasmic reticulum" (Lucca, Italy, 2018, where this work was awarded with a poster prize), and the online conference "Ubiquitin & Friends" (Vienna, Austria, 2020).
To address the question how Hrd1 mediates complete translocation of a soluble substrate across the ER membrane, we also employed reconstituted systems with purified Hrd1 in different model membranes. We showed that Hrd1 alone can form a pore. Opening of this pore is triggered by site-specific autoubiquitination of Hrd1 and interaction with misfolded proteins. De-ubiquitination closes the pore. Furthermore, autoubiquitination creates a binding site for the retrotranslocating polypeptide on the cytoplasmic side of Hrd1. We propose that this interaction provides the initial driving force for translocation until attachment of a polyubiquitin handle onto the substrate and Cdc48 bring about irreversible extraction of the substrate into the aqueous environment (Figure 2). Our work established that autoubiquitination of Hrd1 leads to yet undefined structural changes in Hrd1 that allow for retrotranslocation. However, it remains unclear if and how autoubiquitination regulates retrotranslocation in the context of the full Hrd1 complex. Current research in our laboratory therefore aims to understand the function of Hrd1 in the presence of its binding partners. This work has been published in the journal Nature Cell Biology in 2020 (Vasic et al., 2020, NCB) and has been presented to an international audiences at the EMBO workshop Ubiquitin (Dubrovnik, Croatia, 2019).
To characterize the components of SELMA, we have generated nanobodies to purify protein complexes from parasites. Nanobodies are small antibodies first generated in alpacas that can then be produced in bacteria. Using these nanobodies we isolated protein complexes potentially involved in protein import into a membrane-bound organelle homologous to the apicoplast of parasites. For this purpose we used the alga Chromera velia, a distant relative of the malaria parasite P. falciparum. Some of the proteins that we identified are similar to proteins involved in the translocation process in ERAD, e.g. the derlins. Characterization of these protein complexes is ongoing and the results have not been published.
In addition to the projects described above, we contributed to work that provided the first insights into the molecular structure of the Hrd1 complex (Schoebel et al., 2017, Nature). Furthermore, we contributed to work on the Asi ubiquitin ligase complex of the inner nuclear membrane that showed how this complex helps to maintain the specific proteome of the inner nuclear membrane (Natarajan et al., 2020, Molecular Cell).
