Periodic Reporting for period 4 - ERAD_SELMA (Mechanisms of protein translocation in ER-associated protein degradation and the related protein import into the apicoplast of Plasmodium falciparum) Reporting period: 2020-09-01 to 2021-05-31 Summary of the context and overall objectives of the project Cells contain numerous organelles that are enclosed by membranes that create diffusional barriers. Because most proteins are produced in the cytoplasm of the cell but fulfill their functions in other compartments, the cell requires molecular machineries that transport proteins across membranes, a process called translocation. A particular intriguing translocation reaction occurs at the membrane of the endoplasmic reticulum (ER). In the ER, proteins that are secreted by the cell mature into their folded state and assemble into larger protein complexes. This process is error-prone. Pathways have evolved that clear the cell of misfolded proteins and unassembled subunits of protein complexes. While misfolding occurs in the membrane of the ER or its lumen, degradation is achieved in the cytoplasm by a proteolytic enzyme, the proteasome. Therefore, misfolded proteins need to be translocated across or extracted from the membrane of the ER. This process is called ERAD. The first aim of our project was the molecular characterization of proteins involved in ERAD and an understanding of their mechanism of action. The ERAD machinery appears to have been repurposed in certain parasites, e.g. Plasmodium falciparum, that causes malaria in humans. These parasites contain a plastid-like organelle, called the apicoplast, which imports almost all of its proteins from the cytoplasm. Recent reports suggest that an ERAD-like machinery, called SELMA, is catalyzing the import of proteins into the apicoplast. We aimed to characterize SELMA in molecular detail. We envisioned that comparing the two processes, ERAD and SELMA, on a molecular level will yield important insight into the molecular mechanism by which they function.Apart from working with intact cells, we follow a unique approach to understand the mechanisms of ERAD and SELMA. Following a phrase coined by the Nobel laureate Richard Feynman (“What I cannot create, I do not understand”) we are attempting to re-build the ERAD system using purified components. Such an approach allows us to answer questions that are often difficult to address in the rather complex context of an intact cell.Protein misfolding is associated with many diseases in humans, particularly neurodegenerative diseases in which misfolded proteins accumulate and form aggregates. We hope that characterizing the basic mechanisms through which misfolded proteins are normally discarded will help to better understand pathological conditions. Moreover, we hope that a better understanding of the cell biology of parasites such as P. falciparum will aid in the fight against malaria. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far 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). Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) In Schmidt et al. (2020, elife) we isolated an enzymatic activity in the ubiquitin ligase Doa10: It acts as a membrane protein dislocase that facilitates the removal of a membrane protein from the lipid membrane. To our knowledge this is the first direct demonstration of such an activity. Since similar reaction occur in other organelles, we believe that this finding will have significant impact on future approaches to this question.