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Mechanisms of protein translocation in ER-associated protein degradation and the related protein import into the apicoplast of Plasmodium falciparum

Periodic Reporting for period 3 - ERAD_SELMA (Mechanisms of protein translocation in ER-associated protein degradation and the related protein import into the apicoplast of Plasmodium falciparum)

Reporting period: 2019-03-01 to 2020-08-31

Eukaryotic 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 fulfil their functions in other compartments, the cell requires molecular machineries that transport proteins across membranes. Such a process is 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 and 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 the membrane of the ER. This process is called ERAD (Figure A). The first aim of our project is the molecular characterization of proteins involved in ERAD.

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, that imports almost all of its proteins from the cytoplasm. Recent reports suggest that an ERAD-like machinery, called SELMA, is catalysing the import of proteins into the apicoplast. We aim to characterize SELMA in molecular detail. We envision 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 in 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. E.g. we can answer the problem of which components are minimally required to bring about a particular cellular activity such as translocation of a protein across membranes.

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, a disease that causes one million deaths a year by conservative estimates.
In order to understand the molecular mechanism of ERAD in greater detail, we have developed experimental systems that allow us to recapitulate the translocation reaction using purified components (Figure B). Because the ERAD process is conserved in all nucleated cell, we use the simple model organism Saccharomyces cerevisiae (Baker’s yeast) for our experiments. In the reconstituted system, we can deliver a misfolded protein to the luminal face of the translocation machinery in a controlled fashion. We have devised assays with which we can quantitatively measure translocation across a membrane. Using the ubiquitin ligase Doa10, we found that the membrane embedded domain of this protein facilitates the extraction of a ubiquitinated substrate from a membrane. In this process, folded domains residing on the luminal face of the membrane are unfolded by the cytoplasmic ATPase Cdc48.

To characterize the components of SELMA, we have generated nanobodies to purify protein complexes from parasites (collaboration with Prof. D. Görlich, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany). Nanobodies are small antibodies first generated in alpacas that can then be produced easily in bacteria. Because of the readily available techniques to express proteins in bacteria at low cost, in large amounts and modified in ways that aid in their purification, nanobodies are promising and novel tools in the purification of protein complexes from organisms that are otherwise difficult to manipulate genetically. We are currently in the process of characterizing the protein complexes that we purified from parasites and the related non-parasitic organism Chromera velia.
Compared to other translocation reactions, e.g. translocation of proteins into the ER by membrane-bound ribosomes, investigation of ERAD is complicated by the fact that both faces of the membrane need to be accessible to the experimenter. The system that we have developed, in which ERAD-substrate and ERAD-machinery are first reconstituted into different artificial vesicle populations to be subsequently fused, overcomes this challenge and should allow us to understand ERAD in more detail.

The use of Chromera velia as a model organism for studying the apicoplast of parasites like P. falciparum opens the possibility to do biochemical experiments that have hitherto been impossible due to the very limited amounts of cells that can be cultured.