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Intramembrane chaperones : their role in folding membrane proteins

Periodic Reporting for period 1 - MemCHAPS (Intramembrane chaperones : their role in folding membrane proteins)

Reporting period: 2019-02-01 to 2021-01-31

Membrane proteins constitute about 30 % of the eukaryotic proteome and are involved in crucial processes such as transporting molecules across membranes, mediating intracellular trafficking and functioning as signaling receptors. Membrane proteins have diverse topologies ranging from single pass transmembrane proteins to complex structures like the cystic fibrosis transmembrane regulator (CFTR) with 12 transmembrane helices. The underlying mechanism of how multi-spanning membrane proteins are correctly folded and assembled remains unclear. Being embedded in the hydrophobic lipid bilayer, transmembrane proteins have special requirements to fold correctly. For soluble proteins, exposed hydrophobic patches on the surface of a protein are typically indicative of misfolding. The soluble chaperone machinery responsible for maintaining proteostasis in the cell can detect such misfolded regions. The environment of membrane proteins, however, is hydrophobic itself. It is therefore intriguing how the channels and transporters that have exposed charged or polar residues in the lipid bilayer are folded correctly. We hypothesize that the cell must have an intramembrane chaperone machinery in place that is required for folding intramembrane regions of transmembrane proteins, thereby allowing their correct insertion, orientation and hence function. The main goal of this project is to identify intramembrane chaperones involved in folding of membrane proteins.
Misassembled or misfolded transmembrane proteins can have detrimental effects on the functioning of the cell. Destabilizing mutations in transmembrane helices that cause misassembly have been associated with serious diseases like cystic fibrosis and diabetes mellitus, emphasizing that intramembrane quality control is indispensable. Various proteins interacting with the membrane protein during these stages could serve as chaperones to ensure folding to its functional state. Taken together, this makes it imperative to understand the open question of how multi-spanning membrane proteins are chaperoned in the ER membrane
To address the vast field of intramembrane chaperoning, we defined two overall objectives of the project:
1. Identify membrane proteins interacting with ABC transporters in the ER.
2. Determine whether these interactions play a role in folding/assembly of the ABC transporters
In this reporting period we have successfully established the work-flow of the proximity labelling approach required for mass spectrometry. Since the screen was not completed, we initiated the investigation of two prospective intramembrane chaperone candidates namely Bap31 and the endoplasmic reticulum membrane protein complex (EMC). Our results show that both Bap31 and the EMC influence the folding/assembly of CFTR. Significant changes in the folding pattern are observed in the transmembrane region of CFTR, suggesting that both Bap31 and the EMC might directly be involved in the intramembrane chaperoning of CFTR.
Several proteins associated with the intramembrane proteins in the ER could serve as molecular chaperones. However, given the complex topology of intramembrane proteins it can be anticipated that the list of ‘chaperone candidates’ is far from complete. To systematically fill this gap in knowledge, the first goal of this project was to set up a screen in order to identify novel membrane proteins that interact with a model protein of interest as it folds and assembles in the lipid bilayer of the endoplasmic reticulum.
The cystic fibrosis transmembrane conductance regulator (CFTR) was selected as a model protein, as the Braakman lab has a lot of expertise and a vast wealth of reagents and constructs to study its folding dynamics.

The method of choice was proximity-dependent biotin identification (BioID). The principle of this technique is that CFTR is genetically fused to a prokaryotic biotin ligase molecule (BirA). When expressed in HEK293T cells, the proteins in proximity of the fusion ABC transporter are biotinylated. Streptavidin-based biotin affinity capture can then allow to specifically isolate the biotinylated proteins from the cell lysate. The biotinylated proteins purified in this way can be identified using mass-spectrometry (MS) to give a list of interactors. The MS collaboration has been set up with Dr. J. Demmers in the Erasmus Medical Centre of Rotterdam.

Main results achieved:
1. Tagging CFTR at the N-terminal with BioID led to aggregation.
2. CFTR could be tagged with BioID2 at the C-terminus and within the ECL4 loop.
3. BioID2-CFTR folds and assembles similar to untagged wild type CFTR and is stably expressed at the cell surface.
4. Successful optimization of pull down of Biotinylated proteins
5. Proof-of-principle MS test run completed
6. Generation of stable BioID2 cell lines complete
7. Bap31 and the EMC influence the folding/assembly of CFTR. Significant changes in the folding pattern were observed in the transmembrane region of CFTR, suggesting that both Bap31 and the EMC might directly be involved in the intramembrane chaperoning of CFTR.

Exploitation and dissemination of results:
The knowledge generated through this work was disseminated to the scientific community, future scientists and students using different means and channels:
1. Poster presentation at the Dutch Chaperone Day
2. Poster presentation to UU Master students at the MCLS Networking meet
3. Supervision of a Master’s thesis
4. Scientific discussions at the EMBO leadership course: Project management for Scientist
In the course of the project, we broadened our toolbox of CFTR constructs and reagents that are a valuable resource at the Braakman lab. Tagging CFTR at different positions and studying its effects on the expression, folding and transport of CFTR from ER to the plasma membrane has extended our knowledge. This has been advantageous for another project in the Braakman lab that required tagging of CFTR to follow its course in the cell using fluorescence microscopy.
Moreover, the stable cell lines generated are a valuable resource which will continue to be used in the Braakman group.

After extensive optimizations and troubleshooting, the BioID2 screen is now ready for implementation and we expect to identify novel CFTR interactors. The preliminary results obtained with Bap31 and the EMC are promising leads and will be expanded further in the Braakman group. This research aims at answering a fundamental question in cell biology pertaining to how the folding of ~ 30 % of the proteome is assisted under physiological conditions. The findings of this research could have highly relevant implications for industry in terms of the modulating the assembly of CFTR or designing strategies to inhibit the other similar complex ABC transporters like P-gp and BCRP that cause chemotherapeutic drug resistance.