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.
