Co-Translational Folding of the First Transmembrane Domain of ABC-Transporter CFTR is Supported by Assembly with the First Cytosolic Domain
Kleizen, Bertrand; van Willigen, Marcel; Mijnders, Marjolein; Peters, Floor; Grudniewska, Magda; Hillenaar, Tamara; Thomas, Ann; Kooijman, Laurens; Peters, Kathryn W.; Frizzell, Raymond; van der Sluijs, Peter; Braakman, Ineke
(2021) Journal of Molecular Biology, volume 433, issue 13, pp. 1 - 24
(Article)
Abstract
ABC transporters transport a wealth of molecules across membranes and consist of transmembrane and cytosolic domains. Their activity cycle involves a tightly regulated and concerted domain choreography. Regulation is driven by the cytosolic domains and function by the transmembrane domains. Folding of these polytopic multidomain proteins to their functional state
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is a challenge for cells, which is mitigated by co-translational and sequential events. We here reveal the first stages of co-translational domain folding and assembly of CFTR, the ABC transporter defective in the most abundant rare inherited disease cystic fibrosis. We have combined biosynthetic radiolabeling with protease-susceptibility assays and domain-specific antibodies. The most N-terminal domain, TMD1 (transmembrane domain 1), folds both its hydrophobic and soluble helices during translation: the transmembrane helices pack tightly and the cytosolic N- and C-termini assemble with the first cytosolic helical loop ICL1, leaving only ICL2 exposed. This N-C-ICL1 assembly is strengthened by two independent events: (i) assembly of ICL1 with the N-terminal subdomain of the next domain, cytosolic NBD1 (nucleotide-binding domain 1); and (ii) in the presence of corrector drug VX-809, which rescues cell-surface expression of a range of disease-causing CFTR mutants. Both lead to increased shielding of the CFTR N-terminus, and their additivity implies different modes of action. Early assembly of NBD1 and TMD1 is essential for CFTR folding and positions both domains for the required assembly with TMD2. Altogether, we have gained insights into this first, nucleating, VX-809-enhanced domain-assembly event during and immediately after CFTR translation, involving structures conserved in type-I ABC exporters.
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Keywords: co-translational, domain assembly, helical packing, multispanning membrane proteins, protein folding
ISSN: 0022-2836
Publisher: Academic Press Inc.
Note: Funding Information: We thank Dr. Eric Sorscher (Emory University, Atlanta GA, USA) for the 2.3-5 antibody, Dr. Priyanka Sahasrabudhe (Cellular Protein Chemistry, Utrecht University) for designing and preparing the E1-22 antibody, the Cystic Fibrosis Foundation (CFF) for C4 and C18, Dr. Neil Bulleid (University of Glasgow, UK) for the HT1080 fibrosarcoma cells, and all members of the Braakman-Van der Sluijs lab for effective discussions. We thank Dr. Joseline Houwman for preparing figures and editing and Dr. Hanneke Merkens-Hoelen for Figure 1A. Drs Fredrick van Goor and Jeffrey H. Stack at Vertex Pharmaceuticals are thanked for inspiring discussions and initial funding of the project. This work was funded by CFF (BRAAKM08XX0, BRAAKM14XX0), the Dutch Research Council (NWO 731.016.403, NWO 731.017.420, and NWO 022.004.019; MM), the Netherlands Organization for Health Research and Development (Zon-Mw TOP grant 40-00812-98-14103), by the Netherlands Cystic Fibrosis Foundation (NCFS, HIT-CF grant). The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding Information: We thank Dr. Eric Sorscher (Emory University, Atlanta GA, USA) for the 2.3-5 antibody, Dr. Priyanka Sahasrabudhe (Cellular Protein Chemistry, Utrecht University) for designing and preparing the E1-22 antibody, the Cystic Fibrosis Foundation (CFF) for C4 and C18, Dr. Neil Bulleid (University of Glasgow, UK) for the HT1080 fibrosarcoma cells, and all members of the Braakman-Van der Sluijs lab for effective discussions. We thank Dr. Joseline Houwman for preparing figures and editing and Dr. Hanneke Merkens-Hoelen for Figure 1 A. Drs Fredrick van Goor and Jeffrey H. Stack at Vertex Pharmaceuticals are thanked for inspiring discussions and initial funding of the project. This work was funded by CFF (BRAAKM08XX0, BRAAKM14XX0), the Dutch Research Council (NWO 731.016.403, NWO 731.017.420, and NWO 022.004.019; MM), the Netherlands Organization for Health Research and Development (Zon-Mw TOP grant 40-00812-98-14103), by the Netherlands Cystic Fibrosis Foundation (NCFS, HIT-CF grant). Publisher Copyright: © 2021 The Author(s)
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