Cotranslational Folding of CFTR
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The life of the cystic fibrosis transmembrane conductance regulator (CFTR) protein in the cell is dictated by its biogenesis, cellular trafficking, regulated function, and destruction. Cystic fibrosis (CF) is the direct result of perturbations in these processes. Treatment of CF mandates the understanding of the molecular events leading to CFTR loss-of-function. The mechanisms by which different mutations throughout the CFTR protein result in misfolding are unclear. The correction of these processes and ultimately the treatment of CF require elucidation of these mechanisms. In this report, CF-causing mutations in the first transmembrane (TM) spanning domain (TMD1) that result in CFTR misfolding are examined. First, the G85E and G91R mutations in TM1 are shown to have different molecular pathologies. For G85E, TM1 is destabilized in the membrane by the ionizable side chain, which correlates with temperature insensitive ER accumulation. By contrast, G91R does not destabilize TM1, which correlates with temperature sensitive ER accumulation. Both mutants were then identified to perturb TMD1 in a manner recognizable by the cell. Finally, consistent with propagation of these defects, all multidomain CFTR constructs were recognized and degraded in the cell. Other mutations in the interdomain interface between TMD1 and the cytosolic nucleotide binding domains (NBDs) did not perturb TMD1, but affected multidomain constructs containing four domains, which can traffic from the ER. Notably, the interface mutants that change a hydrophobic residue to a basic residue increased levels of early multidomain constructs, suggesting a tradeoff between transient stability and later formation of interdomain interactions. The major cellular monitoring of most mutants occurs after TMD2 is present. In most current models, CF-causing mutations like ?F508 are shown to perturb interdomain interactions before TMD2 is produced. However, evidence presented here suggests these interactions are not important until after TMD2 production. The comparison of TM1 mutants and other mutants supports specific domain interactions in the hierarchical folding model. Taken together, the data herein generate a model of CFTR folding that begins with TMD1. Interdomain interactions then become important in a four domain construct, and the final domain confers additional stability and increases cellular trafficking. Multidomain misfolding clearly plays a role in the molecular pathology of CF, thus, a more detailed understanding of this process as globally outlined above is required to generate novel ways to rescue mutant CFTR.