Journal of Molecular Biology
Domain Interdependence in the Biosynthetic Assembly of CFTR
Introduction
ATP cassette binding (ABC) transporters, which comprise the largest known family of membrane transport proteins, contain two nucleotide-binding domains (NBDs) and two membrane-spanning domains (MSDs). The former associate to constitute two composite ATP binding sites1., 2., [3] and the latter to form a specific permeation pathway.4 In many eukaryotic ABC proteins, a single polypeptide contains all the domains, which must fold efficiently as or after they are synthesized. At least one human ABC protein, the CFTR chloride channel, assembles inefficiently, with the majority of its nascent chains ubiquitinated and degraded by the proteasome instead of being transported from the endoplasmic reticulum (ER) to the Golgi.5,6 In the case of the most common cystic fibrosis-causing mutation, ΔF508, virtually all chains meet this fate.7 This occurs despite the fact that the 3D structure of isolated NBD1 is apparently unaltered except in a small local surface region around the normal location of Phe508.8 Other recent findings have shown that an interaction between the MSDs is disrupted by the ΔF508 mutation.9 However, it is unknown whether this reflects loss of an interaction of the normal NBD1 surface with an MSD or possibly a more indirect result of disruption of NBD1/NBD2 association, which is believed to be important in CFTR function.10 In support of the first possibility and consistent with the recent report that NBD2 may fold post-translationally,11 we have found now that wild-type CFTR is able to assemble and pass ER quality control in the complete absence of NBD2 and that ΔF508 has the same impact on this truncated protein as on the full-length molecule. As well as furthering understanding of the influence of the ΔF508 mutation, these findings indicate that the structural requirements to satisfy ER quality control and to form a final native structure are distinct. Additionally, in contrast to the independence from NBD2 of the folding of the more N-terminal domains, we confirm the findings of Du et al.11 that achievement of complete NBD2 stability requires the wild-type form of the earlier domains.
Section snippets
CFTR truncated after MSD2 acquires complex oligosaccharide chains
In experiments to investigate the role of NBD2 in CFTR maturation, we initially analyzed a C-terminal truncation terminating at residue 1248 at the beginning of the Walker A motif of that domain (1248X; Figure 1(a)). Because CFTR maturation is quite susceptible to even small perturbations of the structure, we were surprised to observe that the 1248X construct generated a diffuse major band in Western blots more consistent with the presence of complex rather than core N-linked oligosaccharide
Discussion
Several significant conclusions can be drawn from this work. First, the correct inter-domain assembly as well as the local folding of individual CFTR domains is essential to the formation of a stable unit that satisfies ER quality control. Second, this assembly of the wild-type unit requires all CFTR domains from the N terminus to the C-terminal end of MSD2 but, interestingly, not NBD2. Third, the ΔF508 mutation disrupts maturation and ER export of the truncated ΔNBD2 unit just as it does the
CFTR expression
Human CFTR cDNAs coding for full-length and truncated proteins (wild-type and ΔF508) were expressed in BHK21 cells transiently and stably employing pcDNA3 and pNUT plasmid vectors, respectively, by methods described.32 N-glycosylation sites were inserted into the first extracytoplasmic loop (EL1) of C-terminal truncation constructs terminating before the fourth extracytoplasmic loop (EL4), which contains the native glycosylation sites. To do this, DNA fragments coding for a polypeptide similar
Acknowledgements
This research was supported by the NIH and the CFF.
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