Limited proteolysis as a probe for arrested conformational maturation of ΔF508 CFTR

Abstract

Deletion of phenylalanine 508 (ΔF508) in the cystic fibrosis transmembrane-conductance regulator (CFTR) prevents the otherwise functional protein from reaching the plasma membrane and is the leading cause of cystic fibrosis. Indirect evidence suggests that the mutant protein, ΔF508 CFTR, is misfolded. We address this issue directly, using comparative limited proteolysis of CFTR at steady state and during biosynthesis in the native microsomal environment. Distinct protease susceptibilities suggest that cytosolic domain conformations of wild type and ΔF508 CFTR differ, not only near F508, but globally. Moreover, ΔF508 CFTR proteolytic cleavage patterns were indistinguishable from those of the early folding intermediate of wild type CFTR. The results suggest that the ΔF508 mutation causes the accumulation of a form of the protein that resembles an intermediate in the biogenesis of the wild type CFTR, rather than induces the production of non-native variant.

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References

  1. 1

    Rommens, J.M. et al. Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 245, 1059–1065 (1989).

  2. 2

    Kerem, B. et al. Identification of the cystic fibrosis gene: genetic analysis. Science 245, 1073–1080 (1989).

  3. 3

    Riordan, J.R. et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245, 1066–1073 (1989).

  4. 4

    Riordan, J.R. The cystic fibrosis transmemebrane conductance regulator. Annu. Rev. Physiol. 55, 609–630 (1993).

  5. 5

    Quinton, P.M. Cystic fibrosis: A disease in electrolyte transport. FASEB J. 4, 2709–2717 (1990).

  6. 6

    Boucher, R.C. Human airway ion transport. Am. J. Resp. Crit. Care. Med. 150, 271–281 (1994).

  7. 7

    Welsh, M.J. & E, S.A. Molecular mechanism of CFTR channel dysfunction in cystic fibrosis. Cell 73, 1251–1254 (1993).

  8. 8

    Zielenski, J. & Tsui, L.-C. Cystic fibrosis: genotypic and phenotypic variations. Annu. Rev. Genet. 29, 777–807 (1995).

  9. 9

    Cheng, S.H. et al. Defective intracellular transport and processing of CFTR is the moleuclar basis of most cystic fibrosis. Cell 63, 827–834 (1990).

  10. 10

    Lukacs, G.L. et al. Conformational maturation of CFTR but not its mutant counterpart (ΔF508) occurs in the endoplasmic reticulum and requires ATP. EMBOJ. 13, 6076–6086 (1994).

  11. 11

    Ward, C.L. & Kopito, R.R. Intracellular turnover of cystic fibrosis transmembrane conductance regulator. J. Biol. Chem. 269, 25710–25718 (1994).

  12. 12

    Denning, G.M. et al. Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature sensitive. Nature 350, 761–764 (1992).

  13. 13

    Kartner, N., Augustinas, O., Jensen, T.J., Naismith, A.L. & Riordan, J.R. Mislocalization of ΔF508 CFTR in cystic fibrosis sweat gland. Nature Genet. 1, 321–327 (1992).

  14. 14

    Pind, S., Riordan, J.R. & Williams, D.B. Participation of the endoplasmic reticulum chaperone calnexin (p88, IP90) in the biogenesis of the cystic fibrosis transmembrane conductance regulator. J. Biol. Chem. 269, 12784–12788 (1994).

  15. 15

    Yang, Y., Janich, S., Cohn, J. & Wilson, J.M. The common variant of cystic fibrosis transmembrane conductance regulator is recognized by hsp70 and degraded in a pre-Golgi nonlysosomal compartment. Proc. Natl. Acad. Sci. USA 90, 9480–9484 (1993).

  16. 16

    Teem, J.L. et al. Identification of revertants for the cystic fibrosis ΔF508 mutation using STE6-CFTR chimeras in yeast. Cell 73, 335–346 (1993).

  17. 17

    Thomas, P.J., Shenbagamurthi, P., Sondek, J., Hullihen, J.M. & Pedersen, P.L. The cystic fibrosis transmembrane conductance regulator. J. Biol. Chem. 267, 5727–5730 (1992).

  18. 18

    Qu, B.-H. & Thomas, P.J. Alteration of the cystic fibrosis transmembrane conductance regulator folding pathway. J. Biol. Chem. 271, 7261–7264 (1996).

  19. 19

    Beynon, R.J. & Bond, J.S. Proteolytic enzymes (IRL Press, Oxford; 1989).

  20. 20

    Kuznetsov, G., Chen, L.B. & Nigan, S.K. Several endoplasmic reticulum stress proteins, including ERp72, interact with thyroglobulin during its maturation. J. Biol. Chem. 269, 22990–22995 (1994).

  21. 21

    Huovila, A.P., Eder, A.M. & Fuller, S.D. Hepaptitis B Surface antigen assembles in a post-ER pre-Golgi compartment. J. Cell Biol. 118, 1305–1320 (1992).

  22. 22

    Dill, K.A. & Hue, S.C., From Levinthal to pathways to funnels. Nature Struct. Biol. 4, 10–19 (1997).

  23. 23

    Baker, D. & Agard, D.A. Kinetics versus thermodynamics in protein folding. Biochemistry 33, 7505–7509 (1994).

  24. 24

    Qu, B.-H., Strickland, E.H. & Thomas, P.J. Localization and supression of the kinetic defect in CFTR folding. J. Biol. Chem. 272, 15739–15744 (1997).

  25. 25

    Lukacs, G.L. et al. The ΔF508 mutation decreases the stability of CFTR in the plasma membrane. J. Biol. Chem. 268, 21592–21598 (1993).

  26. 26

    Balch, W.E. & Rothman, J.E. Characterization of protein transport between successive compartments of the Golgi apparatus: asymetric properties of donor and acceptor activities in a cell free system. Arch. Biochem. Biophys. 240, 413–425 (1985).

  27. 27

    Lukacs, G.L., Segal, G., Kartner, N., Grinstein, S. & Zhang, F. Constitutive internalization of CFTR is mediate by clathrin-dependent endocytosis and is regulated by protein phosphorylation. Biochem. J. 328, 353–361 (1997).

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Correspondence to Gergely L. Lukacs.

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Zhang, F., Kartner, N. & Lukacs, G. Limited proteolysis as a probe for arrested conformational maturation of ΔF508 CFTR. Nat Struct Mol Biol 5, 180–183 (1998). https://doi.org/10.1038/nsb0398-180

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