ABCMdb

PMID: 15673927

Tsigelny I, Hotchko M, Yuan JX, Keller SH
Identification of molecular determinants that modulate trafficking of DeltaF508 CFTR, the mutant ABC transporter associated with cystic fibrosis.
Cell Biochem Biophys. 2005;42(1):41-53., [PubMed]

Sentences

No. Mutations Sentence Comment

172 ABCC7 p.Tyr517Ala ABCC7 p.Tyr517Ala ABCC7 p.Arg518Ala The putative basic amino acid and tyrosine-based trafficking signals were altered by site-directed mutagenesis to eliminate the canonical trafficking signal sequences at the 516-520 site by changing the Tyr-517 and Arg-518 residues to Ala (this variant designated Y517A:R518A ∆F508 CFTR). Login to comment 190 ABCC7 p.Tyr517Ala ABCC7 p.Tyr517Ala Of the modifications examined, only the double mutation Y517A:R518A in the ∆F508 CFTR template (designated Y517A:R518A ∆F508 CFTR) appeared to enhance the cell surface expression (Fig. 7A). Login to comment 191 ABCC7 p.Tyr517Ala In cells transfected with Y517A:R518A ∆F508 CFTR, both intracellular and the cell boundary regions revealed immunofluorescent stain (Fig. 7A). Login to comment 192 ABCC7 p.Tyr517Ala Fluorescent emission at the cell boundary is consistent with the interpretation that Y517A:R518A ∆F508 CFTR was delivered to the cell surface. Login to comment 197 ABCC7 p.Tyr517Ala Because expression of ∆F508 CFTR and single alterations in the ∆F508 CFTR template did not correspond with fluorescent emission at the cell periphery, whereas expression of wild-type CFTR and Y517A:R518A ∆F508 CFTR was reflected in detection of fluorescent emission at the cell boundary, we conclude that elimination of the specific tyrosine-based and basic amino acid signals elevated delivery of ∆F508 CFTR to the plasma membrane. Login to comment 203 ABCC7 p.Tyr517Ala Western Blot Analysis We expressed wild-type CFTR, ∆F508 CFTR, and Y517A:R518A ∆F508 CFTR in HEK cells using equivalent amount of plasmids and cells, and processed the cell lysates identically for Western blotting. Login to comment 205 ABCC7 p.Tyr517Ala A B-band was associated with ∆F508 CFTR (Fig. 7B, lane 1) and Y517A:R518A ∆F508 CFTR (Fig. 7B, lane 3). Login to comment 207 ABCC7 p.Tyr517Ala Although ∆F508 CFTR lacked a C-band (Fig. 7B, lane 1), Y517A:R518A ∆F508 CFTR revealed a C-band (Fig. 7B, lane 3), albeit significantly fainter than the C-band associated with wild-type CFTR (Fig. 7B, lane 2). Login to comment 208 ABCC7 p.Tyr517Ala The appearance of the C-band associated with Y517A:R518A ∆F508 CFTR indicated that the sequence alteration elevated the upstream trafficking of ∆F508 CFTR. Login to comment 209 ABCC7 p.Tyr517Ala Since the Y517A:R518A alterations did not quantitatively restore trafficking levels comparable to wild-type CFTR (Fig. 7B, compare lanes and 3), addi- 50 Tsigelny et al. Cell Biochemistry and Biophysics Volume 42, 2005 Fig. 6. Login to comment 215 ABCC7 p.Tyr517Ala (A) Confocal-immunofluorescent images of ∆F508 CFTR, wild-type, and Y517A:R518A ∆F508 CFTR expressed transiently in human embryonic kidney cells. Login to comment 217 ABCC7 p.Tyr517Ala Immunofluorescent stain at the cell surface (CS) is apparent in the cells expressing wild-type and Y517A:R518A ∆F508 CFTR. Login to comment 219 ABCC7 p.Tyr517Ala (B) Western blot of lysates from cells expressing ∆F508 CFTR, wild-type, and Y517A:R518A ∆F508 CFTR. Login to comment 225 ABCC7 p.Tyr517Ala Lane 3, Y517A:R518A ∆F508 CFTR. Login to comment