ABCMdb

ABCC7 p.Tyr517Ala

None has been submitted yet.

Publications

PMID: 15673927 [PubMed] Tsigelny I et al: "Identification of molecular determinants that modulate trafficking of DeltaF508 CFTR, the mutant ABC transporter associated with cystic fibrosis."

No. Sentence Comment

172 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 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 In cells transfected with Y517A:R518A ∆F508 CFTR, both intracellular and the cell boundary regions revealed immunofluorescent stain (Fig. 7A). Login to comment
192 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 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 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 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 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 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 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 (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 Immunofluorescent stain at the cell surface (CS) is apparent in the cells expressing wild-type and Y517A:R518A ∆F508 CFTR. Login to comment
219 (B) Western blot of lysates from cells expressing ∆F508 CFTR, wild-type, and Y517A:R518A ∆F508 CFTR. Login to comment
225 Lane 3, Y517A:R518A ∆F508 CFTR. Login to comment

PMID: 16131493 [PubMed] Swiatecka-Urban A et al: "The short apical membrane half-life of rescued {Delta}F508-cystic fibrosis transmembrane conductance regulator (CFTR) results from accelerated endocytosis of {Delta}F508-CFTR in polarized human airway epithelial cells."

No. Sentence Comment

52 To construct the GFP-⌬F508-CFTR Y517A mutant, the GFP-⌬F508-CFTR cDNA sequence in pcDNA3.1 was mutated using the QuikChangeTM XL site-directed mutagenesis kit (Stratagene; La Jolla, CA). Login to comment
54 Transient transfection of the GFP-tagged CFTR cDNAs into parental CFBE41o-cells was performed using LipofectamineTM 2000 according to the manufacturer`s instructions. CFBE41o-cells grown on 40-mm tissue culture plates were incubated for 24 h with the transfection mixture (2 ␮g of cDNA and 4 ␮l of LipofectamineTM 2000 per plate) at 37 °C. Subsequently, cells were cultured in fresh medium at 27 °C for 36 h to increase the expression of GFP-⌬F508-CFTR and GFP-⌬F508-CFTR Y517A in the plasma membrane. Login to comment
64 Parental CFBE41o-cells transiently transfected with either GFP- ⌬F508-CFTR or GFP-⌬F508-CFTR Y517A were grown on plastic tissue culture plates. Login to comment
161 To test this hypothesis, we transiently expressed the GFP-tagged ⌬F508-CFTR or the GFP-⌬F508-CFTR Y517A mutant in parental CFBE41o-cells. Login to comment
164 The apical membrane expression of GFP-⌬F508-CFTR and GFP-⌬F508-CFTR Y517A was similar at steady state (Fig. 7A). Login to comment
165 Furthermore, as illustrated in Figs. 7, B and C, the apical membrane half-life of GFP-⌬F508-CFTR Y517A (1.0 h) did not differ from that of GFP-⌬F508-CFTR (1.1 h). Login to comment
194 FIGURE 7. Summary of experiments performed to determine the effects of the Y517A mutation in ⌬F508-CFTR on the apical membrane expression at steady state and the apical membrane half-life of rescued ⌬F508-CFTR. Login to comment
195 Parental CFBE41o-cells grown on plastic tissue culture plates were transiently transfected with either GFP-⌬F508-CFTR or the GFP-⌬F508-CFTR Y517A mutant. Login to comment
197 A, summary of biotinylation experiments demonstrating that the apical membrane expression of GFP-⌬F508-CFTR and GFP-⌬F508-CFTR Y517A at steady state did not differ. Login to comment
198 B, summary of biotinylation experiments demonstrating that the apical membrane half-life of GFP-⌬F508-CFTR (1.0 h) and GFP-⌬F508-CFTR Y517A (1.1 h) did not differ in CFBE41o-cells. Login to comment
199 Disappearance of GFP-⌬F508-CFTR and GFP-⌬F508-CFTR Y517A from the apical membrane was monitored over time in the presence of 20 ␮g/ml cyclohexamide (CHX) at 37 °C. Login to comment
201 "C,representativeWesternblotsdemonstratingthedisappearance of GFP-⌬F508-CFTR and GFP-⌬F508-CFTR Y517A from the apical membrane over time in CFBE41o-cells. Login to comment