ABCMdb

ABCC7 p.Asn894Asp

Predicted by SNAP2:	A: N (53%), C: D (66%), D: D (59%), E: D (53%), F: D (75%), G: D (53%), H: D (63%), I: D (66%), K: D (53%), L: D (59%), M: D (66%), P: D (66%), Q: N (61%), R: D (53%), S: N (61%), T: N (66%), V: N (53%), W: D (80%), Y: D (59%),
Predicted by PROVEAN:	A: N, C: D, D: N, E: N, F: N, G: N, H: N, I: D, K: N, L: D, M: N, P: N, Q: N, R: N, S: N, T: N, V: D, W: N, Y: N,

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[hide] Role of N-linked oligosaccharides in the biosynthe... J Cell Sci. 2008 Sep 1;121(Pt 17):2814-23. Epub 2008 Aug 5. Chang XB, Mengos A, Hou YX, Cui L, Jensen TJ, Aleksandrov A, Riordan JR, Gentzsch M
Role of N-linked oligosaccharides in the biosynthetic processing of the cystic fibrosis membrane conductance regulator.
J Cell Sci. 2008 Sep 1;121(Pt 17):2814-23. Epub 2008 Aug 5., 2008-09-01 [PMID:18682497]

Abstract [show]
The epithelial chloride channel CFTR is a glycoprotein that is modified by two N-linked oligosaccharides. The most common mutant CFTR protein in patients with cystic fibrosis, DeltaF508, is misfolded and retained by ER quality control. As oligosaccharide moieties of glycoproteins are known to mediate interactions with ER lectin chaperones, we investigated the role of N-linked glycosylation in the processing of wild-type and DeltaF508 CFTR. We found that N-glycosylation and ER lectin interactions are not major determinants of trafficking of wild-type and DeltaF508 from the ER to the plasma membrane. Unglycosylated CFTR, generated by removal of glycosylation sites or treatment of cells with the N-glycosylation inhibitor tunicamycin, did not bind calnexin, but did traffic to the cell surface and exhibited chloride channel activity. Most importantly, unglycosylated DeltaF508 CFTR still could not escape quality control in the early secretory pathway and remained associated with the ER. However, the absence of N-linked oligosaccharides did reduce the stability of wild-type CFTR, causing significantly more-rapid turnover in post-ER compartments. Surprisingly, the individual N-linked carbohydrates do not play equivalent roles and modulate the fate of the wild-type protein in different ways in its early biosynthetic pathway.

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20 Mutation of native N-linked glycosylation sites (N894D/N900D) and treatment of cells with tunicamycin resulted in expression of unglycosylated CFTR (Fig. 2A). Login to comment
25 Inhibition of interactions with ER lectin chaperones does not affect localization of wild-type or ΔF508 CFTR To analyze whether the N-linked oligosaccharides are involved in retention of ΔF508 CFTR at the ER, we expressed ΔF508 with mutations at both N-glycosylation sites (ΔF508 N894D/N900D) (Fig. 3A). Login to comment
26 Similar to non-glycosylated wild-type CFTR, ΔF508 N894D/N900D did not interact with calnexin (Fig. 3B). Login to comment
28 At physiological temperature (37°C), ΔF508 localized only to the ER regardless of whether or not it was glycosylated (ΔF508 or ΔF508 N894D/N900D), as observed by immunolocalization of permeabilized cells (Fig. 3C). Login to comment
29 Staining of the surface CFTR of intact cells with an antibody recognizing an external epitope confirmed that unglycosylated ΔF508 N894D/N900D was not present at the plasma membrane (Fig. 3D). Login to comment
31 When cells were incubated at 27°C, both glycosylated ΔF508 and unglycosylated ΔF508 N894D/N900D could be detected at the cell surface (Fig. 3D). Login to comment
34 Cells expressing unglycosylated CFTR N894D/N900D showed a robust chloride efflux response to cAMP-elevating stimuli, whereas ΔF508 N894D/N900D did not display any activity, reflecting the absence of the channel protein at the plasma membrane. Login to comment
36 At 37°C, the wild-type protein was observed primarily in its native location in the apical membrane regardless of its glycosylation state, whereas neither glycosylated ΔF508 nor unglycosylated ΔF508 N894D/N900D CFTR was able to reach the apical membrane and was instead localized intracellulary (Fig. 3F). Login to comment
50 The single-chain N894D species primarily formed a single band of mobility intermediate between the unglycosylated and wild-type Fig. 1. Login to comment
56 However, the other single-site variant, N900D, generated two strong bands, one with a similar mobility to that of N894D, probably representing the presence of the other single chain, and another more rapidly migrating band, most likely representative of a core rather than complex oligosaccharide chain at this site. Login to comment
57 The identity of the core-glycosylated forms of N894D and N900D was confirmed by digestion with endoglycosidase H, which selectively deglycosylates unprocessed core-glycosylated proteins and thus converted CFTR to a band with the same mobility as the N894D/N900D double mutant (Fig. 5B). Login to comment
62 The oligosaccharide at the N900 site was efficiently attached (N894D Journal of Cell Science 121 (17) in Fig. 5A) and was relatively refractory to removal by the PNGase (Fig. 5C, panel N894D), whereas that at the other site was rapidly removed to yield the unglycosylated species (Fig. 5C, compare panel N900D with N894D/N900D). Login to comment
63 Interestingly, the wild-type protein (Fig. 5C, panel CFTR) behaved similarly to N894D, strongly suggesting that its two chains were differentially sensitive in the same way as when present individually. Login to comment
67 Although unglycosylated N894D/N900D can mature conformationally and reach the cell surface where it functions (Figs 2 and 3), it clearly turns over much more rapidly than the wild-type protein (Fig. 6A). Login to comment
68 By contrast, the single-chain species N894D appeared to mature as effectively as the wild-type protein, with its two chains. Login to comment
71 (A) Western blot of CFTR and unglycosylated CFTR created either by mutation of the glycosylation sites (N894D/N900D) or by expressing CFTR in tunicamycin-treated cells (+ Tunicamycin). Login to comment
72 CFTR and CFTR N894D/N900D variants were stably (left panel) or transiently (right panel) expressed in BHK-21 cells. Login to comment
75 (B) CFTR N894D/N900D does not interact with calnexin. Login to comment
80 Single-channel measurements were performed using membrane vesicles prepared from stably transfected BHK-CFTR, BHK- N894D/N900D cells or from cells transiently transfected with CFTR that were treated with tunicamycin. Login to comment
82 type or N894D proteins, and the mature product appeared to turn over faster than wild-type CFTR. Login to comment
87 The N894D species also turned over at a very similar rate to the wild-type protein in the BFA-exposed cells (Fig. 6B,C). Login to comment
91 ΔF508 N894D/N900D cannot escape ER quality control. Login to comment
92 (A) Western blot of CFTR, CFTR N894D/N900D, ΔF508 and ΔF508 N894D/N900D expressed in BHK-21 cells. Login to comment
94 (B) ΔF508 N894D/N900D does not interact with calnexin. Login to comment
95 ΔF508 N894D/N900D was immunoprecipitated by incubation with anti-CFTR antibody 596 crosslinked to Dynabeads. Login to comment
110 This was further confirmed by the application of the proteasomal inhibitor ALLN, which significantly stabilized CFTR N900D, but neither CFTR N894D nor the double mutant (supplementary material Fig. S4). Login to comment
112 We found that N894D interacted more strongly with calnexin than did N900D (Fig. 7A), but more of the latter was found in association with EDEM (Fig. 7B). Login to comment
113 The oligosaccharide at position 900 might mediate prolonged or preferential association with calnexin, as reflected in the decreased binding of the N894D mutant to EDEM but unaffected interaction with calnexin as compared with wild-type CFTR. Login to comment
181 The CFTR variant with a single oligosaccharide at position 894 (N900D) was much more susceptible to deglycosylation by PNGase, whereas the variant with glycosylation at position 900 (N894D) was surprisingly resistant to this treatment. Login to comment
183 This suggests that N900D CFTR, with a glycan at position 894, might be a much better substrate for this pathway than the N894D variant. Login to comment
185 With just the oligosaccharide chain at position 900 (N894D), turnover was similar to that of the wild-type protein; however, with an oligosaccharide attached only at position 894 (N900D), CFTR maturation was very inefficient. Login to comment
187 The conclusion that N900D CFTR is a better substrate for ERAD than N894D CFTR was further strengthened by the preferential interaction of N900D CFTR with EDEM, which was similar to that of the wild-type protein, but which was reduced in the N894D mutant (Fig. 7), and by the observation Fig. 7. Login to comment
188 Interaction of N894D and N900D single-chain mutants with calnexin and EDEM. Login to comment
189 (A) Cell lysates from BHK-21 cells stably expressing CFTR, CFTR N894D, CFTR N900D or CFTR N894D/N900D were subjected to immunoprecipitation by incubation with anti-CFTR antibody 596 crosslinked to Dynabeads (IP: CFTR, as indicated on the left). Login to comment
192 (B) CFTR was immunoprecipitated from cells that were stably expressing CFTR, CFTR N894D, CFTR N900D or CFTR N894D/N900D and that were also transiently expressing HA-tagged EDEM, by incubation with anti-CFTR monoclonal antibody 596 crosslinked to Dynabeads. EDEM was detected by western blotting using monoclonal antibody HA11 and CFTR using antibody 596. Login to comment
202 The carbohydrate at position 900 in CFTR N894D supports the route to maturation and progress to the Golgi, whereas the oligosaccharide at asparagine residue 894 in N900D promotes degradation. Login to comment
203 that the proteasomal inhibitor ALNN stabilized N900D CFTR, but not N894D CFTR (supplementary material Fig. S4). Login to comment
204 It seems likely that the double mutant behaves in the ER like CFTR N894D rather than like CFTR N900D, because the presence of the oligosaccharide at N894 supports the ERAD pathway, whereas the absence of this oligosaccharide allows the double glycosylation mutant to exit the ER and proceed to the Golgi and plasma membrane. Login to comment
219 Materials and Methods Mutagenesis and stable expression of CFTR Replacement of asparagine residues N894 and N900 with aspartate residues was achieved by replacing a CFTR HpaI-DraIII cDNA fragment (bp 2463 to 3328) in pNUT CFTR with a counterpart generated by PCR coding for N894D, N900D or N894D/N900D, to produce CFTRs with only one or no glycosylation site (Chang et al., 1994). Login to comment
224 The well-differentiated cultures were then transduced with adenoviral vectors as described previously (Coyne et al., 2000) to express Extope-CFTR and Extope-ΔF508 with intact or mutated glycosylation sites (N894D/N900D). Login to comment

[hide] N-glycans are direct determinants of CFTR folding ... J Cell Biol. 2009 Mar 23;184(6):847-62. Glozman R, Okiyoneda T, Mulvihill CM, Rini JM, Barriere H, Lukacs GL
N-glycans are direct determinants of CFTR folding and stability in secretory and endocytic membrane traffic.
J Cell Biol. 2009 Mar 23;184(6):847-62., 2009-03-23 [PMID:19307599]

Abstract [show]
N-glycosylation, a common cotranslational modification, is thought to be critical for plasma membrane expression of glycoproteins by enhancing protein folding, trafficking, and stability through targeting them to the ER folding cycles via lectin-like chaperones. In this study, we show that N-glycans, specifically core glycans, enhance the productive folding and conformational stability of a polytopic membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), independently of lectin-like chaperones. Defective N-glycosylation reduces cell surface expression by impairing both early secretory and endocytic traffic of CFTR. Conformational destabilization of the glycan-deficient CFTR induces ubiquitination, leading to rapid elimination from the cell surface. Ubiquitinated CFTR is directed to lysosomal degradation instead of endocytic recycling in early endosomes mediated by ubiquitin-binding endosomal sorting complex required for transport (ESCRT) adaptors Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and TSG101. These results suggest that cotranslational N-glycosylation can exert a chaperone-independent profolding change in the energetic of CFTR in vivo as well as outline a paradigm for the peripheral trafficking defect of membrane proteins with impaired glycosylation.

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24 Results Cell surface expression of glycosylation-deficient CFTR is severely reduced CFTR glycosylation was abolished by mutagenesis of the two consensus Asn-linked N-glycosylation sites (Kornfeld and Kornfeld, 1985) individually (N894D and N900D) or together (2D-CFTR) in CFTR. Login to comment
26 [ID]FIG1[/ID] The endoglycosidase (endo) H and F sensitivity of N894D- and N900D-CFTR verified that both glycan chains underwent complex glycosylation, whereas the 2D-CFTR failed to obtain N-glycans (Fig. 1 B). Login to comment
27 Indirect immunostaining of the extracellular 3HA tag in nonpermeabilized cells revealed that N894D-, N900D-, and 2D-CFTR were expressed at the cell surface, as proved by colocalization with Alexa Fluor 594-conjugated wheat germ agglutinine (WGA), a plasma membrane marker (Fig. 1 C). Login to comment
28 Quantitative analysis of CFTR cell surface density by anti-HA antibody (Ab) binding to the 3HA tag revealed that the N894D-, N900D-, and 2D-CFTR expression level was decreased by ~37%, ~63%, and ~87%, respectively, relative to wild-type (wt) CFTR, which is in line with the immunostaining results (Fig. 1 D). Login to comment
54 The wt CFTR folding efficiency (34.0 ± 5.6%) was reduced to 15.6 ± 0.5% and 13.8 ± 1.2% in N894D- and N900D-CFTR, respectively (Fig. 2 A). Login to comment
92 The transport-competent native 2D-CFTR has threefold accelerated (t1/2 = ~4 h), whereas the N894D- and N900D-CFTR have approximately twofold faster metabolic turnover rates than the wt channel (t1/2 = ~12 h; Fig. 4, A and B). Login to comment
303 The N894D-/N900D-CFTR (2D-CFTR) was generated using the N894D-CFTR as a template and the N900D mutagenic primers. Login to comment
306 Cell culture and transfection Stably transfected BHK cell lines expressing wt, N894D, N900D, 2D-, 2A-, and 2Q- or ΔF508-CFTR-2D as well as N900D+T908N- and 2D+T908N-CFTR variants were generated as described previously (Du et al., 2005). Login to comment

[hide] Modulation of endocytic trafficking and apical sta... Am J Physiol Lung Cell Mol Physiol. 2010 Mar;298(3):L304-14. Epub 2009 Dec 11. Cholon DM, O'Neal WK, Randell SH, Riordan JR, Gentzsch M
Modulation of endocytic trafficking and apical stability of CFTR in primary human airway epithelial cultures.
Am J Physiol Lung Cell Mol Physiol. 2010 Mar;298(3):L304-14. Epub 2009 Dec 11., [PMID:20008117]

Abstract [show]
CFTR is a highly regulated apical chloride channel of epithelial cells that is mutated in cystic fibrosis (CF). In this study, we characterized the apical stability and intracellular trafficking of wild-type and mutant CFTR in its native environment, i.e., highly differentiated primary human airway epithelial (HAE) cultures. We labeled the apical pool of CFTR and subsequently visualized the protein in intracellular compartments. CFTR moved from the apical surface to endosomes and then efficiently recycled back to the surface. CFTR endocytosis occurred more slowly in polarized than in nonpolarized HAE cells or in a polarized epithelial cell line. The most common mutation in CF, DeltaF508 CFTR, was rescued from endoplasmic reticulum retention by low-temperature incubation but transited from the apical membrane to endocytic compartments more rapidly and recycled less efficiently than wild-type CFTR. Incubation with small-molecule correctors resulted in DeltaF508 CFTR at the apical membrane but did not restore apical stability. To stabilize the mutant protein at the apical membrane, we found that the dynamin inhibitor Dynasore and the cholesterol-extracting agent cyclodextrin dramatically reduced internalization of DeltaF508, whereas the proteasomal inhibitor MG-132 completely blocked endocytosis of DeltaF508. On examination of intrinsic properties of CFTR that may affect its apical stability, we found that N-linked oligosaccharides were not necessary for transport to the apical membrane but were required for efficient apical recycling and, therefore, influenced the turnover of surface CFTR. Thus apical stability of CFTR in its native environment is affected by properties of the protein and modulation of endocytic trafficking.

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268 A: Western blot of lysates of HAE cells adenovirally expressing CFTR N894D N900D, in which asparagine residues N894 and N900 are replaced with aspartate, confirmed lack of glycosylation. Login to comment
269 B: primary HAE cultures were adenovirally transduced to express wild-type CFTR and a nonglycosylated variant (N894D N900D) of CFTR. Login to comment
272 Time course experiments were performed on 3 different HAE cultures; 1 representative example is shown. D: immunofluorescence labeling of sodium-potassium-chloride cotransporter isoform 1 (NKCC1) confirms that internalized CFTR N894D N900D is transported to basolateral compartments. Login to comment
273 HAE cultures were adenovirally transduced to express CFTR N894D N900D. Login to comment
266 A: Western blot of lysates of HAE cells adenovirally expressing CFTR N894D N900D, in which asparagine residues N894 and N900 are replaced with aspartate, confirmed lack of glycosylation. Login to comment
267 B: primary HAE cultures were adenovirally transduced to express wild-type CFTR and a nonglycosylated variant (N894D N900D) of CFTR. Login to comment
270 Time course experiments were performed on 3 different HAE cultures; 1 representative example is shown. D: immunofluorescence labeling of sodium-potassium-chloride cotransporter isoform 1 (NKCC1) confirms that internalized CFTR N894D N900D is transported to basolateral compartments. Login to comment
271 HAE cultures were adenovirally transduced to express CFTR N894D N900D. Login to comment

[hide] Protein quality control at the plasma membrane. Curr Opin Cell Biol. 2011 Aug;23(4):483-91. Epub 2011 May 14. Okiyoneda T, Apaja PM, Lukacs GL
Protein quality control at the plasma membrane.
Curr Opin Cell Biol. 2011 Aug;23(4):483-91. Epub 2011 May 14., [PMID:21571517]

Abstract [show]
Cellular proteostasis (or protein homeostasis) depends on the timely folding and disposal of conformationally damaged polypeptides during their life span at all subcellular locations. This process is particularly important for membrane proteins confined to the cell surface with crucial regulatory role in cellular homoeostasis and intercellular communication. Accumulating evidences indicate that membrane proteins exported from the endoplasmic reticulum (ER) are subjected to peripheral quality control (QC) along the late secretory and endocytic pathways, as well as at the plasma membrane (PM). Recently identified components of the PM QC recognition and effector mechanisms responsible for ubiquitination and lysosomal degradation of conformationally damaged PM proteins uncovered striking similarities to and differences from that of the ER QC machinery. Possible implications of the peripheral protein QC activity in phenotypic modulation of conformational diseases are also outlined.

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31 Structural destabilization of the Pma1 and Gap1 transmembrane domains in strains defective of sphingoid base synthesis could be mechanistically similar to the farnesol-induced 484 Membranes and organelles Table 1 Peripheral protein QC substrates Membrane protein Mutation/condition Degron PM stability Related disease Reference Mammalian bile salt export pump (BSEP) E297G (Cy) D482G (Cy) 2-3 Ub # progressive familial intrahepatic cholestasis type 2 (PFIC2) [36] CFTR rDF508 (Cy) poly/multimono Ub # cystic fibrosis (CF) [44 ,27 ] D70 (Cy truncation) poly/multimono Ub # [27 ] N894D, N900D (Ex) poly/multimono Ub # [22] Na/H exchanger (NHE6) D255-256 (TM) poly/multimono Ub # Angelman syndrome [35] MLC1 multiple (TM or Cy) ND # megalencephalic leukoencephalopathy with subcortical cysts (MLC) [66] HERG low K+ Ub # type 2 long QT syndrome [37] LDL receptor high salt or low pH (Ex) ND # hypercholesterolemia [16] Dopamine D4.4 receptor M345T (TM) poly/multimono Ub # attention deficit hyperactivity disorder [28 ] Vasopressin V2 receptor W164S (TM) poly/multimono Ub # nephrogenic diabetes insipidus [28 ] alpha-2A adrenergic receptor D79N (TM) ND # cardiovascular diseases [14] N422D (TM) ND # [14] Di3loop (Cy) ND # [69] CD4tl-lm L57C (Cy) poly/multimono Ub # model protein [28 ] H+ /K+ -ATPase b subunit N99Q, N130Q, N161Q, N222Q (Ex) ND # gastric, autoimmune diseases [18] k opioid receptor N25/39Q (Ex) ND # pain control, neuronal phenotypes [19] D opioid receptor N18Q/N33Q (Ex) ND # pain control, neuronal phenotypes [20] GLUT1 N45Y, Q or D (ex) ND # GLUT1 deficiency syndrome [70] EGFR L858R (Cy), exon 19 deletion (Cy) poly/multimono Ub # cancer suspectibility [71] ErbB2 Hsp90 inhibition poly/multimono Ub # breast cancer [72] TGFBR2 Hsp90 inhibition poly/multimono Ub # tumor suspectibility [73] Yeast Pma1 Icb1-100 poly/multimono Ub # NA [29] Pma1-7 poly/multimono Ub # NA [7] Pma1-10 poly/multimono Ub # NA [52] Gap1 absence of sphingolipids poly/multimono Ub # NA [9] Abbreviations: Cy, cytosolic; Ex, extracellular; TM, transmembrane; Ub, ubiquitin; ND, not determined; #, decreasing stability; CFTR, cystic fibrosis transmembrane conductance regulator; HERG, human ether-a` -go-go related gene; LDL, low-density lipoprotein; GLUT, glucose transporter; EGFR, epidermal growth factor receptor; ErbbB2, v-erb-b2 erythroblastic leukemia viral oncogene homolog 2; TGFBR2, transforming growth factor (TGF)- beta type II; Pma1, H(+)-ATPase; Gap1, general amino acid permease. Login to comment

[hide] Domain interdependence in the biosynthetic assembl... J Mol Biol. 2007 Jan 26;365(4):981-94. Epub 2006 Nov 10. Cui L, Aleksandrov L, Chang XB, Hou YX, He L, Hegedus T, Gentzsch M, Aleksandrov A, Balch WE, Riordan JR
Domain interdependence in the biosynthetic assembly of CFTR.
J Mol Biol. 2007 Jan 26;365(4):981-94. Epub 2006 Nov 10., [PMID:17113596]

Abstract [show]
The dimerization of their two nucleotide binding domains (NBDs) in a so-called "nucleotide-sandwich" is the hallmark of ATP cassette binding (ABC) proteins and the basis of their catalytic activities. The major disease-causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR or ABCC7), deletion of Phe508 in NBD1, does not grossly alter the structure of that domain but prevents conformational maturation of the whole CFTR protein, possibly by disrupting the native interaction between NBD1 and NBD2. However, the role of inter-domain interactions in CFTR folding has been brought into question by a recent report that all CFTR domains fold independently. Here we show that in addition to domain folding, correct inter-domain assembly is essential to form a stable unit that satisfies endoplasmic reticulum (ER) quality control. N-terminal domains depend on their more C-terminal neighbors, most essentially the second membrane-spanning domain (MSD2) but significantly, not NBD2. Wild-type C-terminal truncation constructs, completely devoid of NBD2 are transported out of the ER and to the cell surface where they form characteristic CFTR chloride channels with low open probability. The DeltaNBD2 wild-type protein matures and has similar stability as its full-length counterpart. Therefore, the catalytically crucial inter-NBD associations are not required to satisfy ER quality control mechanisms. The DeltaF508 mutation arrests the maturation of DeltaNBD2 just as it does full-length CFTR, indicating that DeltaF508 perturbs other portions of the molecule in addition to NBD2. We find that the mutation prevents formation of a compact MSD1, reflected in its susceptibility to protease digestion. This perturbation of MSD1 may in turn prevent its normal integration with MSD2. The dispensability of NBD2 in the folding of more N-terminal domains stands in contrast to the known hypersensitivity to proteolysis of NBD2 in the DeltaF508 protein.

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211 Complex N-glycosylation of EL1 only, was confirmed when EL4 glycosylation sites were mutated14 (substitutions N894D/N900D), and of both EL1 and EL4 when sites were present in both loops. Login to comment
209 Complex N-glycosylation of EL1 only, was confirmed when EL4 glycosylation sites were mutated14 (substitutions N894D/N900D), and of both EL1 and EL4 when sites were present in both loops. Login to comment

[hide] Mapping of cystic fibrosis transmembrane conductan... J Biol Chem. 1994 Jul 15;269(28):18572-5. Chang XB, Hou YX, Jensen TJ, Riordan JR
Mapping of cystic fibrosis transmembrane conductance regulator membrane topology by glycosylation site insertion.
J Biol Chem. 1994 Jul 15;269(28):18572-5., [PMID:7518437]

Abstract [show]
Technical difficulties in obtaining three-dimensional structures of intrinsic membrane proteins continues to limit understanding of their function. However, considerable insight can be gained from their two-dimensional topological arrangement in the lipid bilayer. Efficient molecular genetic approaches are available to discern the topology of prokaryotic but not of eukaryotic membrane proteins. The absolute asymmetry of the sidedness of their N-glycosylation was employed here to develop such a method using the cystic fibrosis transmembrane conductance regulator (CFTR). Insertion by in vitro mutagenesis of N-glycosylation consensus sequences (NXS/T) in predicted cytoplasmic and extracytoplasmic loops between hydrophobic sequences capable of traversing the membrane established the membrane topology of CFTR. This provides the first experimental evaluation of the original topological model of CFTR based solely on hydropathy algorithms and a method which may be generally applicable for the in vivo evaluation of the topology of other mammalian membrane proteins.

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22 A fragment inwild- type pNUT-CFTR (21, 22) was replaced by a PCR fragment (between HpaI site atnucleotide 2463 andDraIII site atnucleotide 3328)coding for the N894D and N900D changes to produce aCFTR with no consensus glycosylation sites on extracytoplasmic loops (designated ELO,Fig. Login to comment