ABCMdb

ABCC4 p.Arg815Ala

Predicted by SNAP2:	A: N (82%), C: N (78%), D: D (63%), E: N (53%), F: D (53%), G: N (53%), H: N (53%), I: N (82%), K: N (87%), L: N (78%), M: N (87%), N: N (82%), P: D (59%), Q: N (82%), S: N (93%), T: N (93%), V: N (93%), W: D (80%), Y: D (75%),
Predicted by PROVEAN:	A: N, C: D, D: N, E: N, F: D, G: D, H: N, I: N, K: N, L: N, M: N, N: N, P: D, Q: N, S: N, T: N, V: N, W: D, Y: D,

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Publications
[hide] Crucial role for phylogenetically conserved cytopl... J Biol Chem. 2013 Aug 2;288(31):22207-18. doi: 10.1074/jbc.M113.476218. Epub 2013 Jun 13. Cheepala SB, Bao J, Nachagari D, Sun D, Wang Y, Zhong TP, Naren AP, Zheng J, Schuetz JD
Crucial role for phylogenetically conserved cytoplasmic loop 3 in ABCC4 protein expression.
J Biol Chem. 2013 Aug 2;288(31):22207-18. doi: 10.1074/jbc.M113.476218. Epub 2013 Jun 13., [PMID:23766510]

Abstract [show]
The ABC transporter ABCC4 is recognized as an ATP-dependent exporter of endogenous substances as well as an increasing variety of anionic chemotherapeutics. A loss-of-function variant of zebrafish Abcc4 was identified with a single amino acid substitution in the cytoplasmic loop T804M. Because this substituted amino acid is highly conserved among ABCC4 orthologs and is located in cytoplasmic loop 3 (CL3), we investigated the impact of this mutation on human and zebrafish Abcc4 expression. We demonstrate that zebrafish Abcc4 T804M or human ABCC4 T796M exhibit substantially reduced expression, coupled with impaired plasma membrane localization. To understand the molecular basis for the localization defect, we developed a homology model of zebrafish Abcc4. The homology model suggested that the bulky methionine substitution disrupted side-chain contacts. Molecular dynamic simulations of a fragment of human or zebrafish CL3 containing a methionine substitution indicated altered helicity coupled with reduced thermal stability. Trifluoroethanol challenge coupled with circular dichroism revealed that the methionine substitution disrupted the ability of this fragment of CL3 to readily form an alpha-helix. Furthermore, expression and plasma membrane localization of these mutant ABCC4/Abcc4 proteins are mostly rescued by growing cells at subphysiological temperatures. Because the cystic fibrosis transmembrane conductance regulator (ABCC7) is closely related to ABCC4, we extended this by engineering certain pathogenic CFTR-CL3 mutations, and we showed they destabilized human and zebrafish ABCC4. Altogether, our studies provide the first evidence for a conserved domain in CL3 of ABCC4 that is crucial in ensuring its proper plasma membrane localization.

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Sentences [show]
No. Sentence Comment
207 Mutation R815A in Human ABCC4 Cytoplasmic Region 3 (CL3) Destabilizes the Protein-Because CLs interact with NBDs in CFTR (26), we hypothesized that a mutation in an amino acid of CL3 in the "coupling helix" (a CL region near the NBD) might disrupt NBD and CL interdomain interactions producing an alteration in stability. Login to comment
209 To investigate this we mutated Arg-815 to Ala. Login to comment
210 The R815A mutation resulted in reduced ABCC4 expression and loss of mature glycosylated band c (Fig. 6, B and C). Login to comment
211 The reduction in mature ABCC4 is consistent with the prediction that CL3 Arg-815 interacts with the NBD to ensure proper folding of the protein To test if reduced temperature enhanced expression of R815A, NIH3T3 cells were transiently transfected with ABCC4, T796M-ABCC4, or R815A-ABCC4 and then incubated at 28 &#b0;C. Login to comment
212 The amount of R815A protein increased, which paralleled an increase in mature glycosylated protein band c (Fig. 6, B and C). Login to comment
257 The increased formation of the mature c-form of ABCC4 R815A at the subphysiological temperature of 28 &#b0;C is consistent with the proposal that CL3/NBD1 interactions are necessary for proper folding of ABCC4. Login to comment
267 A, representation of interdomain interaction between R815A of CL3 region and Asp-522 of NBD1 domain. Login to comment
268 B, immunoblot of whole cell lysates (100 òe;g of protein per lane) prepared from NIH3T3 cells transfected with human ABCC4 WT and ABCC4 mutant (R815A) and incubated at 37 or 28 &#b0;C for 24 h after 24 h of transfection. Login to comment
278 Likewise, we show that the predicted contact between CL3 Arg-815 and NBD1 Asp-522 when disrupted by an R815A substitution reduces the amount of mature ABCC4. Login to comment
206 Mutation R815A in Human ABCC4 Cytoplasmic Region 3 (CL3) Destabilizes the Protein-Because CLs interact with NBDs in CFTR (26), we hypothesized that a mutation in an amino acid of CL3 in the "coupling helix" (a CL region near the NBD) might disrupt NBD and CL interdomain interactions producing an alteration in stability. Login to comment
208 To investigate this we mutated Arg-815 to Ala. Login to comment
256 The increased formation of the mature c-form of ABCC4 R815A at the subphysiological temperature of 28 &#b0;C is consistent with the proposal that CL3/NBD1 interactions are necessary for proper folding of ABCC4. Login to comment
266 A, representation of interdomain interaction between R815A of CL3 region and Asp-522 of NBD1 domain. Login to comment
277 Likewise, we show that the predicted contact between CL3 Arg-815 and NBD1 Asp-522 when disrupted by an R815A substitution reduces the amount of mature ABCC4. Login to comment