title: Crucial role for phylogenetically conserved cytoplasmic loop 3 in ABCC4 protein expression.
abstract: 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 α-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.
authors.affiliation: Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
authors.forename: Satish B
authors.initials: SB
authors.lastname: Cheepala
authors.forename: Ju
authors.initials: J
authors.lastname: Bao
authors.forename: Deepa
authors.initials: D
authors.lastname: Nachagari
authors.forename: Daxi
authors.initials: D
authors.lastname: Sun
authors.forename: Yao
authors.initials: Y
authors.lastname: Wang
authors.forename: Tao P
authors.initials: TP
authors.lastname: Zhong
authors.forename: Tao
authors.initials: T
authors.lastname: Zhong
authors.forename: Anjaparavanda P
authors.initials: AP
authors.lastname: Naren
authors.forename: Jie
authors.initials: J
authors.lastname: Zheng
authors.forename: John D
authors.initials: JD
authors.lastname: Schuetz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D041681:NIH 3T3 Cells; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid; D015027:Zebrafish
chemical_list: D027425:ATP-Binding Cassette, Sub-Family C Proteins; C073492:ABCC4 protein, human
keywords: ABC Transporter; Cytoplasmic Loop 3; Homology Modeling; Molecular Modeling; Mrp4; Multidrug Transporters; Zebrafish