Crucial Role for Phylogenetically Conserved Cytoplasmic Loop 3 in ABCC4 Expression

Cheepala, S.B., Bao, J., Nachagari, D., Sun, D., Wang, Y., Zhong, T., Naren, A.P., Zheng, J., and Schuetz, J.D.
The Journal of biological chemistry   288(31): 22207-18 (Journal)
Registered Authors
Zhong, Tao P.
ABC transporter, homology modeling, molecular modeling, multidrug transporters, zebrafish, Mrp4, cytoplasmic loop 3
MeSH Terms
  • Amino Acid Sequence
  • Animals
  • Cell Line
  • Cytoplasm/metabolism*
  • Humans
  • Mice
  • Models, Molecular
  • Molecular Sequence Data
  • Multidrug Resistance-Associated Proteins/chemistry
  • Multidrug Resistance-Associated Proteins/genetics*
  • Multidrug Resistance-Associated Proteins/metabolism
  • NIH 3T3 Cells
  • Phylogeny*
  • Sequence Homology, Amino Acid
  • Zebrafish
23766510 Full text @ J. Biol. Chem.

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 orthologues 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 methionine substitutions indicated altered helicity coupled with reduced thermal stability. Trifluroethanol 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 is mostly rescued by growing cells at sub-physiologic temperature. Because CFTR (ABCC7) is closely related to ABCC4, we extended this by engineering certain pathogenic CFTR-CL3 mutations and 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 maturation and plasma membrane localization.

Genes / Markers
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Engineered Foreign Genes