Cheepala, S.B., Bao, J., Nachagari, D., Sun, D., Wang, Y., Zhong, T., Naren, A.P., Zheng, J., and Schuetz, J.D. (2013) Crucial Role for Phylogenetically Conserved Cytoplasmic Loop 3 in ABCC4 Expression. The Journal of biological chemistry. 288(31):22207-18.
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.