Crucial role for phylogenetically conserved cytoplasmic loop 3 in ABCC4 protein expression

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• ABCC4 is an ATP-dependent transporter exporting endogenous substances and chemotherapeutics.
• A loss-of-function zebrafish Abcc4 variant (T804M) was identified in cytoplasmic loop 3 (CL3).
• This mutation site is conserved across ABCC4 orthologs.

Purpose of the Study:

• To investigate the impact of the T804M mutation on human and zebrafish Abcc4 expression and localization.
• To elucidate the molecular mechanisms underlying the observed localization defect.

Main Methods:

• Homology modeling of zebrafish Abcc4.
• Molecular dynamics simulations of CL3 fragments.
• Circular dichroism spectroscopy with trifluoroethanol challenge.
• Cellular expression and localization studies at varying temperatures.
• Engineering and testing of related CFTR-CL3 mutations in ABCC4.

Main Results:

• The T804M mutation in both zebrafish Abcc4 and human ABCC4 significantly reduced protein expression and plasma membrane localization.
• Homology modeling and simulations indicated the methionine substitution disrupts side-chain contacts, alters helicity, and reduces thermal stability of CL3.
• Circular dichroism confirmed that the mutation hinders α-helix formation in CL3.
• Lowering cell growth temperature partially rescued expression and localization of mutant proteins.
• Pathogenic mutations in the related CFTR-CL3 destabilized human and zebrafish ABCC4.

Conclusions:

• A conserved domain within CL3 of ABCC4 is essential for its proper plasma membrane localization.
• The T804M mutation destabilizes ABCC4 by disrupting CL3 structure and function.
• Temperature-sensitive mechanisms are involved in ABCC4 trafficking and stability.
• Findings have implications for understanding ABCC4 function and designing therapies involving ABCC4 or related transporters.