Differential Effects of Mutations of Popeye Domain Containing Proteins on Heteromeric Interaction and Membrane Trafficking

Abstract

Background: The Popeye domain containing (POPDC) genes encode sarcolemma-localised cAMP effector proteins. Mutations in BVES (POPDC1) and POPDC2 have been associated with limb-girdle muscular dystrophy and cardiac arrhythmia. Muscle biopsies of affected patients display impaired membrane trafficking of both POPDC isoforms. Methods: Biopsy material of patients carrying mutations in BVES were immunostained with POPDC antibodies. The interaction of POPDC proteins was investigated by co-precipitation, proximity ligation, bioluminescence resonance energy transfer and bimolecular fluorescence complementation. Site-directed mutagenesis was utilised to map the domains involved in protein interaction. Findings: Patients carrying a novel homozygous variant, BVES (c.547G>T, p.V183F) displayed only a skeletal muscle pathology and a mild impairment of membrane trafficking of both POPDC isoforms. This is in contrast to variants such as BVES p.Q153X or POPDC2 p.W188X, which were associated with a greater impairment of membrane trafficking. Co-transfection analysis in HEK293 cells revealed that POPDC proteins interact with each other through a helix-helix interface located at the C-terminus of the Popeye domain. Site-directed mutagenesis of an array of ultra-conserved hydrophobic residues demonstrated that some of them are required for membrane trafficking of the POPDC1-POPDC2 complex. Interpretation: Mutations in POPDC proteins that cause an impairment in membrane localisation affect POPDC complex formation while mutations which leave the protein interaction intact likely affect some other essential function of POPDC proteins. Funding: This study was funded by an EPSRC/British Heart Foundation co-funded Imperial Institute of Chemical Biology (ICB) Centre for Doctoral Training (CDT) PhD studentship (EP/S023518/1), a project grant of the British Heart Foundation (PG19/13/34247) and the Deutsche Forschungsgemeinschaft (DE1482/9-1).