Generation of fully functional fluorescent fusion proteins to gain insights into ABCC6 biology

Abstract

ABCC6 mediates release of ATP from hepatocytes into the blood. Extracellularly, ATP is converted into the mineralization inhibitor pyrophosphate. Consequently, inactivating mutations in ABCC6 give low plasma pyrophosphate and underlie the ectopic mineralization disorder pseudoxanthoma elasticum. How ABCC6 mediates cellular ATP release is still unknown. Fluorescent ABCC6 fusion proteins would allow mechanistic studies, but fluorophores attached to the ABCC6 N- or C-terminus result in intracellular retention and degradation. Here we describe that intramolecular introduction of fluorophores yields fully functional ABCC6 fusion proteins. A corresponding ABCC6 variant in which the catalytic glutamate of the second nucleotide binding domain was mutated, correctly routed to the plasma membrane but was inactive. Finally, N-terminal His¹⁰ or FLAG tags did not affect activity of the fusion proteins, allowing their purification for biochemical characterization.

Keywords: ABC transporter; cellular ATP efflux; fluorescent fusion protein; pseudoxanthoma elasticum; purification.

Figure 1.. Overview of the rAbcc6 fusion protein constructs.

A: Schematic representation of the membrane topology of rAbcc6 with the locations of the fluorophores and tags. B: Schematic overview of the generated fusion proteins indicating the positions where the mNeonGreen or mScarlet fluorophores were introduced. His¹⁰, FLAG and HRV C3 protease cleavage sites are also depicted as is the mutation of the Walker B catalytic glutamate residue (E1426Q).

Figure 2:. Expression of the rAbcc6 fusion proteins in HEK293 cells.

Immunoblot analysis showing relative protein levels of unmodified rAbcc6 and the rAbcc6 fluorescent fusion proteins in HEK293 cells. The polyclonal K14 rabbit anti-rAbcc6 antibody (1:3000x) and HRP-conjugated anti-rabbit secondary antibody (1:5000x) were used to detect rAbcc6.

Figure 3:. Subcellular localization of the rAbcc6 fusion proteins in HEK293 cells.

A: representative image of the subcellular localization of the wild type, unmodified rAbcc6 protein overexpressed in HEK293 cells by immunofluorescence with an 40X objective and 3X optical zoom. B-H: representative images of the subcellular localization of the fluorescent fusion proteins by live cell confocal microscopy Panels B-E show the localization of the green fluorescent rAbcc6 fusion proteins with an 60X objective and 3X optical zoom (B: rAbcc6-mNG_ICL1; C: rAbcc6-mNG_ECL3; D: rAbcc6-mNG_bNBD1; E: rAbcc6-mNG_aNBD1. Panel F: rAbcc6^E1426Q-mNG_aNBD1). Panels G and H show representative images of the red fluorescent rAbcc6 fusion proteins (G: His10-rAbcc6_mSc_aNBD1; H: FLAG-C3-rAbcc6-mSc_aNBD1). All scale bars represent 30 µm.

Figure 4.. Functional analysis of the fluorescent rAbcc6 fusion proteins.

A: ATP efflux was followed in real time for 3 hours using a luciferase/luciferin-based detection system. Data represent the mean of an experiment performed in quadruple. B: PPi accumulation in 24-hour cell culture medium samples are given. Data are presented as mean +/− SE of an experiment performed in quadruple. Representative examples of at least 2 independent experiments are shown in A and B.