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

Szeri, Flóra and Niaziorimi, Fatemeh and Donnelly, Sylvia and Orndorff, Joseph and van de Wetering, Koen (2021) Generation of fully functional fluorescent fusion proteins to gain insights into ABCC6 biology. FEBS LETTERS, 595 (6). pp. 799-810. ISSN 0014-5793

Text SzeriFEBSLetters2020_GenerationoffullyfunctionalfluorescentfusionproteinstogaininsightsintoABCC6biology.pdf Restricted to Registered users only Download (3MB) | Request a copy

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.

Actions (login required)

Edit Item