Generation of fully functional fluorescent fusion proteins to gain insights into ABCC6 biology. Issue 6 (5th November 2020)

• Record Type: Journal Article
• Authors: Szeri, Flora; Niaziorimi, Fatemeh; Donnelly, Sylvia; Orndorff, Joseph; van de Wetering, Koen
• Is Part Of: FEBS letters. Volume 595:Issue 6(2021)
• Page Start: 799
• Page End: 810
• Publication Date: 2020-11-05
• Subjects: ABC transporter -- cellular ATP efflux -- fluorescent fusion protein -- pseudoxanthoma elasticum -- purification; Biochemistry -- Periodicals; Biophysics -- Periodicals; Molecular biology -- Periodicals; Biochimie -- Périodiques; Biochemistry; Biophysics; Molecular biology; Periodicals; 572.05
• DOI: 10.1002/1873-3468.13957
• Languages: English
• ISSNs: 0014-5793

Abstract

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 His 10 or FLAG tags did not affect activity of the fusion proteins, allowing their purification for biochemical characterization. Abstract :