The broad range di- and trinucleotide exchanger SLC35B1 displays asymmetrical affinities for ATP transport across the ER membrane

SCHWARZBAUM, PABLO JULIO; SCHACHTER, J; BREDESTON LM

JOURNAL OF BIOLOGICAL CHEMISTRY

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Lugar: Bethesda, Maryland; Año: 2022

0021-9258

In most eukaryotic cells, uptake ofcytosolic ATP in the endoplasmic reticulum is a critical process for thefunctioning of various chaperones, which assist in the folding of proteinsinside this organelle. However, the underlying molecular mechanism mediatingATP uptake is not completely understood. The human SLC35B1 protein was recentlypostulated to be a transporter of the endoplasmic reticulum membrane mediatingATP/ADP exchange.  Here,we report an extensive kinetic characterization of human SLC35B1, expressed inyeast, purified and reconstituted into liposomes. Using an assay based on [α32P]ATPuptake, we tested the nucleotide concentration dependence of ATP/ADP exchangeactivity on both sides of the membrane and found that the apparent affinitiesof the external side were 13 times higher than those of the internal side. Threedifferent experimental approaches indicated that ATP/ADP exchange by SLC35B1was not strict, and that other di-and trinucleotides can act as suitablecounter substrates for ATP, while mononucleotides and nucleotide sugars werenot transported. Finally, bioinformatic analysis and site-directed mutagenesis identified conserved residues K117,K120 from transmembrane helix 4 and K277 from transmembrane helix 9 playing acritical role in transport.The fact that SLC35B1 can promote ATP import in exchange for ADP or UDPpoint to a more direct coupling between ATP import requirements and the needfor eliminating ADP and UDP, generated as a side product of reactions takingplace in the lumen of the endoplasmic reticulum.