Abstract
In eukaryotic cells, uptake of cytosolic ATP into the endoplasmic reticulum (ER) lumen is critical for the proper functioning of chaperone proteins. The human transport protein SLC35B1 was recently postulated to mediate ATP/ADP exchange in the ER; however, the underlying molecular mechanisms mediating ATP uptake are not completely understood. Here, we extensively characterized the transport kinetics of human SLC35B1 expressed in yeast that was purified and reconstituted into liposomes. Using [α32P]ATP uptake assays, we tested the nucleotide concentration dependence of ATP/ADP exchange activity on both sides of the membrane. We found that the apparent affinities of SLC35B1 for ATP/ADP on the internal face were approximately 13 times higher than those on the external side. Because SLC35B1-containing liposomes were preferentially inside-out oriented, these results suggest a low-affinity external site and a high-affinity internal site in the ER. Three different experimental approaches indicated that ATP/ADP exchange by SLC35B1 was not strict, and that other di- and tri-nucleotides could act as suitable counter-substrates for ATP, although mononucleotides and nucleotide sugars were not transported. Finally, bioinformatic analysis and site-directed mutagenesis identified that conserved residues K117 and K120 from transmembrane helix 4 and K277 from transmembrane helix 9 play critical roles in transport. The fact that SLC35B1 can promote ATP transport in exchange for ADP or UDP suggest a more direct coupling between ATP import requirements and the need for eliminating ADP and UDP, which are generated as side products of reactions taking place in the ER-lumen.
Highlights
Introduction independent of thenucleotide sugar transporter (NST) family could be involved (13).The supply of specific solutes is essentialRegarding the transport of ATP to the ER, for protein folding and maturation taking the information has been scarce
Showing ATP/ADP exchange in proteoliposomes prepared from solubilized hSLC35B1 isoform 1 was cloned in total membrane proteins of bacteria plasmid p426GFP and expressed in expressing hSLC35B1 (20)
After column washing with a buffer containing 30 mM imidazole, the fraction eluted in the same buffer containing 200 mM Imidazole showed a main band of approximately 50 kDa, smaller than the predicted 65 kDa value corresponding to the fusion hSLC35B1-GFP
Summary
PJS and LMB: Conceptualization, Methodology, Data curation, Writing-Original draft preparation, Visualization, supervision, Writing- Reviewing and Editing, Project administration; J.S. and LMB: performed the experiments; J.S.: Methodology, Writing- Reviewing and Editing LMB and PJS: funding acquisition. Bredeston From the Departamento de Química Biológica-IQUIFIB, Facultad de Farmacia y Bioquímica Universidad de Buenos Aires-CONICET, CABA, Argentina. Journal Pre-proof Running title: nucleotide exchange at the endoplasmic reticulum
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