Partial purification of reconstitutively active Sac1p-independent adenine nucleotide transport system (ANTS) from yeast endoplasmic reticulum.

The endoplasmic reticulum (ER) is a membrane network present in all nucleated cells, its lumen is separated from the cytosol by the ER membrane. Hence, the passage of substrates depends on the presence of selective ER membrane transporters. ATP synthesized into the cytosol enters the ER lumen, where it is used as energy source in many biochemical reactions, including dissociation of chaperones-proteins complexes, disulphide bridge formation, protein polymerization, glycosylation and phosphorylation of proteins, proteoglycans or lipids. In the ER of Saccharomyces cerevisiae, a 68-kDa protein (Sac1p) was believed responsible for ATP transport, but it was found unable to catalyse any ATP uptake, rather acting as a transport regulator, possibly controlling ER phosphoinositides (1). Here, we report an extensive biochemical characterization of a S. cerevisiae adenine nucleotide transport system (ANTS), not dependent on the presence of Sac1p. This report also represents the first partial purification of the yeast ER ANTS. Highly purified ER membranes from the wild-type and Δsac1 yeast strains reconstituted into liposomes transported ATP with the same efficiency. Hydroxyapatite chromatography was used to partially purify ANTS from Δsac1 ER extract. The two ANTS-enriched transport activity eluted fractions showed essentially the presence of four bands, one having an apparent MW of 56 kDa, similar to that observed for ANTS identified in rat liver ER. The two fractions reconstituted into liposomes efficiently transported ATP and ADP, by a strict counter-exchange mechanism. ATP transport was saturable with a Km of 0.28 mM. ATP/ADP exchange mechanism and kinetic constants’ values suggest that the main physiological role of ANTS is to catalyse the transport of ATP into the ER lumen, where it is used in several energy-requiring reactions and to export back to the cytosol the produced ADP.