1. Functional characterization of the partially purified Sac1p independent adenine nucleotide transport system (ANTS) from yeast endoplasmic reticulum
- Author
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Giuseppe Fiermonte, Anna Rita Cappello, Loredana Capobianco, Vincenza Dolce, Carmela Piazzolla, Luigina Muto, Paola Lunetti, Yuan Li, Francesco Zaffino, Marcello Maggiolini, Emanuela Martello, Rocco Malivindi, Susanna Raho, Rosamaria Lappano, Marianna Madeo, Rosita Curcio, Luca Frattaruolo, Donatella Aiello, Li, Y., Cappello, A. R., Muto, L., Martello, E., Madeo, M., Curcio, R., Lunetti, P., Susanna Raho, S., Zaffino, F., Frattaruolo, L., Lappano, R., Malivindi, R., Maggiolini, M., Aiello, D., Piazzolla, C., Capobianco, L., and Fiermonte, G. and Dolce V.
- Subjects
0301 basic medicine ,Saccharomyces cerevisiae Proteins ,Sac1p ,Saccharomyces cerevisiae ,Biochemistry ,Mass Spectrometry ,03 medical and health sciences ,Adenosine Triphosphate ,adenine nucleotide transport system ,Molecular Biology ,Liposome ,biology ,ATP transport ,HTP purification ,Chemistry ,Endoplasmic reticulum ,Regular Papers ,Biological Transport ,General Medicine ,biology.organism_classification ,Yeast ,endoplasmic reticulum ,Cytosol ,030104 developmental biology ,Membrane ,transport ,Adenine nucleotide transport - Abstract
Several ATP-depending reactions take place in the endoplasmic reticulum (ER). Although in Saccharomyces cerevisiae ER the existence of a Sac1p-dependent ATP transport system was already known, its direct involvement in ATP transport was excluded. Here we report an extensive biochemical characterization of a partially purified adenine nucleotide transport system (ANTS) not dependent on Sac1p. Highly purified ER membranes from the wild-type and Δsac1 yeast strains reconstituted into liposomes transported ATP with the same efficiency. A chromatography on hydroxyapatite 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, by a strict counter-exchange mechanism, ATP and ADP. ATP transport was saturable with a Km of 0.28 mM. The ATP/ADP exchange mechanism and the kinetic constants suggest that the main physiological role of ANTS is to catalyse the transport of ATP into ER, where it is used in several energy-requiring reactions and to export back to the cytosol the ADP produced.
- Published
- 2018
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