Functional characterization of the partially purified Sac1p independent adenine nucleotide transport system (ANTS) from yeast endoplasmic reticulum

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.

Thioalbamide, A Thioamidated Peptide from Amycolatopsis alba, Affects Tumor Growth and Stemness by Inducing Metabolic Dysfunction and Oxidative Stress

“…For immunoblot analysis, cells were grown to 70–80% confluence and treated with 50 nM thioalbamide or DMSO for 24–72 h. For preparation of total cell lysates, cells were harvested and lysed in 200 µL of lysis buffer, as previously described [14]. For cytosolic fraction isolation, cells were mechanically-lysed in isolation buffer for cells (IBC) (10 mM Tris/MOPS, 1 mM EDTA/Tris, 200 mM sucrose, pH 7.4) using a glass potter homogenizer and lysates were centrifuged at 12,000 rpm for 20 min.…”

5-(Carbamoylmethylene)-oxazolidin-2-ones as a Promising Class of Heterocycles Inducing Apoptosis Triggered by Increased ROS Levels and Mitochondrial Dysfunction in Breast and Cervical Cancer

“…Identical amounts of proteins from total lysate, mitochondrial, or cytosolic fraction were resolved on SDS-polyacrylamide gel, blotted to nitrocellulose membrane, and probed with proper primary antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) [37]. Immunoreactive products were identified by using ECL Western blotting detection system (Bio-Rad Laboratories, Hercules, CA, USA) [38].…”

Antioxidant and Anti-Inflammatory Activities of Flavanones from Glycyrrhiza glabra L. (licorice) Leaf Phytocomplexes: Identification of Licoflavanone as a Modulator of NF-kB/MAPK Pathway

“…RAW 264.7 cells were grown to 70–80% confluence and simultaneously treated for 2 h with LPS (1 µg/mL) and extract or licoflavanone at their IC 50 . Then, cells were lysed as previously described [31], using 200 µL of lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 2 mM sodium fluoride, 2 mM EDTA, 0.1% SDS) containing a mixture of protease inhibitors (aprotinin, phenylmethylsulfonyl fluoride, and sodium orthovanadate; Sigma-Aldrich) useful for total protein extraction. The same amounts of proteins were resolved on 15% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane and probed with p-p38, p-JNK and p-ERK1/2 specific antibodies (Santa Cruz Biotechnology).…”