Characterization of Saccharomyces cerevisiae Atm1p functional studies of an ABC7 type transporter

Saccharomyces cerevisiae Atm1p has been cloned, over-expressed and purified from a yeast expression system. The sequence includes both the soluble ATPase and transmembrane-spanning domains. With the introduction of an N-terminal Kozak sequence and a C-terminal (His)(6)-tag, a yield of 1 mg of Atm 1p was obtained from 3 g wet yeast cells, which is comparable to other membrane-associated proteins isolated from eukaryotic expression systems. The ATPase activity of Atm1p is sensitive to sodium vanadate, a P-type ATPase inhibitor, with an IC50 of 4 PM. MgADP is a product inhibitor for Atm1p with an IC50 of 0.9 mM. The Michaelis-Menten constants V-max K-m and k(cat) of Atm 1p were measured as 8.7 +/- 0.3 mu M/min, 107 +/- 16 mu M and 1.24 +/- 0.06 min(-1) respectively. A plot of ATPase activity versus concentration of Atm1p exhibits a nonlinear relationship, suggesting an allosteric response and an important role for the transmembrane domain in mediating both ATP hydrolysis and MgADP release. The metal dependence of Arm I p ATPase activity demonstrated a reactivity order of Mg2+ > Mn2+ > Co2+, while each divalent ion was found to be inhibitory at higher concentrations. The activation and inhibitory effect of phospholipids suggest that formation of a lipid-micelle complex is important for enzymatic activity and stability. Structural analysis of Atm1p by CD spectroscopy suggested a similarity of secondary structure to that found for other members of this ABC protein family. (c) 2006 Elsevier B.V. All rights reserved.