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 Atm1p 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 IC(50) of 4 muM. MgADP is a product inhibitor for Atm1p with an IC(50) of 0.9 mM. The Michaelis-Menten constants V(max), K(M) and k(cat) of Atm1p were measured as 8.7+/-0.3 muM/min, 107+/-16 muM 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 Atm1p ATPase activity demonstrated a reactivity order of Mg(2+)>Mn(2+)>Co(2+), 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.[1]References
- Characterization of Saccharomyces cerevisiae Atm1p. Functional studies of an ABC7 type transporter. Chen, C.A., Cowan, J.A. Biochim. Biophys. Acta (2006) [Pubmed]
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