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 microM. 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 microM/min, 107+/-16 microM 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.
Chen
Chun-An
CA
Evans Laboratory of Chemistry, Ohio State University, Columbus, OH 43210, USA.
Cowan
J A
JA
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017931:DNA Primers; D005453:Fluorescence; D007700:Kinetics; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
C091561:ATM1 protein, S cerevisiae; D018528:ATP-Binding Cassette Transporters; D017931:DNA Primers; D029701:Saccharomyces cerevisiae Proteins