Characterization of Saccharomyces cerevisiae Atm1p. Functional studies of an ABC7 type transporter |
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Authors: | Chun-An Chen JA Cowan |
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Institution: | Evans Laboratory of Chemistry, Ohio State University, 100 West 18th Avenue, Columbus, OH 43210, USA |
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Abstract: | 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 IC50 of 4 μM. MgADP is a product inhibitor for Atm1p with an IC50 of 0.9 mM. The Michaelis–Menten constants Vmax, KM and kcat of Atm1p were measured as 8.7 ± 0.3 μM/min, 107 ± 16 μM 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 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. |
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Keywords: | ABC ATP-binding cassette NBD nucleotide-binding domain Ni-NTA Ni-nitrilotriacetic acid PC phosphatidylcholine PE phosphatidylethanolamine PMSF Benzenemethanesulfonyl fluoride TM transmembrane YPD yeast extract peptone dextrose |
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