TAILIEUCHUNG - Báo cáo Y học: Expression of the V-ATPase proteolipid subunit of Acetabularia acetabulum in a VMA3-deficient strain of Saccharomyces cerevisiae and study of its complementation

Department of Nutritional Science, Faculty of Health and Welfare Science, Okayama Prefectural University, Soja, Japan; 2Division of Biological Science, Institute of Scientific and Industrial Research, Osaka University, Japan; 3Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Japan The function of the translation products of six different cDNAs for Acetabularia V-ATPase proteolipid subunit (AACEVAPD1 to AACEVAPD6 ) was examined using a Saccharomyces cerevisiae VMA3-deficient strain that lacked its own gene for one of the proteolipid subunits of V-ATPase | Eur. J. Biochem. 268 6097-6104 2001 FEBS 2001 Expression of the V-ATPase proteolipid subunit of Acetabularia acetabulum in a VMA3-de icient strain of Saccharomyces cerevisiae and study of its complementation Mikiko Ikeda1 Misato Hinohara1. Kimiko Umami1 Yuki Taauro1 Yoshio Okada1 Yoh Wada2 Yoichi Nakanishi3 and Masayoshi Maeshima3 1 Department of Nutritional Science Faculty of Health and Welfare Science Okayama Prefectural University Soja Japan 2Division of Biological Science Institute of Scienti c and Industrial Research Osaka University Japan 3Laboratory of Cell Dynamics Graduate School of Bioagricultural Sciences Nagoya University Japan The function of the translation products of six different cDNAs for Acetabularia V-ATPase proteolipid subunit AACEVAPD1 to AACEVAPD6 was examined using a Saccharomyces cerevisiae VMA3-deficient strain that lacked its own gene for one of the proteolipid subunits of V-ATPase. Expression of the cDNAs in the strain revealed that four cDNAs from the six complemented the proton transport activity into the vacuole visualized by fluorescence microscopy. The vacuolar-membrane-enriched fractions from the four transformants showed crossreactivity with antibodies against the subunits a and A of S. cerevisiae V-ATPase. Two translation products from the other two cDNAs were demonstrated not to be localized in vacuolar membranes and thus could not complement the function of the VMA3-deficient strain. As the primary structures deduced from the former four cDNAs are similar but clearly different from those of the latter two the latter two translation products may not be able to substitute for theVMA3 gene product. Keywords proton transport proteolipid subunit V-ATPase Acetabularia acetabulum heterologous expression. The vacuolar H1-ATPase V-ATPase is ubiquitous both in prokaryotes and eukaryotes. This enzyme is composed of two domains a large peripheral domain V1 and a membrane integral domain VO . The major component of the VO portion common to

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