| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2004: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2003: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2002: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
Expression of the V-ATPase proteolipid subunit and V-PPase of Acetabularia acetabulum in a VMA3-deficient stain of Saccharomyces cerevisiae and studies of its complementation
We focused our attention on the function of the translation products of six cDNAs (Vc1 to Vc6) for the proteolipid subunit of A.acetabulum V-ATPase. By heterologous expression in a VMA3-deficient strain of S.cerevisiae and its complementation study, we confirmed that four cDNAs (Vc2,4,5 and 6) encode functional proteolipid subunits in the yeast V-ATPase complex, but two others (Vc1 and 3) did not, when constructed in a yeast expression vector, pKTΔATG with GraPDH promoter. The function of the translation product of cDNA for A.acetabulum V-PPase was examined using the S.cerevisiae VMA3-deficient strain. The acidification of the vacuole was observed by fluorescence microscopy when the cDNA was constructed in pYES2 with GAL1 promoter.
We found that the translation products of Vc1 and Vc3 also complemented the VMA3-defi
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cient strain, when the cDNAs were constructed in pYES2. In addition, the translation products of Vc1 to Vc6 did not complement a VMA11-deficient strain.
In conclusion, the 6 isoforms of the proteolipid subunit of A.acetabulum V-ATPase function as Vma3p but not as Vma11p.
Localization of the 6 isoforms in Acetabularia cells : immunoblot analysis and histochemical staining
Specific polyclonal antibodies against the N-terminal adjacent oligopeptides of the six isoforms were raised, and used for immuno analysis and immunoblot histochemical staining.
The reproductive phase of Acetabularia cells were dissected into caps, stalks and whorl of hairs, from which the microsomal fractions were prepared and subjected to immunoblot analysis. The Vc1,2 and 4 isoforms were detected in the caps. The Vc2 and 4 isoforms were found in stalks, and the Vc2 isoform in whorl of hairs in very small amounts. V-PPase polypeptide was detected in the 3 sections with similar intensities. V-PPase is supposed to be a major proton pump in whorl of heirs.
Immunohistochemical staining was performed using caps of Acetabularia cells. Fluorescence dots were observed for vacuolar membranes, when Vc1,2 and 4 antibodies were used as the first antibodies. These results were coincident with those of immunoblot analysis.
Northern analysis was performed using total RNA prepared from Acetabularia cells. All the six mRNAs were detected as the results of Northern hybridization. The N-terminal peptides of the Vc3,5 and 6 isoforms are possibly processed as signal peptides.
Molecular cloning of Na, K-ATPase cDNA homologu from A.acetabulum
A cDNA coding for a putative Na, K-ATPase was isolated from A.acetabulum. The full-length of the cDNA was 3,968 bp long and encoded a 1192 amino acid protein with a molecular weight of 133,170. The deduced product exhibited around 40% identity with Heterosigma akashiwo Na-ATPase (HANA) and possessed a hydrophilic sequence of 234 amino acid residues in the M7-M8 junction as observed for HANA. Less
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