Cloning and expression of cDNAs encoding plant V-ATPase subunits in the corresponding yeast null mutants.
Complementation of yeast null mutants is widely used for cloning of homologous genes from heterologous sources. We have used this method to clone the relevant V-ATPase genes from lemon fruit and Arabidopsis thaliana cDNA libraries. The pH levels are very different in the vacuoles of the lemon fruit and the A. thaliana, yet both are the result of the activity of the same enzyme complex, namely the V-ATPase. In order to investigate the mechanism that enables the enzyme to maintain such differences in pH values, we have compared the subunit composition of the V-ATPase complex from both sources. Towards this end, we have constructed a cDNA library from lemon fruit and cloned it into a similar shuttle vector to the one of the A. thaliana cDNA library, which is commercially available. In this work, we report the cloning and expression of VMA10 from both sources, two isoforms of the lemon proteolipid (VMA3) and the lemon homologue of yeast VPH1/STV1 subunit, LEMAC.
Aviezer-Hagai
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Department of Biochemistry, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Israel.
Nelson
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Nelson
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D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics*; D002957:Citrus / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis / Q000302:isolation & purification; D015723:Gene Library; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics*; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics; D011510:Proteolipids / Q000737:chemistry / Q000235:genetics; D006180:Proton-Translocating ATPases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment; D025262:Vacuolar Proton-Translocating ATPases
D018076:DNA, Complementary; D010940:Plant Proteins; D020033:Protein Isoforms; D011510:Proteolipids; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases