MOLECULAR-CLONING OF P-TYPE ATPASES ON INTRACELLULAR MEMBRANES OF THEMARINE ALGA HETEROSIGMA-AKASHIWO

Two cDNA clones (HAA13 and HAA1) which include conserved regions of ge nes of P-type ATPases were isolated from the marine alga Heterosigma a kashiwo by a method that included the polymerase chain reaction. The l onger cDNA (3286 bp), HAA13, consisted of an open reading frame that e ncoded a 106 kDa polypeptide of 977 amino acids with several possible transmembrane domains and conserved regions of eukaryotic P-type ATPas es. One transmembrane domain had a leucine zipper structure. HAA1 was not a full-length gene (2054 bp) and lacked the 5' region, but it also included the conserved regions and putative transmembrane domains. An tibodies against. the polypeptides encoded by HAA13 and HAA1 that have been expressed in Escherichia coli reacted with 100 kDa and 95 kDa po lypeptides, respectively, on intracellular membranes of H. akashiwo ce lls. Immunostaining of H. akashiwo cells revealed that the HAA13 antig en was distributed on membranes around chloroplasts and the HAA1 antig en was located on small vesicles.