Abstract
Acetabularia acetabulum (A. a) is a giant unicellular marine alga (Dasycladaceae). We isolated 6 different cDNAs (VHA-c1 to VHA-c6) coding for the proteolipid subunit of A. a V-ATPase. After construction of the 6 cDNAs into yeast expression vectors, pKT10ΔATG and pYES2, heterologous expression of the 6 isoforms was conducted in a yeast VMA3- and in a VMA11-deficient strain with ade mutation. The acidification of the vacuole was visualized by fluorescence microscopy due to accumulation of purine intermediate metabolites with ade mutation. The translation products of the 6 cDNAs complemented vma3-mutation, but could not complement vma11-mutation. Polyclonal antibodies were raised against the N-terminal amino acid sequences, and were shown to specifically recognize the corresponding translation products. The two isoforms (VHA-c2 and VHA-c4) were detected in A. a cells before cap formation. Immunohistochemical studies are in progress.
