PRIMARY STRUCTURE AND EVOLUTION OF THE ATP-BINDING DOMAINS OF THE P-TYPE ATPASES IN TETRAHYMENA-THERMOPHILA

The P-type ATPases (e.g., Na+-K+-ATPase and Ca2+-ATPase) occur widely in living cells of fungi, Protozoa, plants, and animals. These ion pum ps show a high degree of divergence in their primary structures but sh are a limited number of common amino acid residues for their ATP-catal ytic function. Particularly, the amino acid sequences for the phosphor ylation site (DKTGTLT) and the binding site for ATP (and its analogs; GDGVND) are conserved throughout evolution. Using two degenerate oligo nucleotides corresponding to these regions, we applied a polymerase ch ain reaction (PCR) technique to the search for P-type ATPase isoforms, which will provide a clue to the evolutionary mechanisms of ion pumps in Tetrahymena thermophila. A total of 12 distinct P-type ATPase gene s were identified. Sequence comparisons revealed that seven of them ca n be compiled into a multigene family, which is similar to animal Na+- K+- and H+-K+-ATPase genes. One of them is close to the sarco(endo)pla smic reticulum Ca2+- ATPase gene, and the other four share a significa nt homology with the gene encoding Plasmodium ATPase-1 whose function is unknown. A Northern blot analysis and reverse transcriptase-PCR dem onstrated that all identified genes are expressed, but the expression levels vary widely under different culture conditions. A Southern blot analysis after pulse-field gel electrophoresis showed that all of the se genes exist in T. thermophila macronuclei. The Na+-K+- and H+-K+-AT Pase gene family has a high multiplicity (at least 10 different genes detected on genomic Southern blot analysis) and is distributed on four different macronuclear chromosomes. On the basis of a calculation wit h the amino acid sequences of the cloned cytoplasmic loop region (betw een the phosphorylation and the ma-[4-(N-2-chloroethyl-N-methylamino)] -benzylamido ATP sites), the genes with >80% identity form a cognate L inkage group within the same macronuclei chromosome, whereas the genes with <70% identity are separated in different chromosomes. The phylog enetic analysis showed that this multigene family is the result of a s eries of gene duplications.