Primary structure and evolution of the atp-binding domains of
protozoan ion pumps: evidence for multiple isoforms of p-type atpases
in tetrahymena thermophila .
Wang, Shusheng, and Kunio Takeyasu.
Department of Medical Biochemistry and Neurobiotechnology Center,
Program of Molecular, Cell and Developmental Biology, The Ohio State
University, Columbus, Ohio 43210, USA and Department of Natural
Environment Sciences, Faculty of Integrated Human Studies, Kyoto
University, Sakyo-ku, Kyoto 606-01, Japan
APStracts 3:0302C, 1996.
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
pumps show a high degree of divergence in their primary structures,
but share a limited number of common amino-acid residues for their
ATP-catalytic function. Particularly, the amino acid sequences for
the phosphorylation site (DKTGTLT) and the binding site for ATP (and
its analogues) (GDGVND) are conserved throughout evolution. Using two
degenerate oligonucleotides corresponding to these regions, we
applied a PCR technique to the search for isoforms of P-type ATPases,
which will provide a clue to the evolutionary mechanisms of ion-pumps
in Tetrahymena thermophila. A total of 12 distinct P-type ATPase
genes were identified. Sequence comparisons revealed that seven of
them can 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
SERCA-ATPase gene, and other four share a significant homology to the
gene encoding Plasmodium ATPase-1 whose function is unknown. A
Northern blot analysis and RT-PCR demonstrated that all identified
genes are expressed, but the expression levels vary widely under
different culture conditions. A Southern blot analysis following
pulse-field gel electrophoresis showed that all of these genes exist
in Tetrahymena macronuclei. The Na+/K+ and H+/K+-ATPase gene family
has a high multiplicity (at least 10 different genes detected on
genomic Southern blot analysis), and is distributed on 4 different
macronuclear chromosomes. Based on a calculation using the amino-acid
sequences of the cloned cytoplasmic loop region (between the
phosphorylation and the CIR ATP sites), the genes with >80 %
identities form a cognate linkage group within the same macronuclei
chromosome, while the genes with <70 % identities are
separated in different chromosomes. The phylogenetic analysis showed
that this multigene family is the result of a series of gene
duplications.
Received 2 February 1996; accepted in final form 6 September
1996.
APS Manuscript Number C64-6.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 5 November 1996