## Doctoral Dissertations

#### Title

The vacuolar H$\sp+$-ATPase from {\it Giardia lamblia\/}: A potential model for the study of the evolution of the first eukaryotes

January 1998

#### Keywords

Biology, Molecular|Biology, Genetics|Biology, Cell|Biology, Microbiology

Ph.D.

#### Abstract

Changes in the primary and quarternary structure of vacuolar and archaeal type ATPases that accompany the pro- to eukaryote transition are analyzed. The sequence of the genes encoding the proteolipid and catalytic subunit of the vacuolar ATPase from Giardia lamblia is reported. Giardia has a typical vacuolar ATPase as observed from the common motifs shared between its proteolipid subunit and other eukaryotic vacuolar ATPases, suggesting that the former enzyme works as a hydrolase in this primitive eukaryote. The phylogenetic analyses of the V-ATPase catalytic subunit, and the front and back halves of the proteolipid subunit strongly place Giardia as the deepest branch within the eukaryotes. Phylogenetic analysis indicates that at least two independent duplication and fusion events gave rise to the larger proteolipid type found in eukaryotes and in Methanococcus. The spatial distribution of the conserved residues among the vacuolar type proteolipids suggest a zipper type interaction among the transmembrane helices and surrounding subunits of the V-ATPase complex. Important residues involved in the function of the F-ATP synthase proteolipid have been replaced during evolution in the V-proteolipid, but are retained in some archaeal A-ATPase. Their possible implication in the evolution of V/F/A-ATPases is discussed.^ The gene encoding V-ATPase catalytic subunit of Giardia, as well as the proteolipid subunit, share motifs at the 5$\sp\prime$ and 3$\sp\prime$ untranslated regions common to other Giardia genes. The total Mg$\sp{2+}$-ATPase activity in Giardia membranes is sensitive to o-vanadate, N-ethylmaleimide, dicyclohexyl-carbodiimide, KNO$\sb3,$ and KCl. The recombinant catalytic subunit was expressed, purified and used for producing antibodies. Attempts to affinity purify the whole V-ATPase complex were done using the affinity purified antibodies.^ Evidence of horizontal transfer of V/F/A-ATPase genes is presented. An example of a mobile genetic element was found in the A-ATPase catalytic subunit of Thermoplasma, containing an intein lacking the endonuclease domains. The importance of horizontal gene transfer in microbial evolution is discussed. ^

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