A Dominant Trifluoperazine Resistance Gene from Saccharomyces cerevisiae Has Homology with F₀F₁ ATP Synthase and Confers Calcium-Sensitive Growth

Abstract

The antipsychotic drug trifluoperazine has been long considered a calmodulin inhibitor from in vitro studies but may function in vivo as a more general inhibitor by disturbing ion fluxes and altering the membrane potential. Resistance to trifluoperazine can arise in Saccharomyces cerevisiae cells by alterations in at least three distinct genetic loci. One locus, defined by a spontaneous dominant trifluoperazine resistance mutation (TFP1-408), was isolated and sequenced. The sequence of the TFP1-408 gene revealed a large open reading frame coding for a large protein of 1,031 amino acids with predicted hydrophobic transmembrane domains. A search of existing amino acid sequences revealed a significant homology with F0F1 ATP synthase. Mutant TFP1-408 cells did not grow efficiently in the presence of 50 mM CaCl2, whereas wild-type cells did. Wild-type cells became resistant to trifluoperazine in the presence of 50 mM CaCl2 or 50 mM MgCl2. Mutant cells showed a higher rate of calcium transport relative to wild-type cells. These data suggest that the TFP1 gene product codes for a transmembrane ATPase-like enzyme possibly involved in Ca2+ transport or in generating a transmembrane ion gradient between two cellular compartments.

Protein Phosphatase 2A in Saccharomyces cerevisiae: Effects on Cell Growth and Bud Morphogenesis

“…Cloning of three yeast genes encoding PP2A-related phosphatases. In a search for high-copy-number suppressors of the GAL] promoter (37,38), we cloned a plasmid containing the 5' half of the TFPI gene (43). The suppressor activity was mapped to a GAL4 site in TFPI, which inhibits GAL] expression by promoter competition (37).…”

Expression of a Proteolipid Gene from a High-Copy-Number Plasmid Confers Trifluoperazine Resistance to Saccharomyces cerevisiae

“…Strains and plasmids. The wild-type TFP-sensitive S. cerevisiae strains NF134 (MATa his4-539 lys2-801 ura3-52), NF147 (MATot ade2 ura3-52), and NF182 (MATa ade2 his3-A200 leu2-3,112 lys2-801 trpl-J ura3-52) are of the S288c background (23,26). The yeast genomic library in YEp24 contains DNA from the S288c background (5).…”

“…Bacterial strains and phage used for the TnJO LUK hop were provided by W. Raymond and N. Kleckner (14). Other bacterial strains, plasmid vectors, media, and methods were as described before (26).…”

“…Construction of deletions and DNA sequencing. Restriction enzyme reactions, ligations, and DNA preparations were done as described before (26). Unidirectional deletions were generated by using exonuclease III (Exo-II) (19).…”

“…The sequencing primer used to determine the insertion site of the transposon was provided by C. Clark-Adams and F. Winston (6). DNA sequence information was compiled and analyzed as described before (26).…”