The primary target of the killer toxin from Pichia acaciae is tRNA(Gln).
The Pichia acaciae killer toxin (PaT) arrests yeast cells in the S-phase of the cell cycle and induces DNA double-strand breaks (DSBs). Surprisingly, loss of the tRNA-methyltransferase Trm9 - along with the Elongator complex involved in synthesis of 5-methoxy-carbonyl-methyl (mcm(5)) modification in certain tRNAs - conferred resistance against PaT. Overexpression of mcm(5)-modified tRNAs identified tRNA(Gln)((UUG)) as the intracellular target. Consistently, toxin-challenged cells displayed reduced levels of tRNA(Gln) and in vitro the heterologously expressed active toxin subunit disrupts the integrity of tRNA(Gln)((UUG)). Other than Kluyveromyces lactis zymocin, an endonuclease specific for tRNA(Glu)((UUC)), affecting its target in a mcm(5)-dependent manner, PaT exerts activity also on tRNA(Gln) lacking such modification. As sensitivity is restored in trm9 elp3 double mutants, target tRNA cleavage is selectively inhibited by incomplete wobble uridine modification, as seen in trm9, but not in elp3 or trm9 elp3 cells. In addition to tRNA(Gln)((UUG)), tRNA(Gln)((CUG)) is also cleaved in vitro and overexpression of the corresponding gene increased resistance. Consistent with tRNA(Gln)((CUG)) as an additional TRM9-independent target, overexpression of PaT's tRNase subunit abolishes trm9 resistance. Most interestingly, a functional DSB repair pathway confers PaT but also zymocin resistance, suggesting DNA damage to occur generally concomitant with specific tRNA offence.
Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstr. 3, D-48149 Münster, Germany.
Roland
R
Klassen
John P
JP
Paluszynski
Sabrina
S
Wemhoff
Annika
A
Pfeiffer
Julia
J
Fricke
Friedhelm
F
Meinhardt
D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D025141:Drug Resistance, Fungal; D055646:Killer Factors, Yeast; D007716:Kluyveromyces / Q000235:genetics / Q000378:metabolism; D009183:Mycotoxins / Q000494:pharmacology*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D012351:RNA, Transfer, Gln / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D012359:tRNA Methyltransferases / Q000235:genetics / Q000378:metabolism
D018797:Cell Cycle Proteins; D055646:Killer Factors, Yeast; D009183:Mycotoxins; D012351:RNA, Transfer, Gln; D029701:Saccharomyces cerevisiae Proteins; C058975:zymocin; C087906:killer toxin, Pichia; C480488:TRM9 protein, S cerevisiae; D012359:tRNA Methyltransferases