Intracellular localization of the Apnl DNA repair enzyme of Saccharomyces cerevisiae: Nuclear transport signals and biological role

The Apnl DNA repair enzyme of Saccharomyces cerevisiae acts on abasic sites and oxygen radical damages. Apnl is homologous to the repair endonuclease IV of Escherichia coli, but the yeast protein is ∼80 residues longer at the C terminus. The Apnl C terminus is rich in basic amino acids and includes two lysine/arginine clusters related to the nuclear transport signals of some other proteins. We show here by indirect immunofluorescence that Apnl is localized to the yeast nucleus. Mutant Apn1 proteins were engineered with progressive deletions inward from the C terminus. Elimination of just the last 12 residues from Apnl (to yield Apn355) did not alter the stability in yeast cells or the in vitro activity of the enzyme. Greater truncation of Apnl produced proteins of apparently lower (Apn334) or much lower (Apn315 and Apn293) in vivo stability. Both Apn355 and Apn334 failed to concentrate in the yeast nucleus and remained in the cytoplasm. These delocalized derivatives also failed to restore wild-type resistance to oxidative or alkylating agents in a Δapn1 strain. Apn355 and Apn334 complemented repair-deficient E. coli as effectively as did wild-type Apnl. Resistance to these DNA-damaging agents in yeast was restored if Apn355 and Apn334 (but not Apn315 or Apn293) were overproduced ∼20-fold, which suggests either weak active transport or passive diffusion of these derivatives into the nucleus. Replacement of the C-terminal 12 residues of Apnl with the nuclear targeting sequence of SV40 T-antigen did not restore effective function or nuclear localization in yeast.