Identification of the nuclear localization signal in the Saccharomyces cerevisiae Pif1 DNA helicase.

Abstract

Saccharomyces cerevisiae Pif1 is a multi-functional DNA helicase that plays diverse roles in the maintenance of the nuclear and mitochondrial genomes. Two isoforms of Pif1 are generated from a single open reading frame by the use of alternative translational start sites. The Mitochondrial Targeting Signal (MTS) of Pif1 is located between the two start sites, but a Nuclear Localization Signal (NLS) has not been identified. Here we used sequence and functional analysis to identify an NLS element. A mutant allele of PIF1 (pif1-NLSΔ) that lacks four basic amino acids (781KKRK784) in the carboxyl-terminal domain of the 859 amino acid Pif1 was expressed at wild type levels and retained wild type mitochondrial function. However, pif1-NLSΔ cells were defective in four tests for nuclear function: telomere length maintenance, Okazaki fragment processing, break-induced replication (BIR), and binding to nuclear target sites. Fusing the NLS from the simian virus 40 (SV40) T-antigen to the Pif1-NLSΔ protein reduced the nuclear defects of pif1-NLSΔ cells. Thus, four basic amino acids near the carboxyl end of Pif1 are required for the vast majority of nuclear Pif1 function. Our study also reveals phenotypic differences between the previously described loss of function pif1-m2 allele and three other pif1 mutant alleles generated in this work, which will be useful to study nuclear Pif1 functions.