Pif1 helicase plays various roles in the maintenance of nuclear and mitochondrial genome integrity in most eukaryotes. Here, we used a proteomics approach called isotopic differentiation of interactions as random or targeted to identify specific protein complexes of Saccharomyces cerevisiae Pif1. We identified a stable association between Pif1 and a mitochondrial SSB, Rim1. In vitro co-precipitation experiments using recombinant proteins indicated a direct interaction between Pif1 and Rim1. Fluorescently labeled Rim1 was titrated with Pif1 resulting in an increase in anisotropy and a K(d) value of 0.69 ?M. Deletion mutagenesis revealed that the OB-fold domain and the C-terminal tail of Rim1 are both involved in interaction with Pif1. However, a Rim1 C-terminal truncation (Rim1?C18) exhibited a nearly 4-fold higher K(d) value. Rim1 stimulated Pif1 DNA helicase activity by 4- to 5-fold, whereas Rim1?C18 stimulated Pif1 by 2-fold. Hence, two regions of Rim1, the OB-fold domain and the C-terminal domain, interact with Pif1. One of these interactions occurs through the N-terminal domain of Pif1 because a deletion mutant of Pif1 (Pif1?N) retained interaction with Rim1 but did not exhibit stimulation of helicase activity. In light of our in vivo and in vitro data, and previous work, it is likely that the Rim1-Pif1 interaction plays a role in coordination of their functions in mtDNA metabolism.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Annotation Extension||Reference|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Assay||Construct||Conditions||Strain Background||Reference|