title: Physical and functional interaction between yeast Pif1 helicase and Rim1 single-stranded DNA binding protein.
abstract: 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 µM. 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 (Rim1ΔC18) exhibited a nearly 4-fold higher K(d) value. Rim1 stimulated Pif1 DNA helicase activity by 4- to 5-fold, whereas Rim1ΔC18 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 (Pif1ΔN) 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205-7199, USA.
authors.forename: Ramanagouda
authors.initials: R
authors.lastname: Ramanagoudr-Bhojappa
authors.forename: Lauren P
authors.initials: LP
authors.lastname: Blair
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Tackett
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Raney
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D004265:DNA Helicases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017384:Sequence Deletion
chemical_list: D004268:DNA-Binding Proteins; D024101:Mitochondrial Proteins; C489601:RIM101 protein, S cerevisiae; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; C085463:PIF1 protein, S cerevisiae; D004265:DNA Helicases