Physical and functional interaction between yeast Pif1 helicase and Rim1 single-stranded DNA binding protein

저자: Ramanagouda Ramanagoudr-Bhojappa, Lauren P. Blair, Alan J. Tackett and Kevin D. Raney

저널: Nucleic Acids Research, 2013, Vol. 41, No. 2 1029–1046

사용한 제품: Dynabeads M-270 Epoxy (Cat# 14301, 14302D)

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 coprecipitation 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 Kd value of 0.69 mM. 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 Kd 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.

제품 사용법

Immunoisolation

Affinity purification of Pif1-TAP and its associated proteins was performed using IgG-coated Epoxy-270 Dynabeads at 300mM NaCl. Briefly, 10 g of disrupted cell mixture was resuspended in 50 ml immunopurification (IP) buffer (20mM HEPES pH 7.4, 300mM NaCl, 2mM MgCl2, 1 mg DNase I, 0.1% Tween-20, 1/100 protease inhibitor cocktail), incubated with 40 mg of IgG-coated Dynabeads and rotated for 2 h at 4 C. Dynabeads were captured with a magnet and washed five times with IP buffer to reduce nonspecific interactors. Captured Dynabeads were treated with 0.5M ammonium hydroxide to elute associated protein complexes. Immunoisolated protein complexes were resolved on a 4-20% NuPAGE gel and visualized by Coomassie staining with GelCode Blue. The entire gel lane was sliced into 38 equal sections, subjected to tryptic digestion and peptides were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). High resolution spectra of peptides were collected with a PerkinElmerSciex MALDI-prOTOF mass spectrometer, while tandem mass spectra were collected with a Thermo vMALDI-LTQ mass spectrometer. Tandem mass spectrometry data were analyzed using XProteo software. Peptides containing either one or two lysine residues were visualized using M-over-Z software. The appearance of isotopically heavy versions of each peptide was recorded with a 4 or 8 Da higher mass depending on the presence of one or two d4-lysines, respectively. The fraction area under light peptides was calculated as described.

Co-Immunoprecipitation

Purified recombinant SSB proteins (Rim1, Rim1 C18 and HsmtSSB), BSA and glycine were covalently cross-linked onto epoxy-activated Dynabeads M-270 (Invitrogen) as per the manufacturer’s instructions.Saturating amounts of protein were used for coating 2.5mg of epoxy-activated Dynabeads at 37 C for 24 h. Co-precipitation experiments were performed by incubating purified Pif1 protein (20 mg) with SSB-coated beads in a buffer containing 25mM HEPES pH 7.5, 250mM NaCl, 2mM MgCl2, 2mM BME, 0.1mM EDTA, 0.1 mg/ml BSA, 0.1% Tween-20 and 5% glycerol. SSB-coated beads and Pif1 were incubated together in a 300-ml buffer with rotation at 4 C for 2 h. Dynabeads were captured using a magnet and washed five times in 1 ml of buffer. Pif1 protein that co-precipitated with the beads was eluted upon addition of 50 ml of Laemmli sample buffer and heating at 95 C for 5 min. The sample (20 ml) was resolved by 4–20% NuPAGE gel and the proteins were visualized by Coomassie staining with GelCode Blue.