Source was yeast cell line; bait produced as transgenic fusion protein; prey produced as transgenic fusion protein (prey was previously cloned reagent).

Two-hybrid system: yeast GAL4-BD/GAL4-AD

Kmn1, Mis12 and either Nnf1a or Nnf1b were coexpressed in E. coli (otherwise proteins were insoluble), then purified by gel filtration. The complex was then treated with BS2G bi-functional crosslinking reagent, and cross-links were mapped by mass spectrometry.

Within either the Kmn1-Mis12-Nnf1a or Kmn1-Mis12-Nnf1b trimeric complexes, all three subunits make extensive contacts with each other, extending along their entire sequences.

Interaction in vitro; proteins produced as a recombinant fusion proteins in bacterial system.

Kmn1, Mis12 and either Nnf1a or Nnf1b were coexpressed in E. coli (otherwise proteins were insoluble), then analyzed by gel filtration to assess complex formation.

Kmn1 and Mis12 interact as part of a trimeric complex with either Nnf1a (64.5 kDa) or Nnf1b (67.1 kDa).

Interaction in vitro; proteins produced as a recombinant fusion proteins in bacterial system.

Interaction in vitro; proteins produced as a recombinant fusion protein in bacterial system.

A Nnf1a-Mis12-Kmn1 trimer ( 1:1:1 ) was analysed by mass spectrometry to identify regions protected from hydrogen-deuterium (H/D), indicative of interacting surfaces.

Interaction in vitro; proteins produced as a recombinant fusion protein in bacterial system.

A Nnf1a-Mis12-Kmn1 trimer ( 1:1:1 ) was analysed by mass spectrometry to identify regions protected from hydrogen-deuterium (H/D), indicative of interacting surfaces.

Interaction in vitro; proteins produced as a recombinant fusion protein in bacterial system.

Kmn1, Mis12 and Nnf1a form a trimeric complex in this assay. The complex size was calculated by mass spectrometry to be 72.02 kDa, consistent with a 1:1:1 stoichiometry.

Kmn1, Mis12 and Nnf1a were mixed in various combinations, and complex formation was observed by size exclusion chromatography.