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, Nnf1a and Nnf1b were co-expressed (otherwise proteins were insoluble), then pulled down via either tagged Nnf1a or Nnf1b.

Mis12 interacts with either Nnf1a or Nnf1b in distinct complexes.

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

Mis12 interacts with either Nnf1a or Nnf1b to form a dimer. Kmn1 can bind the Mis12-Nnf1a or Mis12-Nnf1b dimer to form a trimeric complex that is 64.5 kDa 67.1 kDa in size, respectively.

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

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

MORATORIUM:This data was made available \'pre-publication\' and, as such, its use is subject to the specific limitations described in FBrf0235788 for the period of 12 months. Moratorium expires on June 13, 2018.

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

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

MORATORIUM:This data was made available \'pre-publication\' and, as such, its use is subject to the specific limitations described in FBrf0235788 for the period of 12 months. Moratorium expires on June 13, 2018.

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