BiFC analysis of interactions among p36 and components of the TOM and SAM complexes. (A) BY-2 cells were co-transformed (via biolistic bombardment) with either p36, the N-terminal mitochondrial matrix targeting presequence of the β subunit of the F1-ATPase (βATPase), porin, p33, or Cb5 fused at either their N or C termini to cVenus (columns) and various constituents of the TOM complex (or an N-terminal mutant version of TOM20; TOM20mut), METAXIN, or p36 fused at either their N or C termini to nVenus (rows). In addition, all cells were co-transformed with either RFP, which served as a convenient means of identifying transformed cells. At ~16 h post-bombardment, cells were formaldehyde fixed and viewed by epifluorescence microscopy. Interactions were scored based on either the presence (+) or absence (-) of a BiFC (Venus) signal relative to reconstitution controls in which individual Venus half fusion proteins were co-expressed with the corresponding "empty" vector containing nVenus or cVenus fragments alone. All BiFC signals observed co-localized with (co)expressed mitochondrial βATPase-GFP. For each pair of plasmids tested, > 25 transformed cells were scored from at least two independent transformation (biolistic bombardment) experiments. ND, not determined. (B) Predicted topologies of mitochondrial outer membrane proteins used in BiFC experiments, the results of which are presented in (A). Also shown is the localization of the soluble βATPase N-terminal mitochondrial targeting presequence in the matrix following import. The topological orientations of mitochondrial outer membrane proteins shown are based on differential permeablization results presented either in Figure 2 (for p36), in Additional file 3 (for TOM proteins, METAXIN, and porin) or published previously in Hwang et al.  (for Cb5). Regions of proteins proposed to be hydrophobic membrane-spanning domains or hydrophilic domains facing the cytosol, intermembrane space or matrix were identified using the TMpred program http://www.ch.embnet.org/software/TMPRED_form.html webcite. The dashed line in the N-terminal portion of TOM20 represents the region of the receptor that was deleted in the corresponding mutant (TOM20mut); specifically, protein's N-terminal 1–142 residues including the tetratricopeptide motif-based receptor domain for mitochondrial precursor proteins . Asterisks indicate the relative position of nVenus or cVenus and the immediately adjacent myc- or HA-epitope tag appended to each protein. IMS, intermembrane space.
Hwang et al. BMC Cell Biology 2008 9:54 doi:10.1186/1471-2121-9-54