Additional file 3.
Localization and topology of nVenus and cVenus fusion proteins used in BiFC assays. (A) Tobacco BY-2 cells were transformed transiently (via biolistic bombardment) with selected individual nVenus (and myc-tagged) or cVenus (and HA-tagged) fusion proteins as shown in Figures 7A and 7B (refer to Methods 'Construction of plasmids: Plasmids used for BiFC' for details on the cloning of individual Venus half and epitope-tagged fusion proteins). With the exception of cell transformed with p33-cVenus, all cells were also co-transformed with βATPase-GFP, consisting of the N-terminal mitochondrial targeting presequence (residues 1–60) of the βATPase fused to the N terminus of GFP, and serving as a well-established mitochondrial marker protein [66,67], and thus confirming their mitochondrial localization. Cells transformed with p33-cVenus were co-transformed with RFP-MFP, consisting of the RFP fused to the N-terminal end of peroxisomal matrix marker protein MFP serving as a peroxisomal matrix marker protein [90,99]. At ~16 h post-bombardment, all cells were then processed for (immuno)epifluorescence microscopy using anti-myc or anti-HA antibodies. Each micrograph is labelled at the top left with the name of the transiently co-expressed fusion protein. Differential interference contrast (DIC) images correspond to the same cells shown to the left. Note that with the exception of peroxisomal-localized p33-cVenus, all individual nVenus and cVenus fusion proteins sorted to mitochondria as evidenced by their co-localization with βATPase-GFP. p33-cVenus, on the other hand, colocalized with RFP-MFP, as expected for this peroxisomal-localized viral protein . Bar = 10 μm. (B) BY-2 cells were transformed with various individual nVenus (and myc-tagged) or cVenus (and HA-tagged) fusion proteins as in (A); however, cells were incubated with digitonin (rather than triton X-100 as in [A]) which permeabilizes only the plasma membrane and not organellar membranes . Permeabilized cells were then processed for immuno(epi)fluorescence microscopy using anti-myc, anti-HA, anti-E1β, anti-βATPase and/or anti-α-tubulin antibodies. Each micrograph is labelled at the top left with the name of the transiently-expressed fusion protein or endogenous E1β, βATPase or α-tubulin. Note that the presence or absence of an immunofluorescence signal attributable to an expressed fusion protein indicates whether or not the protein's appended myc- or HA-epitope tag (immediately adjacent to nVenus or cVenus, respectively) is exposed to the cytosol or not; compare to the absence or presence of an immunofluorescence signal attributable to mitochondrial matrix-localized E1β or cytosolic α-tubulin in the same cells. Note also that the relative position of the myc- or HA-epitope tag (and the immediately adjacent nVenus or cVenus fragment, respectively) for selected fusion proteins localized to the mitochondrial outer membrane are shown in Figure 7B (indicated with asterisks). DIC images correspond to the same cells shown to the left. Bar = 10 μm.
Format: TIFF Size: 4.3MB Download file
Hwang et al. BMC Cell Biology 2008 9:54 doi:10.1186/1471-2121-9-54