Fig. 2

Analysis of subcellular localization and membrane topology of putative plant TAMPs. a Targeting analysis by confocal microscopy of plant proteins expressed as GFP fusion proteins in BY2 cells. The constructs expressed are illustrated schematically and included: wildtype, GFP fused to the N-terminus of the complete coding region indicated above the panel; +tail, GFP fused to the ‘tail’ sequence only, i.e., TMD and CTS; and –tail, GFP fused to the rest of the coding sequence without the ‘tail’ sequence. As localization markers the indicated protein (mCherry-BCAT3, mCherry-Vac) was co-expressed or the cells were stained with mCherry-conjugated ConA, or immunofluorescence stained with antibody recognizing E1β. Scale bar = 10 μm. b Topology assessment of plant TAMPs in BY2 cells by BiFC. Representative confocal microscope images of plant cells co-expressing cCFP fused to the amino-terminus of the indicated putative TAMP, with the cytoplasmic fusion protein, nVenus-CAT. Shown also are the corresponding images of the chlorophyll autofluorescence, ER marker, vacuole marker, and peroxisome targeting signal (Perox) fused to mCherry. APG1 functions as a negative control. Scale bar = 20 μm