Figure 4

Localization of p36-p33 hybrid proteins in BY-2 cells. (A) Alignment of the deduced amino acid sequences of CIRV p36 and TBSV p33. Sequences were obtained from GenBank and aligned using the ClustalW http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_clustalw.html. Identical amino acids in each protein are indicated with asterisks and putative TMDs (underlined) were determined using TMHMM (version 2.0) and visual inspection. Clusters of amino acids in TBSV p33 that were reported previously [25] to constitute the peroxisomal (i.e., -K₁₁K₁₂-, K₇₆RRQR₈₀- and -R₁₂₄PSVPKK₁₃₀-) or pER (i.e., -K₅R₆-) targeting signals are shaded grey or bolded, respectively. Conserved residues in p33 (residues 184–203) and p36 (residues 218–237) that are immunorecognized by a polyclonal antibody raised against this (synthetic) peptide sequence [25] are italized and bolded. (B) Schematic illustrations of C-terminal myc-tagged p36 (p36-myc), p33 (p33-myc) and various p36-p33 hybrid proteins and their corresponding subcellular localizations in transformed (via biolistic bombardment) tobacco BY-2 cells. The numbers in the name of each hybrid construct denote the specific amino acid residues from p36 or p33 that were fused to (trunctated) p33-myc or p36-myc, respectively. Portions of the p36 ORF are colored blue or black, the latter denoting the two putative TMDs in p36 and the numbers shown above p36-myc indicating their relative amino acid positions. Similarly, portions of the p33 ORF are colored white and red, the latter denoting the two putative TMDs in p33 and the numbers shown above p33-myc indicating their relative amino acid positions. The grey colored box for each construct denotes the C-terminal appended myc epitope. Cyt, cytosol; Mito, mitochondria; Perox, peroxisome. (C) Representative (immuno)epifluorescence micrographs illustrating the localizations of the various constructs shown in (B). Each micrograph is labelled at the top left with the name of either the transiently-expressed protein, the endogenous mitochondrial matrix marker protein E1β, or the endogenous peroxisomal matrix marker protein catalase. Hatched boxes represent the portion of the cells shown at higher magnification in the panels to the right. Solid arrowheads indicate obvious colocalizations; open arrowheads indicate obvious non-colocalizations. Bar = 10 μm.