Figure 6

Localization of p36 intervening-loop mutants in BY-2 cells. (A) Deduced amino acid sequence of the intervening loop sequence (residues 120–165) and portions of the immediately-adjacent TMDs (underlined) in p36. The numbers shown above individual amino acids indicate their relative positions in p36. Amino acids predicted to form an α-helix (residues 131–157) are shaded grey and residues that are proposed to constitute the positively-charged face of the α-helix are bolded. Secondary structure prediction was carried out using Jpred http://www.compbio.dundee.ac.uk/~www-jpred/. Numbers next to each amino acid residue shown in the α-helical wheel projection correspond to their relative positions in p36 (residues 121–157) beginning at the N-terminal end of the predicted α-helix. Positively-charged residues in the α-helix are colored red. (B) Schematic illustrations of various p36-myc mutant proteins and their corresponding subcellular localizations in transformed (via biolistic bombardment) tobacco BY-2 cells. The numbers in the name of each p36 mutant denote the specific amino acid residues that were either deleted or replaced with glycine residues. Portions of the p36 ORF are colorized blue or black, the latter denoting the two putative TMDs in p36. Asterisks highlight the relative position of the cluster of positively-charged amino acids that were replaced with glycine residues. Grey boxes denote the C-terminal myc epitope. Cyt, cytosol; Mito, mitochondria. (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 or the endogenous mitochondrial marker protein, E1β. Hatched boxes represent the portion of the cells shown at higher magnification in the panels to the right. Arrowheads indicate obvious colocalizations. Bar = 10 μm.