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First published online 27 May 2003
doi: 10.1242/jcs.00517

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Plastid ultrastructure defines the protein import pathway in dinoflagellates

Département de Sciences Biologiques, Université de Montréal, 4101 Sherbrooke est, Montréal, Québec, Canada H1X 2B2

View larger version (31K): [in a new window]   Fig. 2. The PCP leader contains a signal peptide that targets a reporter gene to canine microsomes in vitro. (A) A schematic view of the G. polyedra PCP leader used (top) shows two hydrophobic regions (numbered black boxes) separated by an S/T-rich region (white box) fused to luciferase (line). The small arrow indicates the potential AXA signal peptidase site. This construct was transcribed and translated in vitro in rabbit reticulocyte lysates (RRL) with the additions as illustrated. A smaller protein is produced after translation in the presence of canine microsomes, confirming cleavage after the first hydrophobic region. The topology of the protein, deduced from its susceptibility to trypsin digestion, is shown schematically on the right (small arrow indicates cleavage by the signal peptidase inside the vesicle). Note that the bulk of the protein remains outside the microsomal membrane. (B) A schematic view of the luciferase reporter fused with a modified G. polyedra PCP leader (top) lacking the first hydrophobic region. This construct was also translated as above. Most of the translation product is found in the pellet (P) rather than the supernatant (S) after ultracentrifugation, suggesting that it may be inserted into the microsomal membrane as found in Euglena. The predicted topology is again shown schematically on the right.

View larger version (46K): [in a new window]   Fig. 4. The leader sequence is defined by plastid ultrastructure rather than by its phylogeny. (A) Alignment of signal peptides from (top to bottom) five dinoflagellate (Amphidinium carterae, Symbiodinium sp and Gonyaulax polyedra) and six Euglena gracilis plastid-directed, nuclear-encoded proteins. Two conserved hydrophobic regions (blue) are interspersed with polar (yellow), acidic (red) or basic (green) residues. Gaps (white) were introduced to align the hydrophobic regions. The AXA signal peptidase site is at the end of the first hydrophobic region (arrow). (B) A phylogenetic reconstruction using glyceraldehyde-3-phosphate dehydrogenase sequences shows significant bootstrap support for the apicomplexan Toxoplasma and the dinoflagellate Gonyaulax as sister clades, using both plastid and cytoplasmic isoforms, as well as strong bootstrap support excluding Euglena. Numbers at the nodes indicate bootstrap support (10,000 trees). (C) Plastid evolution. Dinoflagellates and apicomplexans share a common ancestor both for the host cell and the plastid, and are unrelated to either Euglenoids or their plastids. Gene transfer from the endosymbiont nucleus to the new host cell nucleus must be accompanied by addition of a peptide signal, which allows the plastid-directed proteins to pass the new membranes. (D) Representative hydrophobicity plots of signal peptide sequences from the apicomplexan ribosomal protein S9 (RPS9), showing only a single hydrophobic region, as well as from a dinoflagellate and a Euglenoid glyceraldehyde-3-phosphate dehydrogenase (GAP), showing two distinct hydrophobic regions. All plots are to the same scale, with hydrophobicity increasing above the midline.

View larger version (136K): [in a new window]   Fig. 1. Nuclear-encoded plastid proteins transit through the Golgi. Gonyaulax cell sections were treated with antibodies raised against two nuclear-encoded plastid proteins, Rubisco and peridinin-chlorophyll a-protein (PCP). In one series of experiments, cells were harvested and fixed at LDT 0 (by convention, the start of the light phase), a time when both proteins are actively synthesized in vivo. As a control for the specificity of the Golgi labeling, cells were also harvested and fixed at times when Rubisco or PCP were not actively synthesized in vivo (LDT 6 and 19, respectively). Sections were stained with either anti-Rubisco (A-C) or anti-PCP (D-F) as a primary antibody and a 20 nm gold-conjugated goat anti-rabbit as a secondary antibody. The Golgi is indicated by arrows in all pictures, and all scale bars represent 1 µm. Note that Rubisco and PCP have different sub-organellar locations (stroma and thylakoid lumen, respectively) within the plastids (P). The thylakoid membranes appear white, as osmium tetroxide was not used during fixation to preserve the antigenicity of the proteins.

View larger version (128K): [in a new window]   Fig. 3. Targeting to higher plant plastids by the S/T-rich region in the PCP leader. The luciferase reporter construct lacking the first hydrophobic region (as described in Fig. 2B) was fused with a Solanum chacoense 5'UTR and introduced into a highly regenerable genotype (G4) of S. chacoense. Two independent transformants expressed high levels of luciferase in the chloroplasts (A,B), as shown by immunoelectron microscopy using a commercial anti-luciferase as a primary antibody and a 20 nm gold-conjugated rabbit anti-goat as a secondary antibody. Label is observed over the plastid (P) but not the cell wall (CW), vacuole (V) or nucleus (N). Untransformed plants (C) show only background labeling. (D) Quantification of the label density is shown as number of gold beads per µm2 for the two transformed plants above as well as for an untransformed control. Bars, 1 µm.