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First published online April 24, 2006
doi: 10.1242/10.1242/jcs.02904

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Five Arabidopsis peroxin 11 homologs individually promote peroxisome elongation, duplication or aggregation

School of Life Sciences, Arizona State University, PO Box 874501, Tempe, AZ 85287-4501, USA

View larger version (92K): [in a new window]   Fig. 1. Amino acid sequence alignment of AtPex11a, -b, -c, -d and -e. AtPex11 protein sequences were aligned using the ClustalW algorithm (http://www.ch.embnet.org/software/ClustalW.html). Identical and similar residues were shaded black and gray, respectively, with BOXSHADE (http://www.ch.embnet.org/software/BOX_form.html). Transmembrane domains were predicted using TMpred (http://www.ch.embnet.org/software/TMPRED_form.html), and are overlined in black. Basic clusters of amino acid residues are overlined with dashes. Dilysine motifs are boxed.

View larger version (78K): [in a new window]   Fig. 2. Gene transcripts of AtPEX11a, -b, -c, -d and -e are expressed in Arabidopsis roots, leaves, siliques and suspension culture cells. Total RNA extracted from 5-week-old roots (A), leaves (B) and developing siliques (seed pods) (C), or 3-day-cultured suspension cells (D), was treated with DNase I and reverse transcribed using poly-T primers. AtPEX11a, -b, -c, -d and -e (labeled here 11a-11e) were amplified by PCR using primers complimentary to 5' or 3' termini. Control reactions (E) were designed using the following noncomplementary primer pairs in a leaf cDNA library: foreword primers for AtPEX11c (lanes 1,2), AtPEX11d (lanes 3,4) and AtPEX11e (lanes 5,6), and reverse primers for AtPEX11d (lanes 1,6), AtPEX11e (lanes 2,4) and AtPEX11c (lanes 3,5). No PCR products were observed for any of these reactions. PCR products, shown here, were separated electrophoretically in a 1% agarose gel containing ethidium bromide. As expected, all PCR products were approximately 700 bp in length.

View larger version (93K): [in a new window]   Fig. 3. Immunofluorescence images illustrate that overexpressed, myc-tagged AtPex11a, - b, -c, -d and -e sort directly to peroxisomes during a 2.5 hour post-bombardment period in dual immunolabeled Arabidopsis and tobacco BY-2 suspension cells. The five myc-tagged AtPex11 proteins were introduced individually (biolistic bombardments) into Arabidopsis (A-J) or BY-2 (K-T) cells. Following bombardments (2.5 hours), cells were fixed in formaldehyde, cell walls perforated/digested with pectolyase (and cellulase, Arabidopsis only), and membranes permeabilized in Triton X-100. Cells were then dual immunolabeled with anti-myc plus anti-Cy-2-conjugated antibodies (1:500; 1 hour each) and anti-catalase plus RhodamineX-conjugated antibodies (1:2000; 1 hour each) (labeled columns of cells). Each representative image depicts one transformed cell per panel (labels on left side). In all cases, the myc-AtPex11 protein is colocalized with peroxisomal catalase (arrowheads point to examples in A,F,K,P). Arrows point to peroxisomes in neighboring nontransformed cells. Bars, 10 µm.

View larger version (60K): [in a new window]   Fig. 4. Arabidopsis peroxisomes bearing overexpressed myc-AtPex11c and -d became elongated/tubulated (2.5-72 hours), whereas peroxisomes acquiring myc-AtPex11b became spherical. (A-H) A representative single transformed cell in each panel illustrates elongated peroxisomes possessing myc-AtPex11c (A,C,D) or myc-AtPex11d (E,G,H). Arrows point to examples of perfect colocalization between these myc-tagged proteins (A,E) and endogenous peroxisomal catalase (B,F). Arrowheads point to normal spherical or rod-shaped peroxisomes in neighboring nontransformed cells. (I-L) Single representative transformed cells show spherical/toric peroxisomes bearing overexpressed myc-AtPex11b (I,K,L) perfectly colocalized with endogenous peroxisomal catalase (compare I and J). (M-P) Peroxisomes bearing CAT-SKL (M,O,P) are spherical or rod-shaped and are perfectly colocalized with endogenous catalase (compare M and N). (Q, lower panel) Graphical representation of the percentage of transformed cells bearing peroxisomes 3 µm at various time points post-bombardment (n30 for all data points). Cells were bombarded with myc-AtPEX11a (), -b(), -c (), -d (), -e () or CAT-SKL (), fixed at the indicated time points (x-axis), and immunolabeled with anti-myc plus Cy-2-conjugated antibodies or anti-CAT plus Cy-2-conjugated antibodies. Intercepts at the y-axis (0 hours) were projected to the baseline (3% elongation) for each construct. Bar, 10 µm.

View larger version (73K): [in a new window]   Fig. 5. Arabidopsis peroxisomes bearing overexpressed myc-AtPex11a, myc-AtPex11e or GFP-AtPex11e increase in number per cell between 5 and 45 hours post-bombardment. (A-F, upper panels) Representative myc-AtPex11-transformed cells (left side labels) were fixed at three time points (top labels) and then dual immunolabeled with anti-myc plus Cy-2-conjugated (and anti-catalase, not shown) antibodies. (G-J, lower panels) Single cells expressing chimeric proteins (left and right side labels). (G,H) GFP autofluorescent peroxisomes at 24 and 72 hours post-bombardment. (I,J) Peroxisomal colocalization of anti-CAT/Cy-2 (I) and anti-catalase/Cy-5 (J) antibodies. All panels are confocal epifluorescence projection images. Bar, 10 µm.

View larger version (80K): [in a new window]   Fig. 6. Deletion of the C-terminal dilysine motif in AtPex11d and -e (myc-AtPex11d6 or -e7, respectively) variously affected changes in Arabidopsis peroxisome morphology and duplication. Cells transformed with myc-AtPex11d6 (A-C) or myc-AtPex11e7 (D-F) were fixed at three time points (top labels) and immunolabeled with anti-myc plus Cy-2-conjugated antibodies(dual label with anti-catalase not shown). Bar, 10 µm.

View larger version (49K): [in a new window]   Fig. 7. Both the C- and N-termini of AtPex11b, -c, -d and -e are on the cytosolic side of the peroxisomal membrane, whereas the N- and C-termini of AtPex11a are on the cytosolic and matrix side of the membrane, respectively. (A-T) Bombarded cells were fixed in formaldehyde at 24 (C-H) or 48 (A,B,I,J) hours, treated with pectinase, and then incubated in digitonin to permeabilize plasma (not organellar) membranes. Labels on the left of each pair of panels indicate myc-tagged proteins dual immunolabeled with anti-myc (A-U) and anti-catalase antibodies (K-V). (U-Z) Controls for digitonin permeabilization. (U-X) Anti-myc plus Cy-2-conjugated (U,W) and anti-catalase plus RhodamineX-conjugated (V,X) antibodies applied to cells transformed with myc-AtPex3p and permeabilized with digitonin (U,V) or Triton X-100 (W,X). (Y,Z) A cell from the same batch of AtPex3p-transformed cells permeabilized with digitonin and dual immunolabeled with anti-tubulin plus Cy-2-conjugated (Y) and anti-catalase plus Rhodamine-conjugated (Z) antibodies. Bars, 10 µm.

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