AtPex16p

Lin et al. (1999, 2004) suggested that AtPex16p, also called SSE1p from its shrunken seed mutant phenotype, was essential for ER-dependent protein and oil-body biosynthesis in developing Arabidopsis seeds. An implication for its involvement in peroxisomal biogenesis was the peroxisomal localizations of GFP-AtPex16p in root hairs and embryos of stably transformed Arabidopsis plants. Karnik and Trelease (2005) discovered a definite link between endogenous AtPex16p and ER in suspension-cultured Arabidopsis cells. At-Pex16p coexisted at steady state within ER and peroxisomes. An example of these results is presented in Fig. 3. The immunofluorescence image in panel A shows AtPex16p throughout a reticular compartment in each cell, and within organelles exhibiting a punctate pattern. Panel B shows that the punctate organelles are catalase-containing peroxisomes. The punctate AtPex16p in panel A is co-localized with peroxisomes shown in panel B. Evidence that the reticular pattern (in panel A) is ER is convincingly demonstrated in the comparison of the confocal images presented in panels C and D. Virtually 100% of the reticular AtPex16p in panel C is co-localized with the reticular ER marker BiP in panel D. The extensive co-localization indicates that this peroxin homolog is distributed within most of the reticular ER in the cells. This ER localization is significantly different from the ER localization of APX, i.e., endogenous and transiently expressed APXs are observed only within subdomains of ER, which constitute a relatively small proportion of the total ER in each cell (Mullen et al. 1999).

Fig. 3 Confocal immunofluorescence images reveal coexistence of endogenous At-Pex16p in both ER and peroxisomes in wild-type suspension-cultured Arabidopsis cells. A-D Representative confocal section images of cells that were fixed in formaldehyde, treated with pectolyase and cellulase, permeabilized (membranes) with Triton X-100, and then primary and dye-conjugated secondary antibodies added to the cells adhered to glass microscope slides. A Cells exhibit Cy2 punctate (arrows) and reticular patterns (arrowheads) labeled with rabbit anti-AtPex16p IgGs (1: 500, 1 h). B The same cells as those in A exhibit co-localized Cy5 peroxisomal catalase immunofluorescence due to mouse anti-catalase monoclonal IgGs (1: 500, 1 h). C A different portion of the same population of cells exhibit a Cy2 punctate pattern and a reticular pattern (arrowheads) labeled with rabbit anti-AtPex16p IgGs (1: 500, 1 h). D The same cells as those in C exhibit a co-localized Cy5 reticular pattern (arrowheads) attributable to the mouse monoclonal BiP antibodies (1: 500, 1 h). Bar = 5 |im (from Karnik and Trelease 2005)

Fig. 3 Confocal immunofluorescence images reveal coexistence of endogenous At-Pex16p in both ER and peroxisomes in wild-type suspension-cultured Arabidopsis cells. A-D Representative confocal section images of cells that were fixed in formaldehyde, treated with pectolyase and cellulase, permeabilized (membranes) with Triton X-100, and then primary and dye-conjugated secondary antibodies added to the cells adhered to glass microscope slides. A Cells exhibit Cy2 punctate (arrows) and reticular patterns (arrowheads) labeled with rabbit anti-AtPex16p IgGs (1: 500, 1 h). B The same cells as those in A exhibit co-localized Cy5 peroxisomal catalase immunofluorescence due to mouse anti-catalase monoclonal IgGs (1: 500, 1 h). C A different portion of the same population of cells exhibit a Cy2 punctate pattern and a reticular pattern (arrowheads) labeled with rabbit anti-AtPex16p IgGs (1: 500, 1 h). D The same cells as those in C exhibit a co-localized Cy5 reticular pattern (arrowheads) attributable to the mouse monoclonal BiP antibodies (1: 500, 1 h). Bar = 5 |im (from Karnik and Trelease 2005)

Results from cell-fractionation experiments confirmed the coexistence of AtPex16p in ER and peroxisomes (Karnik and Trelease 2005). Immunoblots revealed the presence of AtPex16p in purified peroxisome and ER fractions obtained from sucrose gradients. AtPex16p also exhibited a definite Mg+2-

induced shift with ER vesicles. AtPex16 seems to be a peripheral, ionically bound membrane protein in both ER and peroxisomes. Topological orientation results were interesting and somewhat surprising in that AtPex16p was found mostly on the matrix side of the peroxisomal boundary membranes, whereas it was mostly on the cytosolic surface of ER membranes. The combined results indicate a bifunctional role for AtPex16p. That is, a portion of the ER-localized protein may function in constitutive oil- and protein-body biogenesis. Another portion of the ER-localized protein and that localized in ER vesicles and peroxisomes may function in the maturation of immature pre-existing peroxisomes, and/or constitutive/induced replication of mature pre-existing peroxisomes (semi-autonomous pathway, Fig. 1).

The trafficking pathway for nascent AtPex16p lends support to the latter function hypotheses (Karnik and Trelease 2006). Time course, cold treatment, and BFA incubation experiments with both wild-type and mycAtPex16p-transformed BY-2 and Arabidopsis cells resulted in unambiguous accumulations of mycAtPex16p in ER. During subsequent time and recovery periods, mycAtPex16p was detected in vesicles (likely derived from ER) en route to pre-existing peroxisomes. These results mirrored published results with the membrane enzyme APX. Interestingly however, the PMPs trafficked from ER in different vesicles en route to the same pre-existing peroxisomes.

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