Arabidopsis peroxin 16 trafficks through the ER and an intermediate compartment to pre-existing peroxisomes via overlapping molecular targeting signals

Journal of Experimental Botany, Vol. 58, No. 7, pp. 1677–1693, 2007 doi:10.1093/jxb/erm018 Advance Access publication 12 April, 2007 This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details) RESEARCH PAPER Arabidopsis peroxin 16 trafficks through the ER and an intermediate compartment to pre-existing peroxisomes via overlapping molecular targeting signals Sheetal K. Karnik and Richard N. Trelease* Arizona State University, School of Life Sciences, PO Box 874501, Tempe, AZ 85287-4501, USA Received 15 September 2006; Revised 8 January 2007; Accepted 22 January 2007 Previously it has been shown that the endogenous Arabidopsis peroxin, AtPEX16, coexisted at steady state in membranes of the endoplasmic reticulum (ER) and peroxisomes. Herein, an ER-to-peroxisome trafficking pathway and the requisite molecular targeting signals for mycAtPEX16 transiently expressed in Arabidopsis and tobacco BY-2 suspension cells are described. Immunofluorescent mycAtPEX16 was observed initially in the cytosol (<2 h) and subsequently (2–4 h) in perinuclear/reticular ER and non-Golgi/nonperoxisome structures termed the ER-peroxisome intermediate compartment. After 4 h, all catalase- and ascorbate peroxidase-containing peroxisomes also possessed mycAtPEX16, indicative of mycAtPEX16 sorting to pre-existing peroxisomes. Incubations of bombarded cells at 15
C, or in brefeldin A at 25
C, resulted in accumulations of mycAtPEX16 within the ER. Following re-equilibration of cold-treated cells at 25
C, or removal of brefeldin A, mycAtPEX16 was observed mainly in the ER-peroxisome intermediate compartment, and later within all of the peroxisomes in both species. Two internal membrane helices and the intervening sequence including the amino acid residues -VRS- were found necessary and sufficient for targeting AtPEX16 first to the ER and then to peroxisomes. Individual targeting signals for these organelles were indistinguishable, indicative of overlapping signal(s). In summary, the trafficking study of AtPEX16 revealed a dynamic link between the ER and preexisting peroxisomes, which provided novel data in support of an upgraded semi-autonomous peroxisome model portraying participation of the ER in the sorting of certain peroxisome membrane proteins, such as AtPEX16, through an intermediate compartment to pre-existing plant peroxisomes. Key words: Arabidopsis thaliana suspension cells, ascorbate peroxidase, brefeldin A, immunofluorescence microscopy, inter-organellar targeting signals, peroxins, peroxisome membrane protein, reticular endoplasmic reticulum, tobacco BY-2 cells, transient protein expression. Introduction An important feature of peroxisomes is their lack of DNA and protein synthesizing machinery, which dictate posttranslational acquisitions from the cytosol of virtually all of their nuclear-encoded matrix and peroxisome membrane proteins (PMPs) (Sparkes and Baker, 2002; Erdmann and Schliebs, 2005). Peroxin (PEX) genes code for peroxins (PEX), which are proteins that mediate multiple aspects of peroxisome biogenesis such as ontogeny, maturation (differentiation), and multiplication (duplication and induced proliferation). A consecutive numbering system was created to identify and compare PEX genes and their corresponding peroxin homologues (Distel et al., 1996). To date, 23 plant peroxins have been identified. One of these homologues, peroxin 16, has been identified in Arabidopsis (AtPEX16) (Lin et al., 1999) and in only two other species, namely humans (HsPEX16) (Kim et al., 2006) and the yeast Yarrowia lipolytica (YlPEX16) (Titorenko and Rachubinski, 1998). Lin et al. (1999) first reported that the Arabidopsis SSE1 (shrunken seed) gene coded for the putative plant peroxin 16 homologue—AtPEX16. More recently, Lin et al. (2004) discovered that normal peroxisomes were not present in sse1 mutant embryos and that a GFP chimeric * To whom correspondence should be addressed. E-mail: ª 2007 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract 1678 Karnik and Trelease AtPEX16 between the ER and peroxisomes in two different plant cells. The preponderance of endogenous AtPEX16 throughout the ‘general’ ER and its coexistence within the peroxisomes in Arabidopisis suspension cells (Karnik and Trelease, 2005) provided a unique opportunity to dissect and elucidate the overall trafficking pathway of this PMP. Transiently expressed myc-epitope-tagged AtPEX16 was traced via immunofluorescence microscopy from its first appearance in the cytosol ultimately to peroxisomes in both Arabidopsis and tobacco BY-2 cells. These two cells types were included in the study to assess the commonality of trafficking pathways within different plant species. Observations were made during normal time-courses and compared with observations made during time-courses in which experimental treatments were imposed to impair trafficking through the ER, i.e. low temperature and brefeldin A (BFA) treatments. These experiments revealed the existence of a perceived, but not previously described, compartment in these cells, generally referred to as an ‘ER-peroxisome intermediate compartment’ (ERPIC) (Titorenko and Mullen, 2006). In addition, site-directed mutagenesis of AtPEX16 was employed to elucidate putative necessary and/or sufficient targeting signals within the pathway. Virtually the same results from all of these experiments with Arabidopsis and BY-2 cells collectively validated and revealed new data relative to PMP trafficking in recent model(s) portraying semi-autonomous peroxisome maturation and replication during plant peroxisome biogenesis. Materials and methods Suspension cell cultures, microprojectile bombardment, and (immuno)fluorescence microscopy Suspension cell cultures of Arabidopsis thaliana var. Landsberg erecta and Nicotiana tabacum L. cv. Bright Yellow (BY-2) were grown and maintained as described previously by Lisenbee et al. (2003a) and Lee et al. (1997), respectively. Cells were harvested 4-d post-subculture for transient transformations accomplished via microprojectile biolistic bombardments, which were done according to procedures described in Karnik and Trelease (2005) and Flynn et al. (2005). Cells were spread on filter paper pre-moistened with transformation buffer, bombarded, and were allowed to transiently express gene products for varied times in the dark between 2 h and 23 h depending upon the experiment. Cells were fixed in 4% formaldehyde (prepared from paraformaldehyde) for 1 h and then processed using the standard tube procedure for immunofluorescence microscopy as described previously (Karnik and Trelease, 2005). Antibody concentrations, room t (...truncated)