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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/non-peroxisome 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 degrees C, or in brefeldin A at 25 degrees C, resulted in accumulations of mycAtPEX16 within the ER. Following re-equilibration of cold-treated cells at 25 degrees 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 pre-existing 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.

Homologs of peroxin 16 genes (PEX16) have been identified only in Yarrowia lipolytica, humans (Homo sapiens), and Arabidopsis (Arabidopsis thaliana). The Arabidopsis gene (AtPEX16), previously reported as the SSE1 gene, codes for a predicted 42-kD membrane peroxin protein (AtPex16p). Lin et al. (Y. Lin, J.E. Cluette-Brown, H.M. Goodman [2004] Plant Physiol 135: 814-827) reported that SSE1/AtPEX16 was essential for endoplasmic reticulum (ER)-dependent oil and protein body biogenesis in peroxisome-deficient maturing seeds and likely also was involved in peroxisomal biogenesis based on localization of stably expressed green fluorescent protein::AtPex16p in peroxisomes of Arabidopsis plants. In this study with Arabidopsis suspension-cultured cells, combined in vivo and in vitro experiments revealed a novel dual organelle localization and corresponding membrane association/topology of endogenous AtPex16p. Immunofluorescence microscopy with antigen affinity-purified IgGs showed an unambiguous, steady-state coexistence of AtPex16p in suspension cell peroxisomes and ER. AtPex16p also was observed in peroxisomes and ER of root and leaf cells. Cell fractionation experiments surprisingly revealed two immunorelated polypeptides, 42 kD (expected) and 52 kD (unexpected), in homogenates and microsome membrane pellets derived from roots, inflorescence, and suspension cells. Suc-gradient purifications confirmed the presence of both 42-kD and 52-kD polypeptides in isolated peroxisomes (isopycnic separation) and in rough ER vesicles (Mg2+ shifted). They were found peripherally associated with peroxisome and ER membranes but not as covalently bound subunits of AtPex16p. Both were mostly on the matrix side of peroxisomal membranes and unexpectedly mostly on the cytosolic side of ER membranes. In summary, AtPex16p is the only authentic plant peroxin homolog known to coexist at steady state within peroxisomes and ER; these data provide new insights in support of its ER-related, multifunctional roles in organelle biogenesis.