title: Peroxisomal membrane ascorbate peroxidase is sorted to a membranous network that resembles a subdomain of the endoplasmic reticulum.
abstract: The peroxisomal isoform of ascorbate peroxidase (APX) is a novel membrane isoform that functions in the regeneration of NAD(+) and protection against toxic reactive oxygen species. The intracellular localization and sorting of peroxisomal APX were examined both in vivo and in vitro. Epitope-tagged peroxisomal APX, which was expressed transiently in tobacco BY-2 cells, localized to a reticular/circular network that resembled endoplasmic reticulum (ER; 3,3'-dihexyloxacarbocyanine iodide-stained membranes) and to peroxisomes. The reticular network did not colocalize with other organelle marker proteins, including three ER reticuloplasmins. However, in vitro, peroxisomal APX inserted post-translationally into the ER but not into other purified organelle membranes (including peroxisomal membranes). Insertion into the ER depended on the presence of molecular chaperones and ATP. These results suggest that regions of the ER serve as a possible intermediate in the sorting pathway of peroxisomal APX. Insight into this hypothesis was obtained from in vivo experiments with brefeldin A (BFA), a toxin that blocks vesicle-mediated protein export from ER. A transiently expressed chloramphenicol acetyltransferase-peroxisomal APX (CAT-pAPX) fusion protein accumulated only in the reticular/circular network in BFA-treated cells; after subsequent removal of BFA from these cells, the CAT-pAPX was distributed to preexisting peroxisomes. Thus, plant peroxisomal APX, a representative enzymatic peroxisomal membrane protein, is sorted to peroxisomes through an indirect pathway involving a preperoxisomal compartment with characteristics of a distinct subdomain of the ER, possibly a peroxisomal ER subdomain.
authors.affiliation: Department of Plant Biology and Graduate Program in Molecular and Cellular Biology, Arizona State University, Tempe, Arizona 85287-1601, USA.
authors.forename: R T
authors.initials: RT
authors.lastname: Mullen
authors.forename: C S
authors.initials: CS
authors.lastname: Lisenbee
authors.forename: J A
authors.initials: JA
authors.lastname: Miernyk
authors.forename: R N
authors.initials: RN
authors.lastname: Trelease
mesh_terms: D000595:Amino Acid Sequence; D060387:Ascorbate Peroxidases; D020126:Brefeldin A / Q000494:pharmacology; D002458:Cell Fractionation; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000201:enzymology; D007425:Intracellular Membranes / Q000201:enzymology; D008969:Molecular Sequence Data; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020675:Peroxisomes / Q000201:enzymology*; D010947:Plants, Toxic; D014176:Protein Biosynthesis; D014026:Nicotiana / Q000201:enzymology*; D014158:Transcription, Genetic
chemical_list: D020126:Brefeldin A; D010544:Peroxidases; D060387:Ascorbate Peroxidases