| Two enzymes of a peroxisomal membrane-associated ascorbate antioxidant system, ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDAR), were examined as model enzyme systems to elucidate the intracellular sorting pathway(s) of plant peroxisomal membrane proteins (PMPs). In Chapter 1, emphasis is given to the discovery and partial characterization of a newly-defined subcellular sorting compartment which is a subdomain of endoplasmic reticulum (ER). This compartment, named peroxisomal ER (pER), was identified via immunofluorescence microscopy as reticular/circular structures in cultured BY-2 cells that were transformed transiently with APX gene constructs. It was concluded that unlike matrix proteins, peroxisomal APX sorted to the boundary membrane of peroxisomes indirectly via pER. In Chapter 2, the ambiguous reticular/circular description of pER is resolved into its correct reticular structure. Transformation of BY-2 cells with a green fluorescent protein (GFP) gene construct (GFP-APX) revealed that the circular component of reticular/circular pER actually was an image of homotypically and heterotypically aggregated mitochondria and plastids that were "zippered" together by oligomerization of cytosolic-facing polypeptide domains. In addition to redefining pER as a reticular fluorescence compartment, the research illuminated a "serious" potential pitfall of using overexpressed GFP chimeric proteins for subcellular localization studies. In Chapter 3, cell fractionation experiments were employed to test the hypothesis that reticular pER is a constitutive (not formed by overexpression of PMPs) subcompartment in cultured Arabidopsis cells. Endogenous APX was found in membranes of peroxisomes, rough ER vesicles, and smooth vesicles, and topologically was bound to the cytosolic side of each membrane (tail-anchored). The results provided strong evidence for a biogenetic relationship between the constitutive pER subcompartment and peroxisomes. The fourth chapter includes a description of a previously unidentified ortholog of MDAR, AtMDAR54p, that, unlike peroxisomal APX, was found to sort directly to peroxisomes independent of an association with pER. In summary, the conclusions derived from this dissertation research greatly extend the knowledge of the intracellular sorting of antioxidant PMPs in plant cells, thereby providing a more complete peroxisomal membrane proteome and sorting machinery involved in delivering proteins to the membrane. |
