Previously we reported (R.T. Mullen, C.S. Lisenbee, J.A. Miernyk, R.N. Trelease [1999] Plant Cell 11: 2167–2185) that overexpressed ascorbate peroxidase (APX), a peroxisomal membrane protein, sorted indirectly to Bright Yellow-2 cell peroxisomes via a subdomain of the endoplasmic reticulum (ER; peroxisomal endoplasmic reticulum [pER]). More recently, a pER-like compartment also was identified in pumpkin (Cucurbita pepo) and transformed Arabidopsis cells (K. Nito, K. Yamaguchi, M. Kondo, M. Hayashi, M. Nishimura [2001] Plant Cell Physiol 42: 20–27). Here, we characterize more extensively the localization of endogenous Arabidopsis peroxisomal APX (AtAPX) in cultured wild-type Arabidopsis cells (Arabidopsis var. Landsberg erecta). AtAPX was detected in peroxisomes, but not in an ER subcompartment, using immunofluorescence microscopy. However, AtAPX was detected readily with immunoblots in both peroxisomal and ER fractions recovered from sucrose (Suc) density gradients. Most AtAPX in microsomes (200,000g, 1 h pellet) applied to gradients exhibited a Mg2  +-induced shift from a distribution throughout gradients (approximately 18%–40% [w/w] Suc) to ≥42% (w/w) Suc regions of gradients, including pellets, indicative of localization in rough ER vesicles. Immunogold electron microscopy of the latter fractions verified these findings. Further analyses of peroxisomal and rough ER vesicle fractions revealed that AtAPX in both fractions was similarly associated with and located mostly on the cytosolic face of the membranes. Thus, at the steady state, endogenous peroxisomal AtAPX resides at different levels in rough ER and peroxisomes. Collectively, these findings show that rather than being a transiently induced sorting compartment formed in response to overexpressed peroxisomal APX, portions of rough ER (pER) in wild-type cells serve as a constitutive sorting compartment likely involved in posttranslational routing of constitutively synthesized peroxisomal APX.

SummaryPlant heterotrimeric G-proteins are involved in a variety of signaling pathways, though only one a and a few bc isoforms of their subunits exist. In isolated plasma membranes of California poppy (Eschscholzia californica), the plant-specific Ga subunit was isolated and identified immunologically and by homology of the cloned gene with that of several plants. In the same membrane, phospholipase A 2 (PLA 2 ) was activated by yeast elicitor only if GTPcS (an activator of Ga) was present. From the cholate-solubilized membrane proteins, PLA 2 was co-precipitated together with Ga by a polyclonal antiserum raised against the recombinant Ga. In this immunoprecipitate and in the plasma membrane (but not in the Ga-free supernatant) PLA 2 was stimulated by GTPcS. Plasma membranes and immunoprecipitates obtained from antisense transformants with a low Ga content allowed no such stimulation. An antiserum raised against the C-terminus (which in animal Gas is located near the target coupling site) precipitated Ga without any PLA 2 activity. Using non-denaturing PAGE, complexes of solubilized plasma membrane proteins were visualized that contained Ga plus PLA 2 activity and dissociated at pH 9.5. At this pH, PLA 2 was no longer stimulated by GTPcS. It is concluded that a distinct fraction of the plasma membrane-bound PLA 2 exists in a detergent-resistant complex with Ga that can be dissociated at pH 9.5. This complex allows the Ga-mediated activation of PLA 2 .

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Summary. This paper describes the first purification method for crystalline inclusions (cores) from plant peroxisomes and an ultrastructural characterization of these isolated cores. 5-day-old sunflower (Helianthus annuus L.) cotyledon fractious which were highly enriched in cores showed negligible activity of the matrix enzyme glycolate oxidase but high catalase activity. As proven by electron microscopy, crystalline particles were surrounded neither by matrix material nor by membranes. Their geometrical outlines and ultrastructure were identical to those of cores in tissue sections, as was their reactivity with three different polyclonal catalase antibodies in the immunogold technique. Three-dimensional reconstruction, based on the geometrical outlines and ultrastructure of sectioned isolated cores from sunflower, suggested that they were quadrangular blocks. Ultrastructural analysis revealed an even periodic arrangement of repeating units which are probably cubes with 20 nm long edges. Isolated peroxisomal cores from potato (Solanum tuberosum L.) tubers had outlines which suggested that they were even rhomboidal prisms. They showed a granular ultrastructure without any repeating units and contained catalase, demonstrated by immunogold labelling and enzyme activity measurement. The results presented here suggested the hypothesis that the structural elements in plant peroxisomal cores are made of enzymatically active catalase, although the substructure may vary from species to species.