The Arabidopsis ascorbate peroxidase 3 is a peroxisomal membrane-bound antioxidant enzyme and is dispensable for Arabidopsis growth and development

Ascorbate peroxidase (APX) exists as several isoforms that are found in various compartments in plant cells. The cytosolic and chloroplast APXs appear to play important roles in antioxidation metabolism in plant cells, yet the function of peroxisomal APX is not well studied. In this study, the localization of a putative peroxisomal membrane-bound ascorbate peroxidase, APX3 from Arabidopsis, was confirmed by studying the green fluorescent protein (GFP)-APX3 fusion protein in transgenic plants. GFP-APX3 was found to co-localize with a reporter protein that was targeted to peroxisomes by the peroxisomal targeting signal 1. The function of APX3 in Arabidopsis was investigated by analysing an APX3 knockout mutant under normal and several stress conditions. It was found that loss of function in APX3 does not affect Arabidopsis growth and development, suggesting that APX3 may not be an important antioxidant enzyme in Arabidopsis, at least under the conditions that were tested, or the function of APX3 could be compensated by other antioxidant enzymes in plant cells.     

Mitochondrial and peroxisomal ascorbate peroxidase of pea leaves

The isoenzyme pattern and the substrate specificity of the membrane-bound mitochondrial and peroxisomal ascorbate peroxidases (APX; EC 1.11.1.11) from pea leaves are studied. The substrate specificity of both APXs was assayed using the electron donors ascorbate and pyrogallol, whereas o-dianisidine, hydroquinone, tetramethylbenzidine and 4-methoxy-α-naphthol were also assayed with mitochondrial APX (mitAPX). In leaf mitochondria, the specific activity of APX was similar with pyrogallol and ascorbate, the activity being inhibited by p-CMS. mitAPX showed low activity with the guaiacol peroxidase (GPX)-type substrates, tetramethylbenzidine and 4-methoxy-α-naphthol. Activity of mitAPX with hydroquinone suggest a potential role of mitAPX in the drainage of electrons from the mitochondrial electron chain at the level of ubiquinone. In peroxisomes, the APX (perAPX) specific activity was much higher with pyrogallol than with ascorbate. This perAPX was more sensitive to incubation with Triton X-100 than the mitAPX. By native PAGE the mitAPX was resolved in 6 isoenzyme bands, and the activity of the 3 main bands (mitAPX III, III′ and IV) was inhibited by p-CMS. These 3 major isozymes were also present in mitochondrial membrane fractions. Staining for GPX activity with 4-methoxy-α-naphthol revealed that the APX detected in mitochondria did not have the capacity to oxidize 4-MN, and therefore cannot be considered as true GPX. When intact peroxisomes and peroxisomal membranes were subjected to native PAGE, no APX activity could be detected and this was probably due to the inactivation of perAPX. Results obtained suggest that pea mitochondrial APX (mitAPX) represent a distinct and novel isozyme different from those APXs of chloroplast and cytosolic origin previously reported. The peroxisomal APX (perAPX), however, appears to ressemble the chloroplast APXs as regards its sensitivity to Triton X-100.     

Pumpkin peroxisomal ascorbate peroxidase is localized on peroxisomal membranes and unknown membranous structures

To investigate the roles of peroxisomal membrane proteins in the reversible conversion of glyoxysomes to leaf peroxisomes, we characterized several membrane proteins of glyoxysomes. One of them was identified as an ascorbate peroxidase (pAPX) that is localized on glyoxysomal membranes. Its cDNA was isolated by immunoscreening. The deduced amino acid sequence encoded by the cDNA insert does not have a peroxisomal targeting signal (PTS), suggesting that pAPX is imported by one or more PTS-independent pathways. Subcellular fractionation of 3- and 5-d-old cotyledons of pumpkin revealed that pAPX was localized not only in the glyoxysomal fraction, but also in the ER fraction. A magnesium shift experiment showed that the density of pAPX in the ER fraction did not increase in the presence of Mg(2+), indicating that pAPX is not localized in the rough ER. Immunocytochemical analysis using a transgenic Arabidopsis which expressed pumpkin pAPX showed that pAPX was localized on peroxisomal membranes, and also on a unknown membranous structure in green cotyledons. The overall results suggested that pAPX is transported to glyoxysomal membranes via this unknown membranous structure.     

The structural elements of hirudin which bind to the fibrinogen recognition site of thrombin are exclusively located within its acidic C-terminal tail

Six lysyl residues of human thrombin (LysB21, LysB52, LysB65, LysB106, LysB107 and LysB154) have been previously shown to participate in the binding site of hirudin, a thrombin-specific inhibitor [(1989) J. Biol. Chem. 264, 7141-7146]. In this report, we attempted to delineate the region of hirudin which binds to these basic amino acids of thrombin. Using the N-terminal core domains (r-Hir1-43 and r-Hir1-52) derived from recombinant hirudins and synthetic C-terminal peptides (Hir40-65 and Hir52-65)--all fragments form complexes with thrombin--we are able to demonstrate that the structural elements of hirudin which account for the shielding of these 6 lysyl residues are exclusively located within the acidic C-terminal region. Since hirudin C-terminal peptides were shown to bind to a non-catalytic site of thrombin and inhibit its interaction with fibrinogen [(1987) FEBS Lett. 211, 10-16], our data consequently imply that these 6 lysyl residues are constituents of the fibrinogen recognition site of thrombin.     

The cytoplasmic tail of CD1d contains two overlapping basolateral sorting signals

CD1d is a member of the CD1 polypeptide family that represents a new arm of host defense against invading pathogens. In our previous work (Rodionov, D. G., Nordeng, T. W., Pedersen, K., Balk, S. P., and Bakke, O. (1999) J. Immunol. 162, 1488-1495) we have shown that CD1d contained a classic tyrosine-based internalization signal (YQGV) in its short cytoplasmic tail. CD1d is expressed in polarized epithelial cells, and we found that the cytoplasmic tail of CD1d also contained information for basolateral sorting. Interestingly, a mutation of the critical tyrosine residue of the endosomal sorting signal did not result in the loss of basolateral targeting of the mutant CD1d. To search for a basolateral sorting signal we have constructed a full set of alanine mutants, but no single alanine substitution inactivated the signal. However, deletions or mutations of either the C-terminal valine/leucine pair or the critical tyrosine residue from the internalization signal and either residue from the C-terminal valine/leucine pair inactivated basolateral sorting. Our data thus suggest that the cytoplasmic tail contains two overlapping basolateral signals, one tyrosine- and the other leucine-based, each being sufficient to direct CD1d to the basolateral membrane of polarized Madin-Darby canine kidney cells.     

Thylakoid membrane-bound ascorbate peroxidase is a limiting factor of antioxidative systems under photo-oxidative stress

To evaluate the physiological importance of thylakoid membrane-bound ascorbate peroxidase (tAPX) in the active oxygen species-scavenging system of chloroplasts, the level of tAPX in tobacco plants was altered by expression of the tAPX cDNA in both sense and antisense orientation. The tobacco plants transformed with constructs of antisense tAPXs from spinach and tobacco could not be obtained, suggesting that the suppression of tAPX in higher plants had a severe effect on the growth even under normal conditions. In contrast, the transgenic tobacco plants (TpTAP-12) overexpressing tAPX, which had approximately 37-fold higher activity than that of the wild-type plants, were generated. The TpTAP-12 plants showed increased tolerance to oxidative stress caused by application of methylviologen (MV, 50 microm) under light intensity (300 and 1600 microE m(-2) sec(-1)) and by chilling stress with high light intensity (4 degrees C, 1000 microE m(-2) sec(-1)). At 24 h after the MV treatment under illumination at 300 microE m-2 sec-1, destruction of chlorophyll was observed in the wild-type plants, but not in the TpTAP-12 plants. The activities of thiol-modulated enzymes in the Calvin cycle, the level and redox status of ascorbate (AsA), and the activity of tAPX in the wild-type plants significantly decreased, while those in the TpTAP-12 plants were hardly changed. These observations suggest that tAPX is a limiting factor of antioxidative systems under photo-oxidative stress in chloroplasts, and that the enhanced activity of tAPX functions to maintain the AsA content and the redox status of AsA under stress conditions.     

The redox properties of ascorbate peroxidase

Reduction potentials for the catalytic compound I/compound II and compound II/Fe3+ redox couples, and for the two-electron compound I/Fe3+ redox couple, have been determined for ascorbate peroxidase (APX) and for a number of site-directed variants. For the wild type enzyme, the values are E degrees '(compound I/compound II) = 1156 mV, E degrees '(compound II/Fe3+) = 752 mV, and E degrees '(compound I/Fe3+) = 954 mV. For the variants, the analysis also includes determination of Fe3+/Fe2+ potentials which were used to calculate (experimentally inaccessible) E degrees '(compound II/Fe3+) potentials. The data provide a number of new insights into APX catalysis. The measured values for E degrees '(compound I/compound II) and E degrees '(compound II/Fe3+) for the wild type protein account for the much higher oxidative reactivity of compound I compared to compound II, and this correlation holds for a number of other active site and substrate binding variants of APX. The high reduction potential for compound I also accounts for the known thermodynamic instability of this intermediate, and it is proposed that this instability can account for the deviations from standard Michaelis kinetics observed for most APX enzymes during steady-state oxidation of ascorbate. This study provides the first systematic evaluation of the redox properties of any ascorbate peroxidase using a number of methods, and the data provide an experimental and theoretical framework for accurate determination of the redox properties of Fe3+, compound I, and compound II species in related enzymes.     

Hydroxyurea and p-aminophenol are the suicide inhibitors of ascorbate peroxidase

Guaiacol peroxidase from spinach catalyzes the oxidation of p-aminophenol to produce the aminophenoxy radical as the primary product which is converted further into a stable oxidation product with an absorption peak at 470 nm. The p-aminophenol radicals oxidize ascorbate (AsA) to produce monodehydroascorbate radicals. Kinetic analysis indicates that p-aminophenol radicals also oxidize monodehydroascorbate to dehydroascorbate. Incubation of AsA peroxidase from tea leaves and hydrogen peroxide with p-aminophenol, p-cresol, hydroxyurea, or hydroxylamine results in the inactivation of the enzyme. No inactivation of the enzyme was found upon incubation of the enzyme with these compounds either in the absence of hydrogen peroxide or with the stable oxidized products of these compounds. The enzyme was protected from inactivation by the inclusion of AsA in the incubation mixture. The radicals of p-aminophenol and hydroxyurea were produced by AsA peroxidase as detected by their ESR signals. These signals disappeared upon the addition of AsA, and the signal characteristic of monodehydroascorbate was found. Thus, AsA peroxidase is inactivated by the radicals of p-aminophenol, p-cresol, hydroxyurea, and hydroxylamine which are produced by the peroxidase reaction, and it is protected from inactivation by AsA via the scavenging of the radicals. Thus, these compounds are the suicide inhibitors for AsA peroxidase. Isozyme II of AsA peroxidase, which is localized in chloroplasts, is more sensitive to these compounds than isozyme I. In contrast to AsA peroxidase, guaiacol peroxidase was not affected by these various compounds, even though each was oxidized by it and the corresponding radicals were produced.     

Overexpression of an Arabidopsis peroxisomal ascorbate peroxidase gene in tobacco increases protection against oxidative stress.

The Arabidopsis gene APX3 that encodes a putative peroxisomal membrane-bound ascorbate peroxidase was expressed in transgenic tobacco plants. APX3-expressing lines had substantial levels of APX3 mRNA and protein. The H2O2 can be converted to more reactive toxic molecules, e.g. .OH, if it is not quickly removed from plant cells. The expression of APX3 in tobacco could protect leaves from oxidative stress damage caused by aminotriazole which inhibits catalase activity that is found mainly in glyoxysomes and peroxisomes and leads to accumulation of H2O2 in those organelles. However, these plants did not show increased protection from oxidative damage caused by paraquat which leads to the production of reactive oxygen species in chloroplasts. Therefore, protection provided by the expression of APX3 seems to be specific against oxidative stress originated from peroxisomes, not from chloroplasts, which is consistent with the hypothesis that APX3 is a peroxisomal membrane-bound antioxidant enzyme.     

Double antisense plants lacking ascorbate peroxidase and catalase are less sensitive to oxidative stress than single antisense plants lacking ascorbate peroxidase or catalase

The plant genome is a highly redundant and dynamic genome. Here, we show that double antisense plants lacking the two major hydrogen peroxide-detoxifying enzymes, ascorbate peroxidase (APX) and catalase (CAT), activate an alternative/redundant defense mechanism that compensates for the lack of APX and CAT. A similar mechanism was not activated in single antisense plants that lacked APX or CAT, paradoxically rendering these plants more sensitive to oxidative stress compared to double antisense plants. The reduced susceptibility of double antisense plants to oxidative stress correlated with suppressed photosynthetic activity, the induction of metabolic genes belonging to the pentose phosphate pathway, the induction of monodehydroascorbate reductase, and the induction of IMMUTANS, a chloroplastic homologue of mitochondrial alternative oxidase. Our results suggest that a co-ordinated induction of metabolic and defense genes, coupled with the suppression of photosynthetic activity, can compensate for the lack of APX and CAT. In addition, our findings demonstrate that the plant genome has a high degree of plasticity and will respond differently to different stressful conditions, namely, lack of APX, lack of CAT, or lack of both APX and CAT.     

The homologous tryptophan critical for cytochrome c peroxidase function is not essential for ascorbate peroxidase activity

The crystal structures of ascorbate peroxidase (APX) and cytochrome c peroxidase (CCP) show that the active site structures are nearly identical. Both enzymes contain a His-Asp-Trp catalytic triad in the proximal pocket. The proximal Asp residue hydrogen bonds with both the His proximal heme ligand and the indole ring nitrogen of the proximal Trp. The Trp is stacked parallel to and in contact with the proximal His ligand. This Trp is known to be the site of free radical formation in CCP compound I and also is essential for activity. However, APX forms a porphyrin radical and not a Trp-centered radical, even though the His-Asp-Trp triad structure is the same in both peroxidases. We found that conversion of the proximal Trp to Phe has no effect on APX enzyme activity and that the mutant crystal structure shows that changes in the structure are confined to the site of mutation. This indicates that the paths of electron transfer in CCP and APX are distinctly different. The Trp-to-Phe mutant does alter the stability of the APX compound I porphyrin radical, by a factor of two. Electrostatic calculations and modeling studies show that a potassium cation located about 8?Å from the proximal Trp in APX, but absent in CCP, makes a significant contribution to the stability of a cation Trp radical. This underscores the importance of long-range electrostatic effects in enzyme catalyzed reactions.     

The dileucine motif within the tail of MPR46 is required for sorting of the receptor in endosomes

The cytoplasmic tail of MPR46 carries a leucine-based motif that is required for the sorting of lysosomal enzymes by the receptor. In addition, it is one of three independent, but functionally redundant, internalization signals present in the cytoplasmic tail of MPR46. We have analyzed a mutant of MPR46, in which the dileucine pair was replaced by alanines (MPR46 LL/AA) with respect to its intracellular distribution and trafficking. Ultrastructural analysis of cells expressing the MPR46 LL/AA mutant revealed that the substitution of the dileucine pair causes a shift of the receptor distribution from the TGN, where it is packaged into AP1-containing vesicles, to vesicular structures distributed throughout the cytoplasm. The vesicles could be identified as early endosomes with internalized BSA-gold and rab5 as markers. By analyzing the receptor trafficking biochemically, we found that return of the LL/AA mutant receptor from the plasma membrane/endosome pool back to the TGN was impaired, while recycling from endosomes to the plasma membrane was enhanced. In conclusion, our data indicate that the dileucine motif in the MPR46 tail is required for a sorting event in endosomes.     

Targeting of the plant vacuolar sorting receptor BP80 is dependent on multiple sorting signals in the cytosolic tail

Although signals for vacuolar sorting of soluble proteins are well described, we have yet to learn how the plant vacuolar sorting receptor BP80 reaches its correct destination and recycles. To shed light on receptor targeting, we used an in vivo competition assay in which a truncated receptor (green fluorescent protein-BP80) specifically competes with sorting machinery and causes hypersecretion of BP80-ligands from tobacco (Nicotiana tabacum) leaf protoplasts. We show that both the transmembrane domain and the cytosolic tail of BP80 contain information necessary for efficient progress to the prevacuolar compartment (PVC). Furthermore, the tail must be exposed on the correct membrane surface to compete with sorting machinery. Mutational analysis of conserved residues revealed that multiple sequence motifs are necessary for competition, one of which is a typical Tyr-based motif (YXXPhi). Substitution of Tyr-612 for Ala causes partial retention in the Golgi apparatus, mistargeting to the plasma membrane (PM), and slower progress to the PVC. A role in Golgi-to-PVC transport was confirmed by generating the corresponding mutation on full-length BP80. The mutant receptor was partially mistargeted to the PM and induced the secretion of a coexpressed BP80-ligand. Further mutants indicate that the cytosolic tail is likely to contain other information besides the YXXPhi motif, possibly for endoplasmic reticulum export, endocytosis from the PM, and PVC-to-Golgi recycling.     

Calcium-induced conformational changes in C-terminal tail of polycystin-2 are necessary for channel gating

Polycystin-2 (PC2) is a Ca²⁺-permeable transient receptor potential channel activated and regulated by changes in cytoplasmic Ca²⁺. PC2 mutations are responsible for ∼15% of autosomal dominant polycystic kidney disease. Although the C-terminal cytoplasmic tail of PC2 has been shown to contain a Ca²⁺-binding EF-hand domain, the molecular basis of PC2 channel gating by Ca²⁺ remains unknown. We propose that the PC2 EF-hand is a Ca²⁺ sensor required for channel gating. Consistent with this, Ca²⁺ binding causes a dramatic decrease in the radius of gyration (R_g) of the PC2 EF-hand by small angle x-ray scattering and significant conformational changes by NMR. Furthermore, increasing Ca²⁺ concentrations cause the C-terminal cytoplasmic tail to transition from a mixture of extended oligomers to a single compact dimer by analytical ultracentrifugation, coupled with a >30 Å decrease in maximum interatomic distance (D_max) by small angle x-ray scattering. Mutant PC2 channels unable to bind Ca²⁺ via the EF-hand are inactive in single-channel planar lipid bilayers and inhibit Ca²⁺ release from ER stores upon overexpression in cells, suggesting dominant negative properties. Our results support a model where PC2 channels are gated by discrete conformational changes in the C-terminal cytoplasmic tail in response to changes in cytoplasmic Ca²⁺ levels. These properties of PC2 are lost in autosomal dominant polycystic kidney disease, emphasizing the importance of PC2 to kidney cell function. We speculate that PC2 and the Ca²⁺-dependent transient receptor potential channels in general are regulated by similar conformational changes in their cytoplasmic domains that are propagated to the channel pore.     

The sorting sequence of the peroxisomal integral membrane protein PMP47 is contained within a short hydrophilic loop

No targeting sequence for peroxisomal integral membrane proteins has yet been identified. We have previously shown that a region of 67 amino acids is necessary to target Pmp47, a protein that spans the membrane six times, to peroxisomes. This region comprises two membrane spans and the intervening loop. We now demonstrate that the 20 amino acid loop, which is predicted to face the matrix, is both necessary and sufficient for peroxisomal targeting. Sufficiency was demonstrated with both chloramphenicol acetyltransferase and green fluorescent protein as carriers. There is a cluster of basic amino acids in the middle of the loop that we predict protrudes from the membrane surface into the matrix by a flanking stem structure. We show that the targeting signal is composed of this basic cluster and a block of amino acids immediately down-stream from it.     

Ca2+ regulation of gelsolin by its C-terminal tail

Gelsolin is activated by Ca(2+) to sever actin filaments. Ca(2+) regulation is conferred on the N-terminal half by the C-terminal half. This paper seeks to understand how Ca(2+) regulates gelsolin by testing the "tail helix latch hypothesis," which is based on the structural data showing that gelsolin has a C-terminal tail helix that contacts the N-terminal half in the absence of Ca(2+). Ca(2+) activation of gelsolin at 37 degrees C occurs in three steps, with apparent K(d) for Ca(2+) of 0.1, 0.3, and 6.4 x 10(-6) m. Tail helix truncation decreases the apparent Ca(2+) requirement for severing to 10(-7) m and eliminates the conformational change observed at 10(-6) m Ca(2+). The large decrease in Ca(2+) requirement for severing is not due to a change in Ca(2+) binding nor to Ca(2+)-independent activation of the C-terminal half per se. Thus, the tail helix latch is primarily responsible for transmitting micromolar Ca(2+) information from the gelsolin C-terminal half to the N-terminal half. Occupation of submicromolar Ca(2+)-binding sites primes gelsolin for severing, but gelsolin cannot sever because the tail latch is still engaged. Unlatching the tail helix by 10(-6) m Ca(2+) releases the final constraint to initiate the severing cascade.