Targeting of Rac1 to the phagocyte membrane is sufficient for the induction of NADPH oxidase assembly.

The superoxide (O(2))-generating NADPH oxidase complex of phagocytes consists of a membrane-associated flavocytochrome (cytochrome b(559)) and four cytosolic proteins, p47(phox), p67(phox), p40(phox), and the small GTPase Rac (Rac1 or -2). NADPH oxidase activation (O(2) production) is elicited as the consequence of assembly of some or all cytosolic components with cytochrome b(559). This process can be reproduced in an in vitro system consisting of phagocyte membranes, p47(phox), p67(phox), and Rac, activated by an anionic amphiphile. We now show that post-translationally processed (prenylated) Rac1 initiates NADPH oxidase assembly, expressed in O(2) production, in a cell-free system containing phagocyte membrane vesicles and p67(phox), in the absence of an activating amphiphile and of p47(phox). Prenylated Cdc42Hs, a GTPase closely related to Rac, is inactive under the same conditions. Results obtained with phagocyte membrane vesicles can be reproduced fully by replacing these with partially purified cytochrome b(559), incorporated in phosphatidylcholine vesicles. Prenylated, but not nonprenylated, Rac1 binds spontaneously to phagocyte membrane vesicles and also to artificial, protein-free, phosphatidylcholine vesicles, a process counteracted by GDP dissociation inhibitor for Rho. Binding of prenylated Rac1 to membrane vesicles is accompanied by the recruitment of p67(phox) to the same location and the formation of an assembled NADPH oxidase complex, producing O(2) upon the addition of NADPH. Amphiphile and p47(phox)-independent NADPH oxidase activation by prenylated Rac1 is inhibited by Rho GDP dissociation inhibitor and by phosphatidylcholine vesicles, both competing with membrane for prenylated Rac1. We conclude that, in vitro, targeting of Rac to the phagocyte membrane is sufficient for the induction of NADPH oxidase assembly, suggesting that the principal or, possibly, the only role of Rac is to recruit cytosolic p67(phox) to the membrane environment, to be followed by the interaction of p67(phox) with cytochrome b(559).

Mutations in the FIE and MEA genes that encode interacting polycomb proteins cause parent-of-origin effects on seed development by distinct mechanisms.

In flowering plants, two cells are fertilized in the haploid female gametophyte. Egg and sperm nuclei fuse to form the embryo. A second sperm nucleus fuses with the central cell nucleus, which replicates to generate the endosperm, a tissue that supports embryo development. The FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) and MEDEA (MEA) genes encode WD and SET domain polycomb proteins, respectively. In the absence of fertilization, a female gametophyte with a loss-of-function fie or mea allele initiates endosperm development without fertilization. fie and mea mutations also cause parent-of-origin effects, in which the wild-type maternal allele is essential and the paternal allele is dispensable for seed viability. Here, we show that FIE and MEA polycomb proteins interact physically, suggesting that the molecular partnership of WD and SET domain polycomb proteins has been conserved during the evolution of flowering plants. The overlapping expression patterns of FIE and MEA are consistent with their suppression of gene transcription and endosperm development in the central cell as well as their control of seed development after fertilization. Although FIE and MEA interact, differences in maternal versus paternal patterns of expression, as well as the effect of a recessive mutation in the DECREASE IN DNA METHYLATION1 (DDM1) gene on mutant allele transmission, indicate that fie and mea mutations cause parent-of-origin effects on seed development by distinct mechanisms.

Mapping of functional domains in p47(phox) involved in the activation of NADPH oxidase by "peptide walking".

The superoxide generating NADPH oxidase of phagocytes consists, in resting cells, of a membrane-associated electron transporting flavocytochrome (cytochrome b559) and four cytosolic proteins as follows: p47(phox), p67(phox), p40(phox), and the small GTPase, Rac(1 or 2). Activation of the oxidase is consequent to the assembly of a membrane-localized multimolecular complex consisting of cytochrome b559 and the cytosolic components. We used "peptide walking" (Joseph, G., and Pick, E. (1995) J. Biol. Chem. 270, 29079-29082) for mapping domains in the amino acid sequence of p47(phox) participating in the molecular events leading to the activation of NADPH oxidase. Ninety-five overlapping pentadecapeptides, with a four-residue offset between neighboring peptides, spanning the complete p47(phox) sequence, were tested for the ability to inhibit NADPH oxidase activation in a cell-free system. This consisted of solubilized macrophage membranes, recombinant p47(phox), p67(phox), and Rac1, and lithium dodecyl sulfate, as the activator. Eight functional domains were identified and labeled a-h. These were (N- and C-terminal residue numbers are given for each domain) as follows: a (21-35); b (105-119); c (149-159); d (193-207); e (253-267); f (305-319); g (325-339), and h (373-387). Four of these domains (c, d, e, and g) correspond to or form parts of regions shown before to participate in NADPH oxidase assembly. Thus, domain c corresponds to a region on the N-terminal boundary of the first src homology 3 (SH3) domain, whereas domains d and e represent more precisely defined sites within the full-length first and second SH3 domains, respectively. Domain g overlaps an extensively investigated arginine-rich region. Domains a and b, in the N-terminal half of p47(phox), and domains f and h, in the C-terminal half, represent newly identified entities, for which there is no earlier experimental evidence of involvement in NADPH oxidase activation. "Peptide walking" was also applied to the identification of domains in p47(phox) mediating binding to p67(phox). This was done by quantifying, by enzyme-linked immunosorbent assay, the binding of p67(phox), in solution, to a series of 95 overlapping biotinylated p47(phox) peptides, attached to streptavidin-coated 96-well plates. A single proline-rich domain (residues 357-371) was found to bind p67(phox) in the absence and presence of lithium dodecyl sulfate.

Mutational analysis of novel effector domains in Rac1 involved in the activation of nicotinamide adenine dinucleotide phosphate (reduced) oxidase.

The small molecular weight GTP-binding protein Rac (1 or 2) is an obligatory participant in the activation of the superoxide-generating NADPH oxidase. Active NADPH oxidase can be reconstituted in a cell-free system, consisting of phagocyte-derived membranes, containing cytochrome b559, and the recombinant cytosolic proteins p47-phox, p67-phox, and Rac, supplemented with an anionic amphiphile as an activator. The cell-free system was used before for the analysis of structural requirements of individual components participating in the assembly of NADPH oxidase. In earlier work, we mapped four previously unidentified domains in Rac1, encompassing residues 73-81 (a), 103-107 (b), 123-133 (c), and 163-169 (d), as important for cell-free NADPH oxidase activation. The domains were defined by assessing the activation inhibitory effect of a series of overlapping peptides, spanning the entire length of Rac1 [Joseph, G., and Pick, E. (1995) J. Biol. Chem. 270, 29079-29082]. We now used the construction of Rac1/H-Ras chimeras, domain deletion, and point mutations, to ascertain the functional relevance of three domains (b, c, and d) predicted by "peptide walking" and to determine the importance of specific residues within these domains. This methodology firmly establishes the involvement of domains b and d in the activation of NADPH oxidase by Rac1 and identifies H103 and K166, respectively, as residues critical for the effector function of these two domains. The functional significance of domain c (insert region) could not be confirmed, as shown by the minor effect of deleting this domain on NADPH oxidase activation. Analysis of the three-dimensional structure of Rac1 reveals that residues H103 and K166 are exposed on the surface of the molecule. Modeling of the activity-impairing point mutations suggests that the effect on the ability to activate NADPH oxidase depends on the side chains of the mutated amino acids and not on changes in the global structure of the protein. In conclusion, we demonstrate the existence of two novel effector sites in Rac1, necessary for supporting NADPH oxidase activation, supplementing the canonical N-terminal effector region.

Inhibition of NADPH oxidase activation by 4-(2-aminoethyl)-benzenesulfonyl fluoride and related compounds.

The elicitation of an oxidative burst in phagocytes rests on the assembly of a multicomponental complex (NADPH oxidase) consisting of a membrane-associated flavocytochrome (cytochrome b559), representing the redox element responsible for the NADPH-dependent reduction of oxygen to superoxide (O-2), two cytosolic components (p47(phox), p67(phox)), and the small GTPase Rac (1 or 2). We found that 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), an irreversible serine protease inhibitor, prevented the elicitation of O-2 production in intact macrophages and the amphiphile-dependent activation of NADPH oxidase in a cell-free system, consisting of solubilized membrane or purified cytochrome b559 combined with total cytosol or a mixture of recombinant p47(phox), p67(phox), and Rac1. AEBSF acted at the activation step and did not interfere with the ensuing electron flow. It did not scavenge oxygen radicals and did not affect assay reagents. Five other serine protease inhibitors (three irreversible and two reversible) were found to lack an inhibitory effect on cell-free activation of NADPH oxidase. A structure-function study of AEBSF analogues demonstrated that the presence of a sulfonyl fluoride group was essential for inhibitory activity and that compounds containing an aminoalkylbenzene moiety were more active than amidinobenzene derivatives. Exposure of the membrane fraction or of purified cytochrome b559, but not of cytosol or recombinant cytosolic components, to AEBSF, in the presence of a critical concentration of the activating amphiphile lithium dodecyl sulfate, resulted in a marked impairment of their ability to support cell-free NADPH oxidase activation upon complementation with untreated cytosol or cytosolic components. Kinetic analysis of the effect of varying the concentration of each of the three cytosolic components on the inhibitory potency of AEBSF indicated that this was inversely related to the concentrations of p47(phox) and, to a lesser degree, p67(phox). AEBSF also prevented the amphiphile-elicited translocation of p47(phox) and p67(phox) to the membrane. These results are interpreted as indicating that AEBSF interferes with the binding of p47(phox) and/or p67(phox) to cytochrome b559, probably by a direct effect on cytochrome b559.

Electron transfer in the superoxide-generating NADPH oxidase complex reconstituted in vitro.

The superoxide (O2-)-generating NADPH oxidase of phagocytic cells is composed of a membrane-bound flavocytochrome (cytochrome b-559) and three cytosolic components, p47-phox, p67-phox, and the small GTPase rac-1 (or 2). Cytochrome b-559 bears the NADPH binding site and the redox centers (FAD and heme). Electron flow through the redox centers, from NADPH to oxygen, is activated consequent to the assembly of the three cytosolic components with cytochrome b-559. We studied the kinetics of electron flow through the redox centers of NADPH oxidase in a cell-free system, consisting of purified relipidated and reflavinated cytochrome b-559 and recombinant cytosolic components, activated by the anionic amphiphile, lithium dodecyl sulphate. The NADPH oxidase complex assembled in vitro exhibited: (a) a high steady-state electron flow (165 electrons/heme/s); (b) low stationary levels of FAD and heme reduction (about 10%), and (c) a high rate constant of heme oxidation by oxygen (1720 s-1). Surprisingly, the kinetic properties of NADPH oxidase assembled in a semi-recombinant cell-free system, lacking p47-phox (found to generate significant amounts of O2-), were similar to those of the complete system, as shown by a steady-state electron flow of 83 electrons/heme/s, low stationary levels of FAD and heme reduction (10%), and a rate constant of heme oxidation by oxygen of 1455 s-1. The kinetic features of NADPH oxidase assembled in vitro from purified and recombinant components differ considerably from those of solubilized enzyme preparations derived from intact stimulated phagocytes. The fast operation of the cell-free system is best explained by the activation-related facilitation of electron flow at both the FAD-->heme and the heme-->oxygen steps.

The cytosolic component p47(phox) is not a sine qua non participant in the activation of NADPH oxidase but is required for optimal superoxide production.

The superoxide (O-2)-generating NADPH oxidase of phagocytes is a multicomponent complex consisting of a membrane-associated flavocytochrome (cytochrome b559), bearing the NADPH binding site and two redox centers (FAD and heme) and three cytosolic activating components: p47(phox), p67(phox), and the small GTPase Rac (1 or 2). The canonical view is that the induction of O-2 generation involves the stimulus-dependent assembly of all three cytosolic components with cytochrome b559, a process mimicked in vitro by a cell-free system activated by anionic amphiphiles. We studied the requirement for individual cytosolic components in the activation of NADPH oxidase in a cell-free system consisting of purified and relipidated cytochrome b559, recombinant p47(phox), p67(phox), and Rac1, and the amphiphile, lithium dodecyl sulfate. We found that pronounced activation of NADPH oxidase can be achieved by exposing cytochrome b559 to p67(phox) and Rac1, in the total absence of p47(phox) (turnover = 60 mol O-2/s/mol cytochrome b559). However, maximal activation (turnover = 153 mol O-2/s/mol cytochrome b559) could only be obtained in the presence of p47(phox). O-2 production, in the absence of p47(phox), was dependent on: high molar ratios of p67(phox) and Rac1 to cytochrome b559, Rac1 being in the GTP-bound form, cytochrome b559 being saturated with FAD, and an optimal concentration of amphiphile. Single cytosolic components or combinations of two cytosolic components, other than p67(phox) and Rac1, were incapable of activation. We conclude that p67(phox) and Rac1 are the only cytosolic components directly involved in the induction of electron transport in cytochrome b559. p47(phox) appears to facilitate or stabilize the interaction of p67(phox) and, possibly, Rac1 with cytochrome b559, and is required for optimal generation of O-2 under physiological conditions.

Establishment of an Agrobacterium-mediated transformation system for grape (Vitis vinifera L.): the role of antioxidants during grape-Agrobacterium interactions.

Very short exposures of embryogenic calli of Vitis vinifera cv. Superior Seedless grape plants to diluted cultures of Agrobacterium resulted in plant tissue necrosis and subsequent cell death. Antibiotics used for Agrobacterium elimination or as plant selectable markers were not responsible for this necrotic response. Rather, cell death seemed to be oxygen-dependent and correlated with elevated levels of peroxides. Therefore, we studied the effects on necrosis of various combinations of antioxidants during and after grape-Agrobacterium cocultivation. The combination of polyvinylpolypyrrolidone and dithiothreitol was found to improve plant viability. Tissue necrosis was completely inhibited by these antioxidants while Agrobacterium virulence was not effected. These treatments enabled the recovery of stable transgenic grape plants resistant to hygromycin.

High levels of photosystem I subunit II (PsaD) mRNA result in the accumulation of the PsaD polypeptide only in the presence of light.

The light-regulated mRNA and polypeptide accumulation of the nuclear encoded subunit II (PsaD) of the photosystem I reaction center was studied during the greening of etiolated spinach seedlings. Upon exposure to continuous white light, the mRNA, detected at low levels in etiolated seedlings, accumulated in a specific pattern. In contrast, the PsaD subunit could not be detected in the etiolated seedlings; the polypeptide could first be detected in thylakoid membranes approximately 4 h after exposure to continuous light. A pulse of red light induced the expression of the PsaD mRNA, but the polypeptide could not be detected unless the seedlings were exposed to light. In the light (but not in the dark), the PsaD mRNA was found associated with the polysomal fraction. Taken together, the data suggest a dual regulatory mechanism in which both the level of mRNA and the presence of light control the accumulation of the PsaD polypeptide.

Subunit III (Psa-F) of photosystem I reaction center of the C4 dicotyledon Flaveria trinervia.

An immunological survey of C3, C4 and C3-C4-intermediate Flaveria species showed that subunit III (PsaF) of the photosystem I reaction center (PSI-RC) is present in all these species. This was confirmed by the isolation of the gene encoding the PSI-RC subunit III (PsaF) from Flaveria trinervia, the first psaF gene to be isolated from a C4 plant. The deduced amino acid sequence showed a high degree of similarity to the corresponding protein of spinach which is a C3 species. A region of 17 hydrophobic amino acids in the C-terminal part of the F. trinervia protein was found to be especially conserved in all PsaF proteins studied so far (cyanobacteria and Chlamydomonas).

Separable growth and migration factors for large-cell lymphoma cells secreted by microvascular endothelial cells derived from target organs for metastasis.

Metastatic variant sublines of the murine large-cell lymphoma cell line RAW117 were tested for their growth and migration properties in vitro in medium conditioned by soluble factors released from syngeneic mouse liver-, lung-, and brain-derived microvessel endothelial cells. Medium conditioned with hepatic sinusoidal endothelial cells stimulated the growth of highly liver-colonising (RAW117-H10) and highly liver- and lung-colonising (RAW117-L17) sublines at higher rates than the poorly metastatic parental line (RAW117-P) (H10 greater than L17 greater than P). Medium conditioned with lung microvessel endothelial cells selectively stimulated the growth of the lung-colonising RAW117-L17 subline. Medium conditioned with brain microvessel endothelial cells showed no growth selectivity, and equivalently stimulated the growth of various RAW117 cell sublines. Medium conditioned with hepatic sinusoidal endothelial cells preferentially promoted the migration of the liver-colonising H10 and L17 sublines, and medium conditioned with lung endothelial cells differentially stimulated the migration of the lung-colonising L17 subline; whereas medium conditioned with brain endothelial cells only slightly stimulated the migration of L17, but not H10 or P cells. Fractionation of medium conditioned with hepatic sinusoidal endothelial cells by DEAE Sephacel anion exchange chromatography revealed that the growth-stimulating activities were clearly separable from migration-stimulating activities. The growth- and migration-stimulating activities released from organ microvessel endothelial cells may be important in determining the ability of RAW117 cells to selectively form metastatic colonies in particular organs.

The composition and organization of photosystem I.

Photosystem I, extensively studied in the past decade, was shown to be homologous in all photosynthetic organisms of the higher plants type. Its core complex was found to be highly conserved through evolution from cyanobacteria to higher plants. The genes coding for the subunits of CCI were isolated and the resulting sequences provided information about secondary structural elements. These suggested secondary structures enabled the prediction of the topology of these subunits in the photosynthetic membrane. Structural studies using both electron microscopy and X-ray crystallography, on isolated particles as well as on the complexes in the photosynthetic membrane, led to a better understanding of the overall structure of CCI. Recently two forms of three dimensional crystals of CCI were obtained. These crystals contain all the original components of CCI (both protein and pigments); these components have not been altered by crystallization. It is expected that a detailed crystallographic analysis of these crystals, together with biochemical, spectroscopical and molecular biology studies, will eventually lead to the elucidation of the high resolution structure of the photosystem I core complex and to the understanding of the exact role and mode of action of this complex in the photosynthetic membrane.