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Journal of Experimental Botany, Vol. 48, No. 313, pp. 1519-1523, August 1997 Journal of Experimental Botany Cofactor requirement of ribosome-inactivating proteins from plants Domenica Carnicelli, Maurizio Brigotti, Paola Alvergna, Alessandra Pallanca, Simonetta Sperti and Lucio Montanaro1 Dipartimento di Patologia sperimentale dell'Universita di Bologna, Via San Giacomo 14,1-40126 Bologna, Italy Received 22 October 1996; Accepted 18 March 1997 Abstract A large number of type 1 ribosome-inactivating proteins (RIPs) from plants (families of Caryophyllaceae, Cucurbitaceae, Euphorbiaceae, Phytolaccaceae, and Poaceae) were examined for their requirement for ATP and supernatant factors for full activity. A marked requirement was observed with agrostin among Caryophyllaceae, gelonin among Euphorbiaceae, and with both barley RIP and tritin-S among Poaceae. The distribution of cofactor requirement in Phytolaccaceae discriminates leaf forms (cofactor-independent) from seed and root forms (cofactor-dependent). The results are discussed on the basis of the present knowledge on the tissue localization of RIPs and on the sensitivity of ribosomes to conspecific RIPs. Key words: Cofactors, ribosome-inactivating proteins, RNA-/V-glycosidase, up-regulation. Introduction All the above-mentioned RIPs are type 1 RIPs, consisting of a single polypeptide chain of approximately 30 kDa, in contrast to type 2 RIPs, many of which are toxic, constituted of a catalytic polypeptide (A-chain) linked to a B-chain able to bind to cell surface receptors on target cells. Type 1 RIPs are widely distributed in higher plants and many plants produce several RIPs, similar but not identical to each other (possibly isoenzymes), either present within the same organ or in different organs (reviewed in Barbieri et al., 1993). Recently two forms of tritin were purified from the seeds (tritin-S) and from the leaves (tritin-L) of Triticum aestivum (Massiah and Hartley, 1995). Of the two RIPs, which showed striking chemical and immunological dissimilarities, tritin-S, unlike tritin-L, had a marked requirement for cofactors in its action. The availability of a large number of type 1 RIPs purified in the department by Professor F Stirpe and co-workers prompted the present study in which RIPs from different plants and from different parts of the same plant are compared for cofactor requirement in an assay containing Artemia salina 80S ribosomes, which are a good model of animal purified ribosomes devoid of tRNA, mRNA and adherent elongation factors (Sierra et al., 1974; Sperti et al., 1991). Assuming that conspecific plant ribosomes show the same pattern of cofactordependence as A. salina ribosomes, the results may help to clarify the relationship between RIP isoforms and help to shed some light on the physiological role of RIPs, which is still poorly understood, although a defensive and antiviral function is largely believed. Several years ago, and long before ribosome-inactivating proteins (RIPs) were identified as RNA-N-glycosidases which remove a specific adenine from a highly conserved loop of the large-subunit rRNA (Endo and Tsurugi, 1987), tritin, the RIP from wheat (Triticum aestivum), and pokeweed antiviral protein (PAP), the RIP from Phytolacca americana, were shown to require, for full activity on isolated ribosomes, the presence of ATP and extra-ribosomal macromolecular cofactors (Coleman and Roberts, 1981; Ready et al., 1983). Such requirement, initially considered a curiosity, was later observed with gelonin, the RIP from Gelonium multiflorum, with barley RIP from the seeds of Hordeum vulgare and with agrostin, Materials and methods the RIP from Agrostemma githago (Sperti et al., 1991; RIPs were purified in the laboratory of Professor F. Stirpe by Carnicelli et al., 1992; Brigotti et al., 1995). previously described methods (references in Barbieri et al., 1 To whom correspondence should be addressed. Fax: +39 51 354746. E-mail: [email protected] © Oxford University Press 1997 1520 Carnicelli et al. 1993). Cofactor requirement was tested in the standard twostep assay described by Carnicelli et al. (1992) using a rabbit reticulocyte post-ribosomal supernatant gel-filtered through Sepharose G-25 ('S-140', Sperti et al., 1991) as source of macromolecular cofactors and high-salt-washed 80S A. salina ribosomes (Sierra et al., 1974) as substrate. The high-saltwashing procedure removes adherent cofactors from ribosomes (Sperti et al., 1991). In the first step of the assay ribosomes (10 pmol in 10/il) are preincubated with the RIP in the absence and in the presence of gel-filtered 'S-140' (3^1) and ATP (1 mM). After 10 min at 28 °C, 2.5 /A samples are withdrawn and the extent of ribosome inactivation is quantitated in 100 JX\ poly(U) translation system requiring only GTP, poly(U), and a crude preparation of elongation factors. In this system RIPs and other components from the first step are made ineffective by extensive dilution (40-fold). lCi0 is calculated from the linear regression between fractional ribosomal activity and log of RIP concentration. A kDa of 30 was assumed for all RIPs (Barbieri et al., 1993). seeds, suggesting that the leaf and seed forms of the RIP have a different regulation and function. A 200-fold stimulation of activity was observed with PAP-R from roots, a 150-fold stimulation with PAP-C produced by cultured cells in vitro (Barbieri et al., 1989) and a 550-fold stimulation with one of the RIPs (PD-S2) isolated from the seeds of Phytolacca dioica. In Poaceae, barley RIP was 43 000-fold and tritin-S 300-fold stimulated by cofactors. A possible antiviral role of the RIPs in the leaves of Phytolacca americana is based on the following evidence, (i) Examination of the cellular localization of PAP shows the RIP heavily sequestered in the cell wall matrix of pokeweed cells (Ready et al., 1986). (ii) Ribosomes in the leaves of Phytolacca americana are sensitive to the conspecific RIP since they are depurinated during preparation and additional treatment with PAP has no further effect (Taylor and Irvin, 1990; Prestle et al., 1992). Results and discussion Depurination is thought to occur because the initial homogenization of the tissue brings ribosomes in contact Type 1 RIP producing species are particularly abundant with the sequestered RIP. The antiviral hypothesis envisin the families of Caryophyllaceae, Cucurbitaceae, Euphorbiaceae, and Poaceae. As shown in Table 1, all ages that damage to the cell by the pathogen releases the the RIPs from Cucurbitaceae are powerful inactivators RIP from the cell wall matrix in the cytosol where it of ribosomes both in the absence and in the presence of inactivates ribosomes thus preventing viral replication cofactors, and only agrostin in Caryophyllaceae and and spreading of infection. gelonin in Euphorbiaceae emerge as RIPs, poorly active The cDNA sequences encoding PAP and PAP II show by themselves, but which are over 100-fold stimulated in an amino-terminal leader extension found in the initial the presence of ATP and 'S-140'. In Caryophyllaceae the translation product, but not in the mature protein (Lin several immunological related isoforms of saporin isolated et al., 1991; Poyet et al., 1994) and this sequence probably from the leaves, roots and seeds of Saponaria officinalis has a role in the targeting of PAPs to the cell wall matrix are either unaffected (saporin-Rl, -R3 and -S9) or only (Irvin and Uckun, 1992). Although studies on the cellular moderately up-regulated by cofactors (leaf saporins and localization of type 1 RIPs as detailed as those carried saporin-R2, -S5, -S6 and -S8). A slight stimulation also out with PAP have only been carried out with saporins, occurred with dianthins (dianthin 30 present throughout which are sequestered in extracellular and intracellular the plant, seeds included, and dianthin 32 found only in locations in the seeds and leaves of Saponaria officinalis leaves and growing shoots) confirming previous observa(Carzaniga et al., 1994), nucleotide sequences coding for tions (Reisbig and Bruland, 1983). an amino-terminal putative targeting peptide for extracelIn the following discussion, an RIP is considered as lular deposition or intracellular sequestration have been cofactor-dependent when, in the experimental conditions, detected in several other RIPs (references in Irvin and it is over 100-fold up-regulated by gel-filtered supernatant Uckun, 1992; Kataoka et al., 1992a, b) from leaves in the presence of 1 mM ATP. As shown in Table 1, (dianthin 30), seeds (saporin 6, momordin and lufnn-a except in the case of the RIP from Hordeum vulgare, ATP and -j8) and roots (a-trichosanthin). The view that riboalone only approximately halves the /C 50 of cofactorsomes of most RIP-producing plants are sensitive to their dependent RIPs, so that the RIP stimulating effect must own RIPs, from which they are protected by spatial refer mainly to macromolecular components of the supersegregation, is now generally held, and, consistently, natant which become active in the presence of ATP ribosomes isolated from many dicotyledons are inactiv(Sperti et al., 1991). ated by their own RIP during preparation (Prestle et al., Particularly interesting is the distribution of cofactor 1992). The same occurs with ribosomes isolated from the requirement among the RIPs isolated from Phytolacleaves of Triticum aestivum which are the source of tritin-L caceae. PAP was the first type 1 RIP to be identi(Massiah and Hartley, 1995). PAP from leaves and fied and isolated, and PAP, PAP II and PAP-S are among saporins, whose sequestration has been directly demonthe most studied and best characterized RIPs. As shown strated by electron microscopy (Ready et al., 1986; in Table 1, neither PAP from spring leaves nor PAP II Carzaniga et al., 1994), are cofactor-independent RIPs. from summer leaves were cofactor-dependent, while Cofactor-independent are also tritin-L (Massiah and cofactors caused a 700-fold stimulation of PAP-S from Hartley, 1995), dianthin 30, momordin, and trichosan- Ribosome-inactivating proteins and cofactors 1521 Table 1. IC 50 of RIPs assayed for the inactivation of ribosomes in the absence and in the presence of gel-filtered 'S-140' and ATP /QofnM) None Caryophyllaceae Agrostemma githago Linn. Dianthus caryophyllus Linn Lychnis chalcedonica Linn. Saponaria officinalis Linn. Petrocoptis glaucifolia (Lag.) Boiss Cucurbitaceae Bryonia dioica Bieb. Citrullus colocynthis Schrad. Luffa aegyptiaca Mill. Momordica charantia Linn. Momordica cochinchmensis Spreng. Trichosanthes kirilowii Maxim. Euphorbiaceae Croton tiglium Linn. Gelomum mulliflorum A. Juss. Jatropha curcas Linn. Manihot palmata Muell. Arg. Manihol aesculenta Crantz Phytolaccaceae Phytolacca americana Linn. Phytolacca dioica Linn Poaceae Hordeum vulgare Linn Triticum aestivum Linn. 'S-140' +ATP Seeds Leaves Leaves Seeds Leaves Leaves Roots Roots Roots Seeds Seeds Seeds Seeds Whole plant Agrostin Dianthin 30 Dianthm 32 Lychnin Saporin-Ll Saporin-L2 Saporin-Rl Saponn-R2 Saporin-R3 Saporin-S5 Saporin-S6 Saporin-S8 Saporin-S9 Petroglaucin Leaves Roots Seeds Seeds Seeds Seeds Seeds Roots Seeds Bryodin-L Bryodin-R Colocin 1 Colocin 2 Luffin Momordin Momorcochin-S Trichosanthin Tnchokinn Seeds Seeds Seeds Seeds Seeds Seeds Crotin 3 Gelonin Curcin 1 Mapalmin Manutin 1 Manutin 2 Leaves Leaves Seeds Cultured cells Roots Seeds PAP PAP II PAP-S PAP-C PAP-R PD-S2 0.05 004 37.70 9.00 6.04 114.00 12.01 4.01 3.16 52.20 0.08 0.06 0.05 0.06 0.03 0.21 Seeds Germ Barley RIP Tritin-S 863.00 903.00 21.00 321 00 0.02 2.90 thin, whose sequestration is deduced by the presence of a targeting peptide and/or by the occurrence of depurination of ribosomes during purification of the RIP. Among cofactor-dependent RIPs, the cDNA for barley RIP (Leah et al., 1991) and the genomic sequence for tritin-S (Habuka et al., 1993), two RIPs located exclusively in the endosperm (Massiah and Hartley, 1995), do not encode an amino-terminal signal peptide, from which it can be inferred that they accumulate in the cytosol in contact with ribosomes. This coexistence requires a striking resistance of ribosomes to the conspecific RIP. Consistently, in contrast to ribosomes prepared from Triticum aestivum leaves (containing tritin-L), ribosomes isolated from the endosperm (containing the immunological distinct tritin-S) and from wheat germ (containing 9.30 1.45 0.10 0.31 3.70 6.90 0.07 1.50 0.04 0.42 0.37 1.15 1.21 15.85 ATP 5.60 0.08 0.23 0.06 0.04 0.25 0.38 0.52 0.05 0.01 2.30 40.00 0.17 0.23 0.05 0.12 0.09 0.43 0.03 0.26 0.30 0.42 0.08 0.60 0.04 0.02 0.03 0.07 0.90 9.60 0.06 0.25 0.03 0.02 0.23 0.43 0.54 0.05 0.01 23.00 1.37 0.27 0.09 0.15 0.03 0.08 tritin-S derived from contaminating endosperm fragments, Massiah and Hartley, 1995) show no detectable depurination (Taylor and Irvin, 1990; Massiah and Hartley, 1995). For these cofactor-dependent RIPs it may be envisaged that metabolic or environmental alterations producing an increased level of macromolecular cofactors, at the cytosolic concentration of ATP (mM range), may break the physiological resistance of the plant ribosomes to the conspecific RIP. Data on the sensitivity of conspecific ribosomes and on the subcellular localization of the RIP are missing to test this hypothesis on the other cofactor-dependent RIPs. The sequence of PAP-S has only been determined by direct amino acid sequencing (Kung et al., 1990), since the genomic clone encoding a pokeweed antiviral protein 1522 Carnicelli et al. isolated by Kataoka et al. (1992c), and containing signalling peptides, does not apparently encode for PAP-S. A cDNA copy of the gene encoding gelonin contains a presequence (Nolan et al., 1993), but it is unusually long (42 or 46 aa) for a typical RIP leader sequence (21-25 aa, Irvin and Uckun, 1992; Kataoka et al., 1992a, b; Poyet et al., 1994). The antiviral hypothesis applied to cofactorindependent RIPs does not exclude a role in stress reactions. PAP from leaves, which is classified as a cofactorindependent RIP, was actually one of the first RIPs for which a cofactor requirement, detectable only in the presence of high salt concentrations, was reported (Ready et al., 1983). By attacking self ribosomes in virally infected cells and by activation in extreme ionic conditions, PAP might behave both as a defence protein and as an inhibitor of protein synthesis in osmotically stressed or desiccated leaf tissue. Besides the classical RNA-iV-glycosidase activity, a DNA deadenylating activity has been recently reported for PAP (Stirpe et al., 1996) and many other RIPs (Barbieri et al., 1994, 1996). Since both activities increase in senescent and stressed leaves of Phytolacca americana, it is suggested that they might co-operate in the arrest of vital functions in cells bound to die. Acknowledgements We thank Professor Fiorenzo Stirpe of this department for the generous supply of RIPs and for helpful comments on the manuscript. 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