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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. This work was supported by grants from CNR,
Regione Emilia-Romagna, MURST, Pallotti's legacy for Cancer
Research and by University of Bologna (Funds for selected
research topics).
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