From the Biotechnology Center for Agriculture and the
Environment and Department of Plant Pathology, Rutgers University, New
Brunswick, New Jersey 08901-8520 and § Department of
Molecular Genetics and Microbiology and The Cancer Institute of New
Jersey and ¶ Graduate Program in Molecular Biosciences at Rutgers,
University of Medicine and Dentistry of New Jersey,
Piscataway, New Jersey 08854
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ABSTRACT |
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Pokeweed antiviral protein (PAP), a 29-kDa
ribosome-inactivating protein, catalytically removes an adenine residue
from the conserved The 29-kDa pokeweed antiviral protein
(PAP)1isolated from the
American pokeweed plant, Phytolacca americana is a
single-chain ribosome-inactivating protein (RIP) that catalytically
removes a specific adenine residue from the highly conserved,
surface-exposed, The rRNA substrate for PAP, the Yeast Strains and Vectors--
The cDNAs encoding PAP
(NT188) and PAPx (NT224) were introduced as
SmaI/BglII fragments into the yeast expression
vector YEp351. PAPx is an active-site mutant of PAP with a point
mutation (E176V) that abolishes enzymatic activity (9). Transcription
of the cDNAs was under the control of a galactose-inducible
GAL1 promoter. Vectors containing PAP or PAPx were
transformed (19) into the yeast strains S. cerevisiae W303
(MATa, ade2-1 trp1-1 ura3-1 leu2-3, 112 his3-11, 15 can1-100), 1906 (MATa, leu2 mak8-1), or the isogenic strains JD980 (MAT Yeast Growth and Time Course Induction--
Yeast cells were
grown in 300 ml of H-Leu medium (20) with 2% raffinose at 30 °C to
an A600 = 0.6. Aliquots for protein analysis (2 ml), RNA extraction (15 ml), and ribosome isolation (25 ml) were
removed and pelleted by centrifugation at 2,000 × g
for 5 min. The remaining culture was pelleted at the same speed, washed
in H-Leu medium, and resuspended in H-Leu medium with 2% galactose to
induce the expression of PAP and PAPx. At various times during
induction (2, 4, 8, 12, and 24 h), aliquots were removed,
pelleted, and stored at Ribonuclease Protection Assay--
RNA from a frozen yeast
aliquot was extracted according to Cui et al. (21). Total
RNA from the time point aliquot of PAP and PAPx induced in
mak8-1 cells was used in the RNase protection assay as
described in Tumer et al. (10).
Protein Expression Analysis--
Frozen pellets of a 2-ml
aliquot of cells harvested during the time course induction of PAP and
PAPx were resuspended in an equal volume of cold (4 °C)
phosphate-buffered saline buffer and 0.3 g of 0.5-mm diameter
glass beads. Cells were vortexed for 2 min and centrifuged at
16,000 × g for 5 min. Supernatant total protein was
quantified by Bradford using bovine serum albumin as a standard. Total
protein (30 µg) from each time point was separated through 12%
SDS-PAGE, transferred to nitrocellulose, and probed with either an
affinity-purified (22) polyclonal antibody to PAP (1:5000) or a
monoclonal antibody to L3 (anti TCM 7.1.1, gift of J. R. Warner)
(1:5000). PAP and L3 were visualized by chemiluminescence using a
Renaissance kit (NEN Life Science Products). To probe proteins with two
separate antibodies, blots were stripped by incubation in 8 M guanidine hydrochloride at room temperature for 30 min.
The nitrocellulose was washed four times in phosphate-buffered saline
with Tween for 15 min each before exposing the blot to another antibody.
Isolation of Yeast Ribosomes--
Yeast cells harvested from a
25-ml aliquot during the time course induction of PAP and PAPx in
wild-type and mak8-1 cells were ground to a fine powder in
liquid N2 with a mortar and pestle. Cold (4 °C) buffer A
(4 ml of 200 mM Tris-HCl, pH 9.0, 200 mM KCl,
200 mM sucrose, 25 mM MgCl2, 25 mM EGTA, 25 mM 2-mercaptoethanol) was added to
the yeast powder and centrifuged at 16,000 × g for 20 min. The resulting supernatant was increased to 13 ml with buffer A and
layered over a 10-ml cushion of 1 M sucrose, 25 mM Tris-HCl, pH 7.6, 25 mM KCl, 5 mM MgCl2. Ribosomes were pelleted by
centrifugation at 311,000 × g for 3.5 h at
4 °C. The pellets were resuspended in 100 µl of 25 mM
Tris-HCl, pH 7.6, 25 mM KCl, 5 mM
MgCl2, aliquoted, and stored at rRNA Depurination Assay--
Total ribosomes (50 µg) isolated
from yeast cells expressing PAP, PAPx, or vector control were
resuspended in RIP buffer (167 mM KCl, 100 mM
Tris-HCl, pH 7.2, 100 mM MgCl2) to a final
volume of 100 µl. Extraction of rRNA and subsequent analysis for
depurination were conducted according to Tumer et al. (23).
A positive control standard for depurination was generated by
incubating 50 µg of ribosomes from wild-type yeast with 100 ng of
purified PAP (Calbiochem) in RIP buffer. The mixture was incubated at
37 °C for 30 min, and RNA was isolated as referenced above.
Depurination of rRNA was confirmed by the presence of a 360-nucleotide
fragment visible on the urea-acrylamide gel.
Co-immunoprecipitation--
PAP and L3 expressed in
vivo in wild-type and mak8-1 cells were
co-immunoprecipitated with the monoclonal antibody to L3 essentially as
described by Otto and Lee (24). Ribosomes (100 µg) from cells induced
to express PAP, PAPx, or vector control were used as substrate for
immunoprecipitation with protein A-Sepharose beads. The pelleted complex of antibody and protein was eluted from the beads with SDS
sample buffer and visualized by immunoblot analysis using the
antibodies to PAP and L3. Co-immunoprecipitation of in vitro synthesized L3 with purified PAP was used to demonstrate a direct interaction between these two proteins. Radiolabeled L3 (3 nM), synthesized by a linked transcription translation
system (TNT-coupled reticulocyte lysate system, Promega) was incubated
with 3 nM purified nonradiolabeled PAP and
immunoprecipitated with the monoclonal L3 antibody. Proteins were
eluted from the Sepharose beads with SDS sample buffer, and the
solution was divided in half. Half was separated through 12% SDS-PAGE,
transferred to nitrocellulose, and probed with the polyclonal antibody
to PAP. The remaining half was also separated through 12% SDS-PAGE,
then incubated with Entensify Solution A and B (NEN Life Science
Products), dried, and exposed to autoradiography.
mak8-1 Cells Are Resistant to PAP--
PAP removes a specific
adenine residue from the PAP Is Expressed in mak8-1 Cells--
Resistance to PAP may have
arisen because either transcript or protein had not accumulated in
mak8-1 cells. To determine whether PAP transcripts were
synthesized, nuclease protection assays were performed to examine the
accumulation of PAP mRNA relative to CYH2 mRNA, an
internal control that encodes the ribosomal protein L29 (25). The
levels of PAP transcript were compared with those of PAPx, the
active-site mutant of PAP. The zero hour time point represents cells
grown in raffinose under noninducing conditions. As shown in Fig.
2, cells grown in raffinose did not
express PAP or PAPx transcripts. However, 2 h after shifting to
galactose-containing medium, transcripts corresponding to both PAP and
PAPx were detected in mak8-1 cells. Quantitation of PAP
mRNA, relative to the CYH2 internal control, indicated
that both PAP and PAPx transcript levels remained constant during the
time course of induction. A protected RNA fragment corresponding to PAP
was not observed in cells containing the vector alone, 8 h after
induction by galactose (Fig. 2, lane VC). When tRNA was used
in place of total cellular RNA as a control, no specific binding by the
radiolabeled probes was detected (Fig. 2, lane tRNA). These
results demonstrated that both PAP and PAPx mRNAs were transcribed
in cells harboring the mak8-1 allele, and no significant
difference in the level of transcripts could be detected in cells
expressing PAP or PAPx.
To test whether PAP was expressed and accumulated in mak8-1
cells, immunoblot analysis was conducted on aliquots harvested from
cells grown on galactose medium through a 24-h time course. The same
experiment was carried out with wild-type yeast cells harboring PAP
(NT188) and PAPx (NT224). Similar amounts of PAP and PAPx were
expressed in mak8-1 cells, suggesting a lack of toxicity
due to PAP accumulation, whereas in wild-type cells, PAPx was expressed
to a greater degree than PAP (Fig. 3).
The higher molecular mass protein reacting with the PAP antibody likely represents the precursor form of PAP, observed previously in yeast (9).
Overexpression of PAPx often results in lower molecular mass proteins,
most likely breakdown products, seen clearly in wild-type cells induced
for 24 h. However, the primary band in each immunoblot is the
29-kDa mature form of PAP.
mak8-1 Ribosomes Are Not Depurinated by PAP--
To determine
whether there were differences between the ability of PAP to depurinate
ribosomes from wild-type and mak8-1 cells in
vivo, ribosomes were isolated from yeast cells induced to express PAP or PAPx for 8 h. rRNA was isolated from these ribosomes,
treated with aniline, and separated on a urea-acrylamide gel.
Depurination of rRNA was revealed by the presence of the 360-nucleotide
fragment produced by removal of a purine residue from the 25 S rRNA and subsequent cleavage at that site by treatment with aniline
(arrow in Fig. 4). A positive
standard for depurination was generated by incubating wild-type
ribosomes with PAP in vitro, extracting the rRNA, and
treating it with aniline (Fig. 4, lane std). Ribosomes isolated from wild-type cells harvested 8 h after induction of PAP
expression were depurinated, whereas ribosomes of cells harboring the
mak8-1 allele were not depurinated during PAP expression
in vivo (Fig. 4.). Ribosomes isolated from both cell types
expressing PAPx were not depurinated, which was consistent with the
prior observation that PAPx lacks enzymatic activity.
PAP Does Not Associate with Ribosomes in mak8-1 Cells--
A
possible reason for the lack of depurination of rRNA in
mak8-1 cells was that PAP may not be able to access its
rRNA substrate in these cells. To determine whether PAP associated with
ribosomes in wild-type cells, ribosomes examined for depurination were
also assessed by immunoblot analysis with the affinity-purified
antibody against PAP. As illustrated in Fig.
5A, both PAP and PAPx were associated with ribosomes in wild-type cells. In contrast, neither PAP
nor PAPx could be detected with ribosomes isolated from
mak8-1 cells. The higher levels of PAPx associated with
ribosomes of wild-type cells likely reflected the increased level of
expression of enzymatically inactive PAPx relative to the enzymatically
active PAP (Fig. 3). The immunoblots of ribosomal proteins were
stripped and reprobed with a monoclonal antibody against L3 to
illustrate that L3 or its mutant form was detected on both types of
ribosomes and that similar amounts of protein were loaded from both
cell types (Fig. 5B).
PAP Binds Free L3 and Mak8-1p--
Results described above
indicated that PAP is associated with ribosomes in wild-type cells but
not in mak8-1 cells, suggesting that PAP may interact with
L3. To test the hypothesis of direct interaction with L3, purified PAP
was mixed with in vitro synthesized L3 or Mak8-1p and
co-immunoprecipitated with the monoclonal L3 antibody. Purified PAP
co-immunoprecipitated with L3 or Mak8-1p when it was mixed with either
protein and not when it was incubated alone (Fig.
6A). As expected, L3 and
Mak8-1p were immunoprecipitated with L3 antibody when they were each
mixed with PAP or incubated alone (Fig. 6B). These results
indicated that PAP binds directly to L3 or Mak8-1p in its free form
(Fig. 6A).
Co-immunoprecipitation of PAP and L3 from Ribosomes--
To
determine whether PAP interacts with L3 and Mak8-1p incorporated into
ribosomes, ribosomes from wild-type or mak8-1 cells expressing either PAP, PAPx, or vector alone were immunoprecipitated with the monoclonal L3 antibody. As shown in Fig.
7A, PAPx was co-immunoprecipitated with L3 from ribosomes of wild-type, but not
mak8-1 cells, indicating that PAPx does not interact with the mutant form of L3 in ribosomes. The lack of co-immunoprecipitation of PAP with L3 from ribosomes of wild-type cells may reflect the previous observation that wild-type protein is not synthesized as
abundantly as the active-site mutant (Fig. 3). The difference may also
be the result of variation in the kinetics of association between PAP
and PAPx, namely PAP may dissociate more readily from its substrate,
the ribosomes, than PAPx. Fig. 7B illustrates that L3 or
Mak8-1p was immunoprecipitated from ribosomes of both cell types and
that similar amounts of protein were loaded on the gel. These results
suggest that the absence of association between PAP and Mak8-1p in
ribosomes may be the result of a conformational change such that the
peptide sequence or tertiary structure required is not accessible when
Mak8-1p is incorporated into ribosomes. The lack of
co-immunoprecipitation of PAP with Mak8-1p in ribosomes substantiates
earlier results that showed the absence of PAP in ribosomes from
mak8-1 cells and lack of rRNA depurination.
We have reported previously that in vivo
induction of PAP expression in yeast had a cytostatic effect (9). This
work demonstrates that cells containing the mak8-1 allele
are resistant to PAP. The lack of growth inhibition observed in
mak8-1 cells is because of the fact that ribosomes from
these cells are not associated with PAP, and consequently, are not depurinated.
The observation that PAP expressed in wild-type yeast depurinates
ribosomes but does not when expressed in mak8-1 cells
indicates that wild-type L3 is required for depurination of ribosomes.
Co-immunoprecipitation experiments with isolated proteins illustrated
that PAP directly binds to L3 and Mak8-1p in vitro.
However, when the experiments were repeated using intact ribosomes, PAP
co-immunoprecipitated only with wild-type L3 from ribosomes and not
with Mak8-1p, indicating that PAP does not interact with Mak8-1p in
ribosomes. The quaternary structure of a ribosome containing Mak8-1p
may differ from a wild-type ribosome such that the binding site for PAP
may be masked in the mutant ribosomes. Alternatively, a difference in
post-translational modifications between L3 and Mak8-1p may affect its
interaction with PAP in vivo. The hypothesis for altered
binding by the mutant L3 is supported by the observation that we did
not detect PAP or PAPx associated with ribosomes in mak8-1
cells; however, both proteins were associated with ribosomes in
wild-type cells.
The evidence presented here demonstrates a link between L3 and
the These data lead us to propose a model to explain the interaction
between PAP and L3. Co-immunoprecipitation studies demonstrate that PAP
binding to ribosomes requires wild-type L3. Therefore, we suggest that
PAP accesses its substrate, the Although the mechanism underlying the catalytic activity of RIPs is
understood, very little is known about how RIPs gain access to the
ribosome. Although all RIPs have the same specificity for adenine 4324 of naked 28 S rRNA, they show very different levels of activity against
ribosomes of different species. For example, ricin is 23,000 times more
active on rat liver ribosomes than on plant ribosomes (29), whereas PAP
is equally active on ribosomes from all five kingdoms. These data
suggest that the differences in sensitivity of ribosomes to RIPs may
reflect differences in interactions of RIPs with ribosomal proteins.
Endo and Tsurugi (30) showed that the ricin A chain depurinated rat
rRNA at adenine 4324 in intact ribosomes much more efficiently than
naked 28 S rRNA. Conversely, the ricin A chain depurinated naked 23 S
rRNA of E. coli at the homologous adenine 2660 and did not
depurinate intact E. coli ribosomes. Formation of a covalent
complex between saporin and a component of the 60 S subunit of yeast
ribosomes was shown by chemical cross-linking (31). Similarly, the
ricin A chain has been cross-linked to mammalian ribosomal proteins L9
and L10e (32). Despite some evidence for the dependence of RIP activity
on the type of ribosomal substrate, the functional significance of the
association between RIPs and ribosomal proteins has not been reported.
Nevertheless, these observations support the hypothesis for a molecular
recognition mechanism involving one or more ribosomal proteins that
could provide receptor sites for toxins and favor optimal binding to
the target adenine. The results reported here demonstrate that PAP
gains access to the ribosome by recognizing L3. Because L3 is highly
conserved among ribosomes from different species, the interaction
between PAP and L3 may be the underlying reason for the broad spectrum
activity of PAP on ribosomes from different organisms.
-sarcin loop of the large rRNA, thereby
preventing the binding of eEF-2·GTP complex during protein
elongation. Because the
-sarcin loop has been placed near the
peptidyltransferase center in Escherichia coli ribosomes,
we investigated the effects of alterations at the peptidyltransferase
center on the activity of PAP. We demonstrate here that a chromosomal
mutant of yeast, harboring the mak8-1 allele of
peptidyltransferase-linked ribosomal protein L3 (RPL3), is
resistant to the cytostatic effects of PAP. Unlike wild-type yeast,
ribosomes from mak8-1 cells are not depurinated when PAP
expression is induced in vivo, indicating that wild-type L3
is required for ribosome depurination. Co-immunoprecipitation studies
show that PAP binds directly to L3 or Mak8-1p in vitro but
does not physically interact with ribosome-associated Mak8-1p. L3 is
required for PAP to bind to ribosomes and depurinate the 25 S rRNA,
suggesting that it is located in close proximity to the
-sarcin
loop. These results demonstrate for the first time that a ribosomal
protein provides a receptor site for an ribosome-inactivating protein
and allows depurination of the target adenine.
INTRODUCTION
Top
Abstract
Introduction
References
-sarcin loop in the large rRNA of eukaryotic and
prokaryotic ribosomes (1, 2). PAP displays broad-spectrum antiviral activity against plant and animal viruses, including influenza virus
(3), poliovirus (4), herpes simplex virus (5), and human
immunodeficiency virus (6). PAP removes an adenine base by cleavage of
the N-glycosidic bond at A4324 in rat 28 S rRNA
and at homologous sites on ribosomes from other organisms. Ribosomes
depurinated in this manner are unable to bind the eEF-2·GTP complex,
and protein synthesis is blocked at the translocation step (7, 8). We
previously reported that in vivo induction of PAP expression
in yeast had a cytostatic effect. Growth was strongly inhibited when
PAP expression was induced, possibly because of inhibition of
translation (9). Because ribosomal frameshifting occurs during the
elongation phase of protein synthesis, we investigated the role of
translocation inhibition by PAP in programmed ribosomal frameshifting
by utilizing two different viral systems (the L-A and M1
"killer" system and the Ty1 retrotransposable element)
of Saccharomyces cerevisiae. We demonstrated that expression
of PAP in yeast leads to specific inhibition of programmed ribosomal
frameshifting in the +1 direction and interferes with the ability of
Ty1 to retrotranspose (10).
-sarcin loop, has been localized in
close proximity to the peptidyltransferase center within the 50 S
subunit of Escherichia coli ribosomes (11). We hypothesized that yeast cells harboring mutations in ribosomal proteins that have
been linked to the peptidyl transfer reaction may be resistant to the
cytostatic effects of PAP. One of these mutants, mak8-1, was discovered by its inability to maintain the M1
satellite virus (12). mak8-1 is an allele of
RPL3/TCM1 (13), which encodes the large ribosomal subunit
protein L3 (14). L3 has been shown to participate in the formation of
the peptidyltransferase center (15, 16) and identified as an essential
protein in the catalysis of peptide bond formation (17). Recent results
indicate that strains harboring the mak8-1 allele of
RPL3 exhibit increased programmed frameshifting
efficiencies, supporting the notion that events at the
peptidyltransferase center play a critical role in programmed
1
ribosomal frameshifting (18). The current study demonstrates for the
first time that ribosomal protein L3 is essential for binding of PAP to
ribosomes and subsequent depurination of the
-sarcin loop, providing
direct evidence that PAP accesses its rRNA substrate by binding to a
ribosomal protein.
EXPERIMENTAL PROCEDURES
lys2 his3 ura3 leu2
trp1
RPL3
::hisG), containing either pRPL3 or pmak8-1
(18). YEp351 transformed into all cell types was used as a negative control.
80 °C. Pellets for ribosome isolation were
washed twice in water and quickly frozen in liquid N2.
80 °C.
RESULTS
-sarcin loop of yeast 25 S rRNA. Because
this loop is located near the peptidyltransferase center, we introduced
PAP into the strain harboring the mak8-1 allele to
determine whether this mutation conferred resistance to the cytostatic
effects of PAP. Both wild-type and mak8-1 cells were
transformed with the LEU2-based vector, pNT188, containing the PAP cDNA under the control of a GAL1 promoter. As
shown in Fig. 1, galactose induction of
PAP expression did not have a cytostatic effect on the growth of the
strain harboring the mak8-1 allele. In contrast, growth of
wild-type cells was significantly inhibited when PAP expression was
induced by galactose. To confirm this observation, isogenic
RPL3::hisG strains (18) were tested for their
sensitivity to PAP. Cells harboring pmak8-1 were able to grow under
PAP induction, whereas cells containing pRPL3 encoding wild-type L3 did
not grow (data not shown).
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Fig. 1.
Effect of PAP expression on the growth of
wild-type and mak8-1 cells. Both strains were
transformed with NT188 containing PAP under the control of a
GAL1 promoter. Cells were either plated onto H-Leu medium
containing raffinose ( leu raf) or galactose (
leu
gal). wt, wild type.
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Fig. 2.
Transcript levels of PAP and its active-site
mutant (PAPx) in mak8-1 cells. Expression of PAP
and PAPx was induced by growing cells in H-Leu medium containing
galactose. Cells transformed with vector alone (VC) and
grown on galactose medium for 8 h were used as a negative control.
Approximately 15 µg of total RNA was extracted from
mak8-1 cells harvested during the 24-h time course
induction and analyzed by RNase protection. Lanes marked PAP
and CYH2 indicate the position of the ( ) strand probes alone, and
tRNA lane represents RNase protection analysis using tRNA as
a control.
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Fig. 3.
Immunoblot analysis indicating the time
course of PAP expression in wild-type (wt) and
mak8-1 cells. Cells containing PAP or the
active-site mutant of PAP (PAPx) were induced in H-Leu medium
containing galactose. Total protein (15 µg) loaded for each time
point after induction was separated through 12% SDS-PAGE. Proteins
were transferred to nitrocellulose and probed with an affinity-purified
polyclonal PAP antibody (1:5000). Purified PAP, 20 ng, (Calbiochem) was
used as a standard. std, standard.
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Fig. 4.
In vivo depurination of ribosomes from
wild-type (wt) and mak8-1
cells. Expression of PAP or its active-site mutant (PAPx)
was induced by growing the cells on H-Leu medium containing galactose.
Wild-type cells containing vector alone (VC) were used as a
negative control. Aliquots of cells (25 ml) were harvested for
isolation of ribosomes after an 8-h induction. Total RNA extracted from
50 µg of ribosomes was divided in half, and one-half was treated with
aniline (+), the other half was not ( ). After aniline treatment, RNA
was separated on a 7 M urea, 6% acrylamide gel and
visualized with ethidium bromide. A positive control was made by
incubating 50 µg of wild-type ribosomes with 100 ng of PAP in
vitro and treating with aniline (std), resulting in the
360-nucleotide rRNA fragment, shown by the arrow.
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Fig. 5.
Immunoblot analysis indicating association of
PAP with ribosomes from wild-type (wt) but not from
mak8-1 cells. A, ribosomes were
isolated from both wild-type and mak8-1 cells after
induction of PAP or its active-site mutant (PAPx) for 8 and 24 h.
Ribosomes isolated from cells containing vector alone (VC)
were used as a negative control. Total ribosomal protein (50 µg) was
separated through 12% SDS-PAGE, transferred to nitrocellulose, and
probed with an affinity-purified polyclonal antibody to PAP (1:5000).
Purified PAP, 20 ng (Calbiochem), was used as a standard. B,
the same blot as in A stripped with 8 M
guanidine hydrochloride and reprobed with a monoclonal antibody to L3
(1:5000).
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Fig. 6.
Co-immunoprecipitation of purified PAP with
in vitro synthesized L3. 35S-labeled
L3 or Mak8-1p (3 nM) was incubated with 3 nM
purified PAP (Calbiochem) and immunoprecipitated with a monoclonal
antibody to L3. A, immunoprecipitated proteins were
separated through 12% SDS-PAGE, transferred to nitrocellulose, and
probed with the affinity-purified PAP antibody. B,
immunoprecipitated proteins were separated through 12% SDS-PAGE,
dried, and exposed to autoradiography. PAP std denotes 20 ng
of PAP (Calbiochem). Lanes marked PAP, L3, and Mak8-1p
represent purified PAP and 35S-labeled L3 and Mak8-1p,
respectively, immunoprecipitated alone with the L3 antibody. Protein
A-Sepharose beads were used as a background control (Bead).
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Fig. 7.
Co-immunoprecipitation of PAP with L3 from
ribosomes. Ribosomes (100 µg) isolated from wild-type
(wt) and mak8-1 cells induced to express PAP,
PAPx, or vector control (VC) for 8 h were incubated
with a monoclonal L3 antibody and immunoprecipitated with protein
A-Sepharose beads. Immunoprecipitated proteins were separated through
12% SDS-PAGE, transferred to nitrocellulose, and probed with the
affinity-purified PAP antibody (1:5000) (A) or with a monoclonal L3
antibody (1:5000) (B). Standard (std) in
A denotes 40 ng of purified PAP (Calbiochem), and
std in (B) represents the endogenous L3 protein in yeast
total cellular lysates.
DISCUSSION
-sarcin loop in eukaryotic ribosomes. Experiments designed to
reconstitute the minimal ribosomal particle still capable of enzyme
activity have established that L3 is essential for maintaining peptidyltransferase activity (15). A photolabile cDNA probe targeted to the central loop of domain V was shown to cross-link to L3
(26). With the use of a photolabile oligodeoxynucleotide probe
complimentary to the
-sarcin region of E. coli,
Muralikrishna et al. (11) recently demonstrated the
proximity of the
-sarcin region to domains IV and V of E. coli rRNA. tRNA localization experiments further demonstrated the
mutual proximity of domains IV, V, and VI within the 50 S subunit (27).
Chemical and enzymatic footprinting have shown that L3 binds in region
VIA of 23 S rRNA near the
-sarcin loop (28). Preliminary results
from our laboratory indicate that both PAP and PAPx bind to the
-sarcin loop.2 The data
presented here substantiate these observations by suggesting that L3 is
in close proximity of the
-sarcin loop in yeast 25 S rRNA.
-sarcin loop, by recognizing and
binding to L3. Once bound, the close proximity of L3 to the
-sarcin
loop would facilitate the subsequent depurination of the 25 S rRNA by
PAP. Because PAPx does not interact with ribosomes from
mak8-1 cells, we contend that the PAP binding site may be masked in mak8-1 ribosomes. The mak8-1 gene
product encodes a mutant L3 that differs from the wild-type by only two
amino acid substitutions, W255C and P257S, which may be sufficient to
alter the shape of the protein product (18), affecting its interaction with other components of the ribosome. If rRNA is necessary to place
the ribosomal proteins in a proper conformation to facilitate PAP
binding, the point mutations in Mak8-1p may alter the interaction between rRNA and Mak8-1p.
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ACKNOWLEDGEMENTS |
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We thank Drs. Peter Day and Terri Kinzy for critical reading of the manuscript, John Warner for the gift of the monoclonal antibody against L3, and Reed Wickner for strain 1906. We also thank Dr. Annette Chang for constructing pNT188, Bijal Parikh for constructing pNT224, Amy Hammell for constructing pRPL3 and pmak8-1, and Jason Yazenchak for isogenic RPL3/mak8-1 strains.
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FOOTNOTES |
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* This work was supported by National Science Foundation Grants MCB96-31308 (to N. E. T.), MCB97-27941 (to N. E. T. and J. D. D.), and in part by grants from the Foundation of the University of Medicine and Dentistry of New Jersey (16-98) and the State of New Jersey Commission on Cancer Research (97-60-CR) (to J. D. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 732-932-8165 (ext. 215); Fax: 732-932-6535; E-mail: tumer{at}aesop.rutgers.edu.
The abbreviations used are: PAP, pokeweed antiviral protein; RIP, ribosomal-inactivating protein; eEF-2, eukaryotic elongation factor 2; PAGE, polyacrylamide gel electrophoresis.
2 P. Wang and N. E. Tumer, unpublished data.
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REFERENCES |
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