Download Degradation signals within both terminal domains of the cauliflower

The Plant Journal (2001) 27(4), 335±343
Degradation signals within both terminal domains of the
cauli¯ower mosaic virus capsid protein precursor
Aletta Karsies, Thomas Hohn* and Denis Leclerc²
Friedrich-Miescher Institute, CH-4002 Basel, Switzerland
Received 19 March 2001; revised 8 May 2001; accepted 24 May 2001.
*
For correspondence (fax +41 61 6973976; e-mail [email protected]).
²
Present address: Universite Laval, Centre de Recherches en Infectiologie, Sainte Foy, QueÂbec GIV 4G2, Canada.
Summary
Targeted protein degradation plays an important regulatory role in the cell, but only a few protein
degradation signals have been characterized in plants. Here we describe three instability determinants in
the termini of the cauli¯ower mosaic virus (CaMV) capsid protein precursor, of which one is still present
in the mature capsid protein p44. A modi®ed ubiquitin protein reference technique was used to show
that these motifs are still active when fused to a heterologous reporter gene. The N-terminus of p44
contains a degradation motif characterized by proline, glutamate, aspartate, serine and threonine
residues (PEST), which can be inactivated by mutation of three glutamic acid residues to alanines. The
signals from the precursor do not correspond to known degradation motifs, although they confer high
instability on proteins expressed in plant protoplasts. All three instability determinants were also active
in mammalian cells. The PEST signal had a signi®cantly higher degradation activity in HeLa cells,
whereas the precursor signals were less active. Inhibition studies suggest that only the signal within the
N-terminus of the precursor is targeting the proteasome in plants. This implies that the other two
signals may target a novel degradation pathway.
Keywords: degradation signals, cauli¯ower mosaic virus, proteolysis, capsid protein, ubiquitin protein
reference technique, PEST motif.
Introduction
Protein degradation plays an important role in many
cellular processes: it allows much faster alteration of the
amount of regulatory proteins than transcriptional or
translational regulation, and is important for the relocation
of biochemical resources. Although protein degradation
has not been extensively studied in plants, accumulating
evidence indicates that it is a complex process involving a
multitude of proteolytic pathways, with each cellular compartment likely to use one or more of them (Vierstra, 1997).
The most widely used pathway in both nucleus and
cytoplasm is the 26S proteasome, a multicatalytic protease
complex that is conserved in all eukaryotes. Proteins are
targeted to the 26S proteasome mostly by covalent linking
of ubiquitin chains. This attachment is catalysed by three
enzymes: ubiquitin-activating enzyme (E1); one or more
ubiquitin-conjugating enzymes (E2); and, usually, a ubiquitin protein ligase (E3) (Hershko and Ciechanover, 1998).
In animals and yeast, the ubiquitin/26S proteasome
pathway plays a role in a number of cellular processes,
ã 2001 Blackwell Science Ltd
primarily by controlling the degradation of short-lived
enzymes and regulatory proteins. Understanding of the
role of this pathway in plants is still rudimentary. Although
numerous components have been identi®ed, only phytochrome A and the A- and B-type mitotic cyclins are known
to be natural targets in plants (Clough et al., 1999;
Genschik et al., 1998), whereas the tobacco mosaic virus
movement protein is the ®rst example of a plant virus
using this pathway (Reichel and Beachy, 2000).
Little is known about other degradation pathways in
either the nucleus or cytoplasm of plant cells. In animals
and fungi, a family of Ca2+-activated neutral proteases,
calpains, is involved in targeted proteolysis in the
cytoplasm (Shumway et al., 1999). However, so far no
plant homologues have been identi®ed, making it unlikely
that this pathway exists in plants.
Viruses can use the degradation pathways of their host
for their own purposes (Mulder and Muesing, 2000). We
have observed previously that although cauli¯ower
335
336
Aletta Karsies et al.
mosaic virus (CaMV) particles are stable, its capsid protein
(CP) precursor is very unstable in plant protoplasts if
expressed outside the viral context (Leclerc et al., 1999).
This protein might therefore be a good model for the study
of targeted protein degradation in plants.
CaMV is the type member of the caulimoviruses, which
contain a circular double-stranded DNA genome and
replicate via reverse transcription (Rothnie et al., 1994).
Virions are approximately 53 nm in diameter with a T = 7
icosahedral symmetry (Cheng et al., 1992). The capsid is
built from three related proteins: p44, p39 and p37. These
are derived from bi-terminal processing of the 57 kDa CP
precursor, the product of ORF IV. Both terminal domains of
the CP precursor are very acidic (Figure 1), and the function
of these domains is not yet known. The N-terminus of p44
results from proteolytic cleavage of the CP precursor
between amino acids 76 and 77 by the viral protease
(Martinez-Izquierdo and Hohn, 1987; Torruella et al., 1989).
The smaller CP forms p39 and p37 are created from p44 by
further processing on both termini, but how this processing occurs has not been elucidated to date.
The sequence between amino acids 32 and 97 of the CP
precursor is characterized by prolines, glutamic and
aspartic acids, serines and threonines, and is not interrupted by basic amino acids. Such motifs are termed PEST
motifs ± these have been found in many unstable mammalian proteins, and it has been shown in several cases
that degradation is mediated by the 26S proteasome
(Rechsteiner and Rogers, 1996). It is still unknown if
PEST motifs also play a role as degradation signals in
plants. In phytochrome A, a PEST motif has been implicated in protein degradation, but in this case the PEST
domain is not suf®cient to confer protein instability
(Clough et al., 1999).
Degradation signals are often studied by fusion to a
stable reporter gene. To prove that differences in protein
accumulation result from degradation, and are not due to
changes in production, pulse-chase analysis followed by
immunoprecipitation is generally used. To reduce the error
in such studies, the ubiquitin protein reference (UPR)
system was developed (Levy et al., 1996), in which the
protein under investigation is produced as a translational
fusion to a stable reference protein separated by a
ubiquitin monomer. Such fusions are rapidly cleaved by
ubiquitin-speci®c processing proteases, yielding equimolar amounts of test and reference proteins.
Measuring protein half-lives directly by pulse-chase
labelling is dif®cult in plant cells due to the low abundance
of transiently expressed proteins, therefore previous
studies of differential stability of plant proteins have
depended on analysis of steady-state levels of test proteins
(Worley et al., 1998). However, with the UPR system
differences in synthetic rates or transcript stability are
Figure 1. The CP precursor.
(a) Schematic drawing of the CP precursor. The acidic and basic
domains, the nuclear localization domain (NLS), and the zinc ®nger (ZnF)
are indicated. The extent of p44 and the position of the instability
domains (ID) are shown underneath.
(b) The amino acid sequence of both termini. The fragments containing
instability signals are boxed, and the region in which mutations were
introduced is underlined.
corrected for, making pulse data suf®cient to prove
instability.
In this study we adapted the UPR technique by using
reporter genes that can easily be measured in cell extracts
as reference and test proteins, and show that this modi®ed
UPR system can be used in both plant and animal systems.
We used this technique to show that the instability of the
CP and its precursor can be localized to certain transferable
instability motifs. We show that these signals are also
active in mammalian cells. Furthermore, we tested
whether the proteasome degradation pathway might
control CP and CP precursor degradation, and discuss
the role of this instability in the context of the virus life
cycle.
Results
Stabilization of CP and CP precursor fragments by
mutations in the PEST signal
ORF IV fragments corresponding to amino acid positions
1±332, 77±332 and 125±332 were fused in-frame to an
N-terminal hemagglutinin epitope (HA11). The resulting
plasmids, p(1±332), p(77±332) and p(125±332), were transiently expressed under CaMV 35S promoter control in
Nicotiana plumbaginifolia leaf protoplasts. As mentioned
above, position 77 corresponds to the start of mature
capsid protein p44. The protoplasts were harvested after
12 h and total protein extracted. Proteins were separated
by SDS±PAGE and detected by Western blotting with antiHA11 monoclonal antibodies. Signi®cant amounts of
p(125±332) could be isolated and easily identi®ed by
Western blotting (Figure 2). However, under similar conã Blackwell Science Ltd, The Plant Journal, (2001), 27, 335±343
Degradation signals in CaMV capsid protein
Figure 2. Accumulation of CP fragments in plant protoplasts.
Western blot analysis of CP levels in protoplasts expressing HA11-tagged
CP constructs, named by their start and end positions in the CP
precursor, with substitutions as indicated. The amino acids mutated to
alanine were serines 82, 86 and 88 (3ScA) or glutamic acids 83, 84 and
85 (3EcA). CP was detected using HA11 Mab 16B12. The expected
positions of the proteins are indicated by arrows.
ditions very little p(77±332) and no p(1±332) at all could be
detected, indicating that these proteins were unstable. To
test whether this instability correlated with an acidic
domain that contains the phosphorylatable serines (the
sequence between amino acids 82 and 91), two mutations
were produced: serines 82, 86 and 88 were replaced by
alanines in mutant 3ScA, and glutamic acids 83, 84 and 85
were replaced by alanines in mutant 3EcA. p(77±332) was
stabilized considerably by the 3ScA mutation, as had been
demonstrated earlier (Leclerc et al., 1999). However, an
even more profound stabilization was achieved by introducing the 3EcA mutation (Figure 2).
This suggests that the cluster of serines and acidic
amino acids between positions 82 and 91 is in fact an
instability element. In the context of the CP precursor [p(1±
332)], the effect of 3ScA was not visible (not shown), but a
small stabilization effect of the 3EcA mutation became
evident. We concluded that additional instability elements
are located between positions 1 and 76.
The UPR technique
To further characterize the putative degradation signals in
the CP and CP precursor, the UPR technique (Levy et al.,
1996) was employed (Figure 3a). In this system, a test
protein is produced as a translational fusion to a stable
reference protein separated by a ubiquitin monomer. This
ubiquitin contained a K48R substitution to prevent the
conjugation of further ubiquitin monomers, which would
lead to protein degradation. Such fusions are rapidly and
precisely cleaved at the C-terminus of ubiquitin by Ubspeci®c processing proteases (UBPs), yielding equimolar
amounts of the test and reference proteins. This proteolytic activity is not affected by the nature of the downã Blackwell Science Ltd, The Plant Journal, (2001), 27, 335±343
337
stream amino acid. The UPR technique avoids the need for
pulse-chase labelling to study protein degradation signals,
as determining the steady-state molar ratios of test and
reference proteins in cell extracts can yield a direct ranking
of their metabolic stabilities (Levy et al., 1996). We adapted
the UPR technique by using two stable reporter genes. The
b-glucuronidase (GUS) gene served as the internal control,
and the CP fragments, fused to either the N- or the Cterminus of chloramphenicol acetyl transferase (CAT), as
the test protein (Figure 3a).
All CAT fusions started with a methionine to exclude
degradation by the N-end rule, which relates the in vivo
half-life of a protein to the identity of its N-terminal residue
(Varshavsky, 1996). These reporter proteins could be
directly quanti®ed in crude extracts of plant protoplasts
or HeLa cells. For transient expression studies in N.
plumbaginifolia protoplasts expression was driven by the
duplicated 35S promoter of CaMV, and in HeLa cells by the
human cytomegalovirus (hCMV) immediate early promoter.
Characterization of the protein instability element in the
mature CP p44
To test whether the PEST signal identi®ed at the Nterminus of p44 functions is a transferable degradation
motif, the ®rst 53 amino acids of p44 were fused to CAT in
the UPR system (Figure 3a). Western blot analysis revealed
a >99% cleavage ef®ciency (Figure 3b), indicating that the
yeast ubiquitin is recognized ef®ciently by plant UBPs.
Con®rming the observations in Figure 2, the CP fusion 77±
120::CAT accumulated to a steady-state level of only 20%
compared to the non-fused CAT control. This level was
increased to 40% by introducing the 3ScA mutation. For
unknown reasons, this value was highly variable.
However, the 3EcA mutation consistently produced a
more stable protein. An additional triple replacement
(D87A/E90A/E91A) or the combination of the 3EcA with
the 3ScA mutation gave rise to proteins that were almost
as stable as the control.
To study the effect of the 3ScA and 3EcA mutations on
virus replication, mutant virus genomes carrying these
modi®cations were used to inoculate turnip plants. The
3ScA mutant virus was non-infectious, con®rming earlier
results (Leclerc et al., 1999). Symptoms originating from
the 3EcA mutant virus appeared with a 7-day delay (after
17 days compared to 10 days for the wild type). Virus
particles were isolated from plants infected with mutant
and wild-type viruses, and analysed by SDS±PAGE and
silver staining. The ratio between the three forms of the
capsid protein was similar in both samples, indicating that
this mutation has no apparent in¯uence on the processing
of p44 to p39 and p37 (data not shown). After three
passages, a partial revertant was obtained in which A85,
338
Aletta Karsies et al.
Figure 3. Mapping of degradation signals within the N-terminus of CaMV p44.
(a) The UPR technique. Reference and test proteins are separated by a ubiquitin moiety. This fusion is cleaved by ubiquitin (Ub)-speci®c proteases at the
Ub±test protein junction, yielding equimolar amounts of both proteins (Levy et al., 1996). For this study the system was adapted by using two stable
reporter genes, the products of which could be quanti®ed in crude extracts. The CP fragment was fused between a methionine and the N-terminus of the
CAT reporter gene. This fragment is shown as an open bar, with mutations in the PEST signal shown underneath. Fragments are named by their start and
end positions in the CP precursor and their substitutions.
(b) Near-complete processing of the GUS-Ub-CAT protein by ubiquitin-speci®c proteases. Proteins were extracted, separated on SDS±PAGE, blotted, and
detected using CAT IgG. The position of the uncleaved polypeptides is indicated by an arrow.
(c) The stability of CAT fusion proteins was measured using the UPR technique. The relative amount of CAT was determined by ELISA and divided by the
GUS internal control after 22 h transient expression in N. plumbaginifolia protoplasts. The results are aligned with the fragments under (a) and shown as
percentages of the control, a GUS-Ub-CAT without fusion. Each bar represents the average of 13±20 samples spread over four independent experiments.
Error bars indicate the range of possible means according to Student's t test.
but not A83 and A84, was changed back to the original
glutamic acid.
Mapping of further destabilization signals in the CP
precursor
In the UPR system, 1±120::CAT (containing the N-terminus
of the CP precursor) was as unstable as the 77±120::CAT
protein (containing the N-terminus of the largest capsid
protein) (Figures 3 and 4). However, introduction of the
3EcA replacement into 1±120::CAT did not lead to signi®cant stabilization of the fusion, in contrast to its effect in
77±120::CAT (see above). This indicates the presence of
additional instability elements between amino acids 1 and
76 of the CP precursor. Separate analysis of this sequence
and two subfragments, 1±31 and 32±76 (Figure 4), revealed
that in fact a strong instability element is located within the
®rst 31 amino acids of the CP precursor.
To determine if the acidic C-terminus of the CP precursor
protein also contains a destabilizing element, C-terminal
fragments of decreasing length were fused to the Cterminus of CAT in the UPR system. CAT::439±489 and
CAT::447±489 were very unstable; CAT::455±489 was still
moderately unstable; but CAT::463±489 was stable (Figure
4). This shows that the C-terminus also contains a
degradation signal.
Thus we have identi®ed three transferable instability
determinants within the CP precursor (Figure 1), one of
which is still present in the mature CP p44.
CP precursor instability signals also function in HeLa
cells
To see if the three mapped degradation signals are plantspeci®c, we studied the degradation of CAT containing
these signals in HeLa cells. All three signals destabilized
ã Blackwell Science Ltd, The Plant Journal, (2001), 27, 335±343
Degradation signals in CaMV capsid protein
339
Figure 4. Mapping of degradation signals at both termini of the CaMV CP precursor.
(a) N-terminal fragments were fused between a methionine and the N-terminus of CAT, and C-terminal fragments were fused to the C-terminus of CAT.
Fragments of the CP precursor that were fused to CAT are shown as open bars, with the mutation in the PEST signal shown underneath. Fragments are
named by their start and end positions in the CP precursor and their substitutions.
(b) The stability of the CAT fusion proteins was measured using the UPR technique as in Figure 3(c).
the reporter protein, but the CP-based signal was much
stronger in the human system than in plant protoplasts,
whereas degradation of CAT fusions containing the CP
precursor-based signals was less pronounced (Table 1).
This correlated with the high score of the CP-based signal
using the PEST-®nd algorithm (Rechsteiner and Rogers,
1996) which was developed, based on motifs frequently
occurring in rapidly degraded proteins in mammalian
cells, to determine objectively whether a protein contains a
PEST region.
Proteasome inhibition reduces degradation of 1±31::CAT
To study whether the CP precursor degradation signals
target the proteasome, we employed proteasome inhibitors. As a positive control in our system, we created GUSub-CAT fusions with phenylalanine or arginine at the start
of CAT. According to the N-end rule (Varshavsky, 1996),
these amino acids should target the CAT protein for
degradation through the ubiquitin pathway in mammalian
systems and also in plants (Worley et al., 1998). However,
these constructs were unexpectedly stable (data not
shown), probably because the N-terminus of CAT was
not exposed, or no lysine in CAT was accessible to the E3±
ã Blackwell Science Ltd, The Plant Journal, (2001), 27, 335±343
Table 1. Activity of CP and CP precursor degradation signals in
mammalian cells
Sequence
1±31
32±76
77±120
77±120,3EcA
1±120
1±120,3EcA
439±489
Control
PEST
score
HeLa
cells
Plant
protoplasts
na
na
17.6
6.3
11.3
7.8
na
11
62
0.6
96
36
59
17
100
2
50
16
75
15
17
2
100
The activity of CP and CP precursor degradation signals was
measured using the UPR technique after 30 h expression in HeLa
cells and compared with plant cells. Values are shown as
percentages of the control. PEST scores were calculated using the
PEST algorithm (Rechsteiner and Rogers, 1996), available at
http://www.at.embnet.org/embnet/tools/bio/PEST®nd
na; Not applicable as the fragment does not contain a valid PEST
signal.
E2 complex (Suzuki and Varshavsky, 1999). This problem
was solved by using a construct with a 31 amino-acid
lysine-free linker between the arginine at the start and CAT
(R-CAT) (Levy et al., 1999).
340
Aletta Karsies et al.
Figure 5. In¯uence of clasto-lactacystin b-lactone on stability of CAT
fusion constructs.
For each construct, nine to ten batches of protoplasts were transfected
and each divided into two tubes. To one of these, 4 mM clasto-lactacystin
b-lactone was added. After 22 h expression, the CAT : GUS ratio of
inhibitor-treated and control samples was compared. Error bars indicate
the range of possible means according to Student's t test.
We analysed the effect of the proteasome inhibitors
MG132, lactacystin and clasto-lactacystin b-lactone.
MG132 is a reversible, competitive inhibitor, whereas
lactacystin and clasto-lactacystin b-lactone are irreversible
inhibitors that modify all catalytic beta subunits (Craiu
et al., 1997). Lactacystin had no effect, as was also reported
for tobacco BY2 protoplasts (Reichel and Beachy, 2000).
Both MG132 and clasto-lactacystin b-lactone had a strong
inhibitory effect on protein production in the protoplasts.
However, after correcting for this effect using the GUS
internal control and by using a low inhibitor concentration,
we could see a fourfold stabilization of R-CAT using clastolactacystin b-lactone (Figure 5), while the CP precursor
fragment 1±31::CAT fusion was stabilized threefold.
The ratio for 77±120::CAT and CAT::455±489 was similar
to that of the CAT control and a stabilized mutant, showing
that their degradation was not inhibited. This indicates that
the sequence 1±31 activates degradation by the proteasome, whereas the two other instability sequences may be
subject to a different degradation pathway.
Discussion
Variations in protein stability play an important role in the
regulation of cellular processes. In eukaryotes, several
motifs have been correlated with instability, including
certain N-terminal residues: the N-end rule (Varshavsky,
1996); the cell cycle-related destruction (D) box (Glotzer
et al., 1991); the KEN-box (P¯eger and Kirschner, 2000);
and the PEST motif (Rechsteiner and Rogers, 1996). In
many, but not all, cases instability correlates with ubiquitination and degradation by the proteasome. Instability
motifs have been studied, mainly in yeast and mammalian
systems, but the N-end rule and the D box have also been
shown to function in plants (Genschik et al., 1998; Worley
et al., 1998). However, no data showing the involvement of
a PEST motif in plant protein degradation have been
reported so far.
Here we describe three separate, transferable instability
elements that contribute to the instability of the CaMV CP
precursor. One of these is still present in the mature CP
p44. The highest destabilization effect in plant protoplasts
was achieved with polypeptides including the ®rst 31 or
the last 49 amino acids of the CP precursor, although
neither of these sequences contains any of the established
instability motifs. On the other hand, the N-terminal
sequence of the mature CP protein, fragment 77±120,
scores highly in the PEST algorithm, but ranks only third in
destabilizing activity. Those motifs behaved differently
when experiments were performed in HeLa cells, where
sequence 77±120 caused the highest destabilization of the
reporter protein (Table 1).
Phosphorylation often plays a role in the regulation of
PEST signal-targeted degradation. Three serines at positions 82, 86 and 88 have been shown to be the major
phosphorylation targets in the N-terminal part of p44
(Leclerc et al., 1999). Mutation of these serines to alanine in
p(77±332) as well as in 77±120::CAT increases stability of
the proteins, but to a lesser extent than mutation of
glutamic acids 83, 84 and 85 to alanines. These data
suggest that phosphorylation enhances, but is not essential for, targeting the proteins for degradation.
The degradation signal within the C-terminal part of the
CP precursor (amino acids 439±489) is also acidic, but does
not correspond to a valid PEST motif, because the prolines
at positions 449 and 453 are separated from the acidic
domain by a basic residue. However, we show that the
domain containing the prolines still contributes to protein
instability, indicating that the degradation signal may well
be PEST-related. Based on these data, we suggest that
PEST-related signals do act as degradation signals in
plants, but that the PEST algorithm as developed for
mammalian proteins needs to be adapted for plant
systems.
The N-terminal sequence of the CaMV CP precursor
includes a hydrophobic cluster embedded between
charged residues. In this respect, it resembles the Cterminal signals involved in ER protein quality control via
retrograde transport into the cytoplasm and degradation
by the proteasome (Bonifacino et al., 1991; Wiertz et al.,
1996). It is possible that such signals also act as degradation signals for proteins located in the cytoplasm.
The ubiquitin±proteasome pathway represents the
main protein-degradation machinery in eukaryotic cells
(Hershko and Ciechanover, 1998), and many PEST-containing proteins have been shown to be degraded by this
pathway (Rechsteiner and Rogers, 1996). However, from
ã Blackwell Science Ltd, The Plant Journal, (2001), 27, 335±343
Degradation signals in CaMV capsid protein
our data it appears that the signal contained in the ®rst 31
amino acids of the CP precursors in fact targets the
proteasome, whereas the two PEST-like instability determinants induce a different degradation pathway. Recent
data show that there are many differences in the mechanisms by which PEST motifs can be targeted for degradation. For example, ODC is targeted to the proteasome by
antizyme instead of ubiquitin (Murakami et al., 1992), and
C-Myc contains a PEST that seems to in¯uence stability
after the ubiquitination step (Gregory and Hann, 2000). It
has also been shown that PEST sequences can affect
protein stability by targeting proteins to alternative
degradation pathways that are different from the proteasome. For example, IkBa contains a PEST degradation
signal that targets degradation by m-calpain (Shumway
et al., 1999). Calpains are Ca2+-activated cysteine proteases. They have been described in animals and fungi,
but so far no plant homologues have been identi®ed,
making it unlikely that this pathway exists in plant cells.
Thus two out of three of the instability determinants
characterized here may target a novel, possibly plantspeci®c, degradation mechanism.
Although the instability elements de®ned here are
transferable, their effects vary depending on the context
and are not simply additive. For instance, in the UPR
system amino acids 1±31 alone bring more instability to
the fusion than the larger fragments 1±76 or 1±120.
Improper folding of the smaller fragment may also
enhance degradation disproportionately. However, in the
context of the CP, where proper folding is likely to occur
due to the presence of the assembly core (Chapdelaine and
Hohn, 1998), the precursor fragment clearly confers
instability on the protein, thus corroborating conclusions
drawn from the heterologous UPR system. A similar
con®rmation of the effect of the C-terminus on CP stability
is revealed by Western analysis: although a CP antiserum
readily detects a C-terminal truncated CP precursor carrying the stabilizing mutation in the N-terminal degradation
signal [CP p(1±332), 3EcA], the full-length precursor
carrying the same mutation [p(1±489), 3EcA] cannot be
detected, showing that the C-terminus also confers
instability in the CP context (unpublished results).
In CaMV-infected cells, most of the CP precursor is
protected from degradation by the viral inclusion bodies
(Kobayashi et al., 1998). Virus particles themselves are also
very stable, although the proportion of p44 relative to the
smaller CP variants decreases upon storage of isolated
particles. Capsid assembly probably protects p44 from
degradation, but still allows it to be processed to yield p39
and p37.
This raises the question of the instability signals' role in
the virus life cycle. Viral genomes carrying the stabilizing
3EcA mutation were infectious, but the appearance of
symptoms was clearly delayed, and the mutant partially
ã Blackwell Science Ltd, The Plant Journal, (2001), 27, 335±343
341
reverted. Although it remains possible that other functions
of CP are affected by this mutation, these data nevertheless suggest a requirement for this degradation signal, and
a role for CP / CP precursor instability in the virus life cycle.
Although the amino acid sequence of the termini of
caulimovirus CP precursors is not conserved, all contain
PEST-like domains at both ends.
Some evidence that the precursor sequences are important for the virus is that deletion of 76 amino acids from the
N-terminus, or 21 amino acids from the C-terminus, of the
CP precursor resulted in non-replicating virus in a protoplast replication system (K. Kobayashi, Friedrich-Miescher
Institute, personal communication). A regulatory function
for the C-terminal CP precursor sequences has been
proposed (Guerra-Peraza et al. 2000), namely to prevent
viral RNA binding to the basic domain and zinc ®nger
motif, thereby guaranteeing its availability as mRNA until
enough viral proteins (including the protease) are produced. We suggest that the N-terminal acidic domain may
also have a regulatory function in the inhibition of nuclear
targeting (Leclerc et al., 1999; unpublished results).
The degradation signals might ensure removal of the Nand C-terminal propeptides created by proteolytic processing of the CP precursor by the viral protease, to avoid
their charge-mediated association with the mature CP.
Furthermore, the instability signals may also be important
for degradation of free forms of the CP precursor and CP in
the infected plant (resulting from over-production or
misfolding of the protein). These might interfere with the
virus life cycle, and/or cause damage to the plant cell due
to their nucleic acid-binding activity (Chapdelaine and
Hohn, 1998; Daubert et al., 1982). Such effects may explain
the toxicity of the CP precursor expressed in Escherichia
coli (FuÈtterer et al., 1988). Using the data presented here,
we can further characterize the proposed regulatory functions and precisely map the precursor degradation signals,
after which mutations affecting only degradation can be
made, allowing its function for the virus to be elucidated.
Experimental procedures
Plasmids
To construct the UPR cassette, the b-glucuronidase (GUS) gene
was modi®ed by PCR to remove the stop codon. The HA11ubiquitin (K48R) fragment was ampli®ed by PCR from pRc/
dhaUbMbgal (Levy et al., 1996). The chloramphenicol acetyl
transferase (CAT) gene was modi®ed by PCR to remove an
internal NcoI site. The UPR cassette was placed in plasmid
pTAGNLS (Leclerc et al., 1999) replacing the HA11 epitope for
transient expression in plant protoplasts, and in the mammalian
expression vector pcDNA3.1 (Invitrogen, Carlsbad, CA, USA) for
expression in HeLa cells. Fragments of ORF IV and mutants
thereof were generated by PCR, using pCa37 as a template, which
is the CAMV genome cloned in the SalI site of pBR322 (Lebeurier
et al., 1980). These were cloned into pTAGNLS or in the UPR
342
Aletta Karsies et al.
cassette. Details of PCR primers, PCR conditions and cloning
steps are available upon request. To introduce the 3EcA mutation
into the viral genome, the KpnI±HpaI fragment of pCa37 was
subcloned and mutated using the Quikchange method from
Stratagene (La Jolla, CA, USA). The mutant fragment was cloned
back into pCa37.
Transient expression in plant protoplasts and HeLa cells
Leaf mesophyll protoplasts of Nicotiana plumbaginifolia were
transfected using polyethylene glycol (Goodall et al., 1990). DNA
(10 or 20 mg) was added to 6 3 105 protoplasts. For the experiment shown in Figure 2 cells were harvested after 12 h incubation
in the dark at 27°C, and for Figures 3 and 4 after 22 h incubation.
HeLa cells were plated in six-well plates, transfected with 1 mg
DNA per well using Fugene (Roche Molecular Biochemicals,
Indianapolis, IN, USA), and harvested after approximately 30 h.
To both types of cells, 200 ml GUS extract buffer (Jefferson
et al., 1987) was added and cells were lysed by three cycles of
freeze±thawing (liquid nitrogen, ±37°C). Samples were centrifuged for 10 min at 15.000 g and the supernatant used for the
quanti®cation of GUS activity and CAT ELISA levels.
Inhibitor studies
Protoplast were transfected as above. Proteasome inhibitors were
dissolved in water (lactacystin) or DMSO (others), and added to
the protoplast culture 30 min after transfection. The ®nal DMSO
concentration in the cultures was 0.1%. We obtained MG132 from
Peptides International (Louisville, USA), and lactacystin and
clasto-lactacystin-b-lactone from Calbiochem (San Diego, USA).
Protoplasts were harvested after 22 h and processed as above.
Detection of proteins
For Western blotting, proteins were isolated from protoplast
extracts with phenol and precipitated with methanol (Hurkman
and Tanaka, 1986). The proteins were separated on SDS±PAGE
and electroblotted onto nitrocellulose at 80 V for 1 h. Blots were
probed using HA11 monoclonal antibodies 16B12 (BAbCO,
Richmond, CA, USA) or antibodies from the CAT ELISA kit
(Roche Molecular Biochemicals). CAT and CAT fusion proteins
were quanti®ed using the CAT ELISA kit. GUS activities were
assayed in 96-well microtitre plates in 200 ml reaction volumes
containing 1 mM 4-methylumbelliferyl b-D-glucuronide (Sigma,
St Louis, MO, USA) (Jefferson et al., 1987). 50 ml aliquots were
taken after approximately 15 and 45 min, and the quantity of
¯uorescent product was recorded using a Titertek Fluoroskan II
apparatus (Jefferson et al., 1990). The relative amounts of CAT
and GUS were calculated relative to the average of the no-fusion
control.
Inoculation of plants
Cloned virus was digested from the plasmid using SalI. Turnip
plants (Brassica rapa var. Just Right) were grown from seed to the
three- to four-leaf stage in a phytotron with 16 h light at 25°C. The
youngest leaf was dusted with Celite, and 10 mg DNA in 20 ml H2O
was gently applied. Plants were kept in the phytotron until
symptoms developed. Virus was isolated from infected tissue
(Gardner and Shepherd, 1980). Leaf sap of infected plants was
used to infect new plants, and after three passages virus mutants
were analysed by amplifying the DNA fragment containing the
mutation and sequencing. The CP content of puri®ed wild-type
and mutant virus was analysed by SDS±PAGE and silver staining.
Acknowledgements
We thank M. MuÈller for the preparation of protoplasts, W. Krek for
HeLa culture facilities, and F. Levy for providing the HA11ubiquitin (K48R) and lysine free linker fragments. We also thank
H. Rothnie, D. Kirk, K. Wiebauer, L. Stavalone and F. Levy for
critical reading of the manuscript. This work was supported by the
Novartis Research Foundation.
References
Bonifacino, J.S., Cosson, P., Shah, N. and Klausner, R.D. (1991)
Role of potentially charged transmembrane residues in
targeting proteins for retention and degradation within the
endoplasmic reticulum. EMBO J. 10, 2783±2793.
Chapdelaine, Y. and Hohn, T. (1998) The cauli¯ower mosaic virus
capsid protein: assembly and nucleic acid binding in vitro. Virus
Genes, 17, 139±150.
Cheng, R.H., Olson, N.H. and Baker, T.S. (1992) Cauli¯ower
mosaic virus: a 420 subunit (T = 7), multilayer structure.
Virology, 186, 655±668.
Clough, R.C., Jordan-Beebe, E.T., Lohman, K.N., Marita, J.M.,
Walker, J.M., Gatz, C. and Vierstra, R.D. (1999) Sequences
within both the N- and C-terminal domains of phytochrome A
are required for PFR ubiquitination and degradation. Plant J. 17,
155±167.
Craiu, A., Gaczynska, M., Akopian, T., Gramm, C.F., Fenteany, G.,
Goldberg, A.L. and Rock, K.L. (1997) Lactacystin and clastolactacystin beta-lactone modify multiple proteasome betasubunits and inhibit intracellular protein degradation and
major histocompatibility complex class I antigen presentation.
J. Biol. Chem. 272, 13437±13445.
Daubert, S.D., Richins, R.D., Shepherd, R.J. and Gardner, R.C.
(1982) Mapping of the coat protein gene of cauli¯ower mosaic
virus by its expression in a prokaryotic system. Virology, 122,
444±449.
FuÈtterer, J., Gordon, K., Pfeiffer, P. and Hohn, T. (1988) The
instability of a recombinant plasmid, caused by a prokaryoticlike promoter within the eukaryotic insert, can be alleviated by
expression of antisense RNA. Gene, 67, 141±145.
Gardner, R.C. and Shepherd, R.J. (1980) A procedure for rapid
isolation and analysis of cauli¯ower mosaic virus DNA.
Virology, 106, 159±161.
Genschik, P., Criqui, M.C., Parmentier, Y., Derevier, A. and Fleck,
J. (1998) Cell cycle-dependent proteolysis in plants.
Identi®cation of the destruction box pathway and metaphase
arrest produced by the proteasome inhibitor mg132. Plant Cell,
10, 2063±2076.
Glotzer, M., Murray, A.W. and Kirschner, M.W. (1991) Cyclin is
degraded by the ubiquitin pathway. Nature, 349, 132±138.
Goodall, G.J., Wiebauer, K. and Filipowicz, W. (1990) Analysis of
pre-mRNA processing in transfected plant protoplasts. Meth.
Enzymol. 181, 148±161.
Gregory, M.A. and Hann, S.R. (2000) c-Myc proteolysis by the
ubiquitin±proteasome pathway: stabilization of c-Myc in
Burkitt's lymphoma cells. Mol. Cell. Biol. 20, 2423±2435.
Guerra-Peraza, O., de Tapia, M., Hohn, T. and HemmingsMieszczak, M. (2000) Interaction of the cauli¯ower mosaic
ã Blackwell Science Ltd, The Plant Journal, (2001), 27, 335±343
Degradation signals in CaMV capsid protein
virus coat protein with the pregenomic RNA leader. J. Virol. 74,
2067±2072.
Hershko, A. and Ciechanover, A. (1998) The ubiquitin system.
Ann. Rev. Biochem. 67, 425±479.
Hurkman, W.J. and Tanaka, C.K. (1986) Solubilization of
membrane proteins for analysis by two-dimensional gel
electrophoresis. Plant Physiol. 81, 802±806.
Jefferson, R.A., Kavanagh, T.A. and Bevan, M.W. (1987) GUSfusion: b-glucuronidase as a sensitive and versatile gene fusion
marker in higher plants. EMBO J. 6, 3901±3907.
Jefferson, R.A., Goldsbrough, A. and Bevan, M.W. (1990)
Transcriptional regulation of a patatin-1 gene in potato. Plant
Mol. Biol. 14, 995±1006.
Kobayashi, K., Tsuge, S., Nakayashiki, H., Mise, K. and Furusawa,
I. (1998) Requirement of cauli¯ower mosaic virus open reading
frame VI product for viral gene expression and multiplication in
turnip protoplasts. Microbiol. Immunol. 42, 377±386.
Lebeurier, G., Hirth, L., Hohn, T. and Hohn, B. (1980) Infectivities
of native and cloned DNA of cauli¯ower mosaic virus. Gene, 12,
139±146.
Leclerc, D., Chapdelaine, Y. and Hohn, T. (1999) Nuclear targeting
of the cauli¯ower mosaic virus coat protein. J. Virol. 73, 553±
560.
Levy, F., Johnsson, N., Rumenapf, T. and Varshavsky, A. (1996)
Using ubiquitin to follow the metabolic fate of a protein. Proc.
Natl Acad. Sci. USA, 93, 4907±4912.
Levy, F., Johnston, J.A. and Varshavsky, A. (1999) Analysis of a
conditional degradation signal in yeast and mammalian cells.
Eur. J. Biochem. 259, 244±252.
Martinez-Izquierdo, J. and Hohn, T. (1987) Cauli¯ower mosaic
virus coat protein is phosphorylated in vitro by a virion
associated protein kinase. Proc. Natl Acad. Sci. USA, 84,
1824±1828.
Mulder, L.C. and Muesing, M.A. (2000) Degradation of HIV-1
integrase by the N-end rule pathway. J. Biol. Chem. 275, 29749±
29753.
Murakami, Y., Matsufuji, S., Kameji, T., Hayashi, S., Igarashi, K.,
ã Blackwell Science Ltd, The Plant Journal, (2001), 27, 335±343
343
Tamura, T., Tanaka, K. and Ichihara, A. (1992) Ornithine
decarboxylase is degraded by the 26S proteasome without
ubiquitination. Nature, 360, 597±599.
P¯eger, C.M. and Kirschner, M.W. (2000) The KEN box: an APC
recognition signal distinct from the D box targeted by Cdh1.
Genes Dev. 14, 655±665.
Rechsteiner, M. and Rogers, S.W. (1996) PEST sequences and
regulation by proteolysis. Trends Biochem. Sci. 21, 267±271.
Reichel, C. and Beachy, R.N. (2000) Degradation of tobacco
mosaic virus movement protein by the 26S proteasome. J.
Virol. 74, 3330±3337.
Rothnie, H.M., Chapdelaine, Y. and Hohn, T. (1994)
Pararetroviruses and retroviruses: a comparative review of
viral structure and gene expression strategies. Adv. Virus Res.
44, 1±67.
Shumway, S.D., Maki, M. and Miyamoto, S. (1999) The PEST
domain of IkBa is necessary and suf®cient for in vitro
degradation by m-calpain. J. Biol. Chem. 274, 30874±30881.
Suzuki, T. and Varshavsky, A. (1999) Degradation signals in the
lysine±asparagine sequence space. EMBO J. 18, 6017±6026.
Torruella, M., Gordon, K. and Hohn, T. (1989) Cauli¯ower mosaic
virus produces an aspartic proteinase to cleave its polyproteins.
EMBO J. 8, 2819±2825.
Varshavsky, A. (1996) The N-end rule: functions, mysteries, uses.
Proc. Natl Acad. Sci. USA, 93, 12142±12149.
Vierstra, R.D. (1997) Proteolysis in plants ± mechanisms and
functions. Plant Mol. Biol. 32, 275±302.
Wiertz, E.J., Tortorella, D., Bogyo, M., Yu, J., Mothes, W., Jones,
T.R., Rapoport, T.A. and Ploegh, H.L. (1996) Sec61-mediated
transfer of a membrane protein from the endoplasmic
reticulum to the proteasome for destruction. Nature, 384, 432±
438.
Worley, C.K., Ling, R. and Callis, J. (1998) Engineering in vivo
instability of ®re¯y luciferase and Escherichia coli betaglucuronidase in higher plants using recognition elements
from the ubiquitin pathway. Plant Mol. Biol. 37, 337±347.