Plant Physiol. (1999) 119: 713-724
Use of Ubiquitin Fusions to Augment Protein Expression in
Transgenic Plants1
David Hondred2, 3,
Joseph M. Walker2,
Dennis
E. Mathews4, and
Richard D. Vierstra*
Cellular and Molecular Biology Program and the Department of
Horticulture, University of Wisconsin, Madison, Wisconsin 53706
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ABSTRACT |
A
major goal of plant biotechnology is the production of genetically
engineered crops that express natural or foreign proteins at high
levels. To enhance protein accumulation in transgenic plants, we
developed a set of vectors that express proteins and peptides as
C-terminal translational fusions with ubiquitin (UBQ). Studies of
several proteins in tobacco (Nicotiana tabacum) showed that: (a) proteins can be readily expressed in plants as UBQ fusions; (b) by the action of endogenous UBQ-specific proteases (Ubps), these fusions are rapidly and precisely processed in vivo to release the fused protein moieties in free forms; (c) the synthesis of a
protein as a UBQ fusion can significantly augment its accumulation; (d)
proper processing and localization of a protein targeted to either the
apoplast or the chloroplast is not affected by the N-terminal UBQ
sequence; and (e) single amino acid substitutions surrounding the
cleavage site can inhibit in vivo processing of the fusion by Ubps.
Noncleavable UBQ fusions of 
-glucuronidase became extensively
modified, with additional UBQs in planta. Because multiubiquitinated
proteins are the preferred substrates of the 26S proteasome,
noncleavable fusions may be useful for decreasing protein half-life.
Based on their ability to augment protein accumulation and the sequence
specificity of Ubps, UBQ fusions offer a versatile way to express plant
proteins.
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INTRODUCTION |
Current biotechnological strategies for improving crop plants
often require the high-level expression of natural and "foreign" proteins and peptides. Most of these polypeptides are intended to
confer beneficial agronomic traits such as improved nutritional quality
or resistance to herbicides, viruses, and insects (Shah, 1997
; Yuan and
Knauf, 1997). In addition, transgenic plants have also shown promise
for the economic biosynthesis of pharmaceuticals, vaccines, and
industrial proteins (Goddijn and Pen, 1995; Haq et al., 1995). A
primary obstacle to protein expression in plants is low yields.
Although the use of strong promoters has partially overcome
transcriptional limitations to expression, barriers still exist with
regard to the various posttranscriptional steps required to produce a
fully active, mature protein. Numerous, largely anecdotal examples
exist wherein proteins failed to accumulate to adequate levels in
transgenic plants even though the introduced genes were actively
transcribed (e.g. Odell et al., 1990; Ohtani et al., 1991; Cherry et
al., 1993).
One strategy for augmenting protein expression involves the synthesis
of the protein as a translational fusion to another (LaValle and McCoy,
1995). The fusion partner appears to boost expression by increasing
translation of the mRNA and/or by enhancing solubility and folding of
the protein, presumably by acting as a covalently linked chaperone. An
additional proteolytic step is usually required after translation to
release the protein from the fused partner.
A fusion partner that has received considerable attention in recent
years is UBQ, a highly conserved, stable, and abundant protein in
eukaryotes that functions in selective protein degradation (for review,
see Vierstra, 1996). Its application stems from its unusual method of
synthesis. Unlike most other eukaryotic proteins, UBQ is not
synthesized as individual 76-amino-acid monomers but as fusions. The
corresponding genes either encode a poly-UBQ precursor in which UBQ
monomers are linked in tandem, or UBQ extension proteins in which a UBQ
monomer is linked to the N terminus of an unrelated protein, some of
which are ribosomal subunits. The initial translation products of these
genes are accurately and rapidly cleaved in vivo by Ubps (UBQ
C-terminal hydrolases or de-ubiquitinating enzymes), a family of novel,
sequence-specific proteases that release the UBQ monomers (Wilkinson,
1997). Cleavage occurs irrespective of the amino acid immediately
following UBQ, with the exception of P, which is processed
inefficiently (Varshavsky, 1997).
When examined in microorganisms, fusion of UBQ to proteins has been
shown to substantially enhance accumulation (Butt et al., 1989; Ecker
et al., 1989; Baker, 1996) (see Fig. 1).
This approach was especially beneficial for short peptides and gene
products that were expressed poorly, if at all. For example, the levels of some recalcitrant proteins could be increased several hundred times
in yeast and could account for up to 20% of the soluble protein in
Escherichia coli simply by cotranslation with UBQ. In yeast,
intact, nonfused proteins accumulate after cleavage by endogenous Ubp;
the only exceptions are fused proteins beginning with a P residue.
Because prokaryotes lack Ubps, unprocessed UBQ-fusion products
accumulate in E. coli. These fusions can be processed in
vivo by coexpression with Ubps or in vitro following the addition of
purified Ubps. More recent studies suggest that the UBQ fusion approach
could work in plants as well (Hondred and Vierstra, 1992; Garbino et
al., 1995; Worley et al., 1998). Plants naturally express UBQ fusions
(Callis et al., 1995) and have an array of Ubps, some of which can
process UBQ fusions in vitro and in vivo (Sullivan et al., 1990;
Chandler et al., 1997; N. Yan, T. Falbel, and R.D. Vierstra,
unpublished data).
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| Figure 1.
Various strategies for expressing proteins as UBQ
fusions in plants. 1, Genes are created that express chimeric UBQ
fusion proteins in which the N terminus of the candidate protein is
joined in-frame to the C-terminal G-76 of UBQ. Unique restriction sites
in the fusion vector define a small insertional cassette in which
codons at or near the UBQ-protein junction can be exchanged by swapping
short oligonucleotide bridges. When expressed in plants, endogenous
Ubps specifically cleave the UBQ-protein fusion after G-76 to generate
both the candidate protein and UBQ in free forms. Processing is
expected to occur regardless of the nature of the amino acid following
the C-terminal G of UBQ; P is the only exception. 2, By altering the
N-terminal codon(s) for the candidate protein, proteins with N-terminal
residues other than M can be expressed and released in intact forms by
Ubps. 3, By substituting G-76 of UBQ for A or by including P as the
first amino acid of the candidate protein, processing of the UBQ fusion
by Ubps can be effectively inhibited. 4, The UBQ conjugation pathway
will assemble a chain of UBQs onto these noncleavable UBQ moieties
provided that amino acid residue 48 is a K. Theoretically, these
multiubiquitinated proteins can become substrates for degradation by
the 26S proteasome. 5, If K-48 is replaced by R, the noncleavable UBQ
moiety is protected from subsequent ubiquitination and the fusion
protein may be stable. N, N terminus; C, C terminus.
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Given the potential of UBQ fusions for enhancing protein production in
transgenic plants (Hondred and Vierstra, 1992), we created a series of
vectors for their expression. In this paper we show that chimeric
UBQ-protein fusions can be synthesized in tobacco, accurately processed
to yield unmodified active proteins, and correctly localized to their
appropriate subcellular compartments. For two test proteins, GUS and
LUC, we found that expression as a UBQ fusion can significantly
increase accumulation. As a result, the UBQ fusion approach may have
broad utility for enhancing protein production in plants.
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MATERIALS AND METHODS |
Construction of Chimeric UBQ Fusion Vectors
We generated the various constructions with standard cloning
techniques, using replacement with appropriate oligonucleotide bridges
and/or PCR with mutagenic primers to alter the DNA sequence. The design
of each gene was verified by DNA sequence analysis. Diagrams of the
completed vectors appear in Figures 1, 2, 5, and 6. More complete
descriptions of the constructions are available upon request.
Expression of all genes was directed by the CaMV 35S
promoter obtained as a 645-bp XhoI/SpeI fragment
from the plasmid pAMVBTS (Barton et al., 1987) and designed to contain ApaI, EagI, and NheI sites at the 5
end. The promoter sequence was followed by the 39-bp 5-UTR from AMV
(Gehrke et al., 1983). Polyadenylation signals were obtained from the
3 end of the Agrobacterium tumefaciens NOS gene
(Bevan et al., 1983). The UBQ coding region was obtained from the third
UBQ coding repeat within the Arabidopsis AtUBQ11 gene
(Callis et al., 1995) and engineered to contain a translationally
silent BglII site 10 bp downstream from the start codon, and
a translationally silent SacII site 10 bp upstream from the
terminal G codon. We used PCR to convert UBQ codon K-48 to that for R.
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| Figure 2.
Structures of the various gene constructions used
for expressing GUS and LUC as UBQ fusions. The UBQ fusion vector was
designed to contain the CaMV 35S promoter, the
AMV 5-UTR, and the sequence encoding UBQ. Appended to
the UBQ sequence is DNA encoding GUS or LUC, followed by the
polyadenylation signals from NOS. The nucleotide
sequences surrounding the translational initiation site (arrows) and
the UBQ/protein junction are shown. Convenient restriction sites used
for assembly are indicated; the restriction sites within the 5-coding
region of GUS and LUC are XhoI and KasI,
respectively. The structure and sequences of the respective nonfused
genes that were used as controls are also shown. Arrowheads indicate
the predicted cleavage site by Ubps. UBQ-(P)GUS contains GUS with its
N-terminal Met changed to Pro; UBQ(A)-GUS contains UBQ with its
C-terminal Gly changed to Ala.
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| Figure 5.
Expression and chloroplastic localization of
spinach ACPII after synthesis as a UBQ fusion in transgenic tobacco.
A, Diagram of the ACP expression cassette. ACP bearing its
N-terminal TS and a C-terminal c-Myc epitope tag was expressed either
alone (TS-ACP) or as a UBQ fusion (UBQ-TS-ACP). Arrows indicate the
predicted cleavage sites of the proteins after expression and import to
the chloroplast. B, Immunodetection of ACP proteins expressed in
tobacco. Protein was extracted and subjected to SDS-PAGE, and the ACP
protein was visualized by immunoblot analysis with the c-Myc antibody
9E10. Tot represents 10 µg of total leaf protein. Chloro represents
equal aliquots of protein extracted from the chloroplast stromal
fraction following Percoll gradient centrifugation. Migration position
of ACP alone (arrowhead), TS-ACP, and UBQ-TS-ACP was determined using
the corresponding proteins expressed in E. coli. Lane
NT, Sample from a nontransformed plant. (The approximately 15-kD
protein [*] detected in the tobacco samples was the small
subunit of Rubisco nonspecifically interacting with the c-Myc antibody.
The approximately 15-kD protein in the E. coli-expressed
ACP sample is a bacterial breakdown product of ACP.) C,
Co-localization of ACP with the chloroplast protein CH-42 subunit of
the Mg2+ protoporphyrin chelatase. Total leaf protein (Tot)
and protein from the chloroplast stromal fraction (Chloro) were
extracted from plants expressing UBQ-TS-ACP and subjected to SDS-PAGE
and immunoblot analysis with antibodies against CH-42, c-Myc, or the
cytosolic enzyme UBC1.
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| Figure 6.
Expression and apoplastic localization of
B. licheniformis AMY after synthesis as a UBQ fusion in
transgenic tobacco. A, Diagram of the AMY expression cassette. AMY
bearing the N-terminal signal sequence from the soybean VSP was
expressed either alone or as a UBQ fusion (UBQ-AMY). Arrows indicate
the predicted cleavages sites of the proteins after expression and
export to the apoplast. B, Immunodetection of AMY proteins
expressed in tobacco. Protein was extracted and subjected to SDS-PAGE,
and the AMY protein was visualized by immunoblot analysis with either
AMY antibodies (left) or UBQ antibodies (right). Tot represents 10 µg
of total soluble leaf proteins. Apo represents equal aliquots of
protein extracted from the leaf apoplast. The migration positions of
AMY (without the VSP signal sequence) and UBQ-AMY were determined using
the corresponding proteins expressed in E. coli.
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Using appropriate oligonucleotide bridges, GUS, LUC, AMY, and ACP
coding regions were appended in-frame to the 35S/AMV/UBQ vector at the
SacII site. DNA encoding GUS or LUC and the NOS 3 end was isolated from pCMC1100 (McCabe et al., 1988) or pAB14016LBS (de Wet et al., 1987), respectively, and engineered to contain either a
XhoI site (GUS) or a KasI site
(LUC) at the 5 end. The LUC-coding region retained its
peroxisomal targeting sequence (Gould et al., 1990). We altered the
amino acid sequence at the junction between UBQ and GUS by replacing
the spanning DNA sequence between the SacII and
XhoI sites with appropriate 31-bp oligonucleotide bridges.
Coding DNA for the mature portion of Bacillis licheniformis AMY (minus the N-terminal 29-amino acid secretion peptide), obtained by
PCR amplification of genomic DNA (Pen et al., 1992), was modified to
include the coding sequence for a H-6 tag (RYLHHHHHH) appended in-frame
to the 3 end. The sequence encoding the N-terminal, 22-residue ER
signal sequence from the soybean VSP -subunit (Mason et al., 1988)
was recreated by an oligonucleotide bridge and appended in-frame to the
5 end of the AMY coding region. DNA containing the 48-amino acid TS
and 131-amino acid coding region for spinach (Spinacia
oleracea) root ACPII was obtained from the cDNA pKS21 (Schmid and
Ohlrogge, 1990). We used PCR to add codons for the c-Myc epitope
(EQKLISEEDL [Kolodzeij and Young, 1991]), followed by a stop codon
in-frame to the 3 end. We also generated a control plasmid for each
gene that was identical in all aspects, except for the absence of the
UBQ-coding region. The completed genes were inserted into the binary
A. tumefaciens vector BIN19 at the KpnI and
SalI sites (Bevan, 1984).
Plant Transformations
The completed BIN19 plasmids were introduced directly into
A. tumefaciens strain LBA4404 to transform tobacco
(Nicotiana tabacum cv Xanthi) leaf discs (Cherry et al.,
1993). Stably transformed plants were selected by kanamycin resistance.
Transgenic plants were transferred to soil and grown to maturity in a
greenhouse. We performed transformations for each pair of UBQ fusion
and control vectors simultaneously under identical conditions.
Enzyme Extraction and Analysis
Except as noted, proteins were isolated from the youngest fully
expanded leaves of transformed plants. We measured the total soluble
protein by the Bradford method (Bradford, 1976). For plants expressing GUS, leaf tissue was homogenized in 50 mM sodium
phosphate (pH 7.0), 10 mM -mercaptoethanol, 10 mM Na4EDTA, 20 mM sodium metabisulfite, 0.1% (w/v) sodium lauryl sarcosine, and 0.1% (v/v) Triton X-100. We used a fluorometric assay with 4-methylumbelliferyl -D-glucuronide as the substrate (Gallagher, 1992) to
determine the enzymatic activity from the clarified extracts. GUS
protein was purified using ammonium sulfate precipitation (40%-50%
saturation), followed by BioGel A1.5M size-exclusion
chromatography (Bio-Rad), saccharo-1,4-lactone agarose (Sigma) affinity
chromatography (Harris et al., 1973), and, finally, Mono-Q ion-exchange
chromatography (Pharmacia). Purified GUS protein (50-100 µg) was
subjected to N-terminal amino acid sequence analysis using an Applied
Biosystems model 470A protein sequencer.
We homogenized leaf tissue expressing LUC in 25 mM
Tris-phosphate (pH 7.8), 2 mM DTT, 2 mM
1,2-diaminocyclohexane-N,N,N,N-tetraacetic acid, 10%
(v/v) glycerol, and 1% (v/v) Triton X-100. The amount of LUC protein
was determined by a chemiluminescence assay (Promega) using purified
LUC as the standard (Sigma).
The apoplastic fluid from young tobacco leaves was collected by
centrifugal extraction of intercellular fluid (Pen et al., 1992).
Tobacco leaf strips were washed, blotted dry, and vacuum infiltrated
with 20 mM Hepes (pH 6.9), 3 mM
MgCl2, and 3 mM DTT. Afterward, the
strips were blotted dry, rolled between strips of Parafilm, and
centrifuged at 4°C for 10 min at 200g. We isolated chloroplasts from 15-d-old plants by Percoll density-gradient centrifugation (Falbel and Staehelin, 1994), with the inclusion of 1 mM MgCl2 in the sorbitol
buffer.
Immunoblot Analysis
Proteins were separated by SDS-PAGE, electrotransferred onto
either nitrocellulose or Immobilon-P membranes (Millipore), and subjected to immunoblot analysis as previously described (van Nocker et
al., 1993). Rabbit polyclonal antibodies were prepared against GUS
(Clontech, Palo Alto, CA), LUC (Millar et al., 1992), plant UBQ (van
Nocker et al., 1993), Arabidopsis CH42 (Guo et al., 1998), UBC1
(Sullivan et al., 1994), and B. licheniformis AMY (D. Mathews, unpublished data). The c-Myc epitope was detected with the
monoclonal antibody 9E10 (Kolodziej and Young, 1991). We identified the
immunoreactive proteins with the appropriate alkaline
phosphatase-coupled goat anti-rabbit or goat anti-mouse IgGs
(Kirkegaard & Perry Laboratories, Gaithersburg, MD). The UBQ-GUS
conjugates were immunoprecipitated with GUS antibodies and Protein A
Sepharose (Sigma).
To generate Escherichia coli-expressed versions of ACP and
AMY, we amplified the coding regions by PCR from the appropriate BIN19
plasmids and inserted them into the pET28a or pET29a (Novagen, Madison,
WI) vectors. Induced bacteria (strain BL21) were sonicated and the
clarified cell lysates were used directly for the immunoblot analysis.
Nucleic Acid Extraction and Analysis
We extracted DNA from tobacco leaves using the
hexadecyltrimethylammonium bromide method (Doyle and Doyle, 1987). The
presence of introduced DNA sequences was confirmed by genomic DNA
gel-blot analysis using 32P-labeled,
sequence-specific DNA probes or by PCR of genomic DNA using primers
specific for the coding sequence or the CaMV 35S promoter.
Using the procedure of Wadsworth et al. (1988), we isolated total RNA
from the youngest fully expanded leaves. RNA was electrophoresed under
denaturing conditions in 2.2 M
formaldehyde-containing agarose gels, transferred onto a Zetaprobe
membrane (Bio-Rad), and subsequently hybridized with
32P-labeled RNA probes created using T3 or T7 RNA
polymerase (Stratagene). The GUS-specific probe comprised
the 2.3-kb XhoI-BamHI fragment from pCMC1100. The
LUC-specific probe used an internal 1.2-kb EcoRI
fragment from pAB14016LBS. We made a UBQ-specific probe from
a 210-bp BglII-SacII DNA fragment from
AtUBQ11.
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RESULTS |
Construction of the UBQ Fusion Vector
To examine the UBQ fusion approach in plants (Fig. 1), we created
a cassette vector for expressing proteins/peptides as in-frame C-terminal fusions to UBQ (Hondred and Vierstra, 1992), a diagram of
which appears in Figure 2. The coding
sequence for UBQ was provided by the last UBQ repeat within the
Arabidopsis UBQ11 poly-UBQ gene (Callis et al., 1995); its
derived amino acid sequence is 100% identical to the canonical UBQ
sequence present in higher plants. Two silent restriction sites
(BglII and SacII) were introduced to facilitate
the interchange of 5-regulatory elements, 3-coding regions, and the
amino acid sequence at the UBQ/protein junction through replacement of
short oligonucleotide bridges. Regulatory sequences for effective
expression included the CaMV 35S promoter, the AMV 5-UTR,
and the NOS 3-UTR. The nucleotide sequence immediately 5
to the initiation codon conformed to the optimal Kozak sequence for
mammalian translation initiation (CCACC ATG [Kozak,
1986]), although this sequence may be less than optimal in plants
(Gallie, 1993). For comparison, a control vector was also created that was identical in all respects to the UBQ fusion vector, except that it
was missing the UBQ-coding region (Fig. 2). We gave special attention
to maintaining the same nucleotide sequence 5 to the initiation codon
to avoid any differences in translation efficiency not associated with
the presence of the UBQ-coding sequence. We introduced all of the genes
into tobacco and analyzed a number of independent, stable transformants
for expression.
GUS Expression
We first compared the expression of GUS either alone or as a UBQ
fusion. For tobacco expressing GUS alone, RNA gel-blot analysis with a
GUS-specific RNA probe detected a 2.3-kb mRNA, consistent with the
predicted size of the GUS transcript (Fig.
3A). In plants expressing the UBQ-GUS
fusion (UBQ-GUS), a larger, 2.5-kb mRNA was present, in accord with the
additional 228-bp coding region for UBQ. This 2.5-kb mRNA also
hybridized with a UBQ-specific probe, confirming that the
UBQ sequence was transcribed (Fig. 3A). mRNAs of 1.6 and 0.8 kb were
also detected with the UBQ probe; they probably represent
poly-UBQ and UBQ-extension genes endogenous to tobacco (Callis et al.,
1995).
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| Figure 3.
Expression and processing of UBQ-GUS fusions in
transgenic tobacco. A, RNA gel-blot analysis of 10 µg of total RNA
isolated from young leaves expressing GUS alone or as a UBQ fusion
(lanes UBQ-GUS). Left, RNA blot hybridized with a
GUS-specific probe. Right, RNA blot hybridized with a
UBQ-specific probe. The arrowhead indicates migration
position of the UBQ-GUS transcript. RNAs of 1.6 and 0.8 kb
(*) that also hybridize with the UBQ probe
correspond in size to transcripts derived from endogenous tobacco
UBQ genes. B, Immunoblot analysis of plants expressing
GUS or UBQ-GUS. Soluble protein (10 µg) from young leaves was
subjected to SDS-PAGE, and GUS protein was visualized by immunoblotting
with GUS antibodies. Lane UBQ-GUS + GUS, Equal amounts of leaf extract
from plants expressing UBQ-GUS and GUS mixed prior to electrophoresis.
Lane NT, Sample from a nontransformed plant. C, Accumulation of GUS
protein in plants transformed with either the GUS or the UBQ-GUS
vector. Top and middle, Immunoblot analysis with GUS antibodies of
soluble leaf protein extracted from randomly selected T0
plants independently transformed with either the UBQ-GUS or the GUS
vector. Arrowheads indicate the position of mature GUS. NT, Sample from
a nontransformed plant. Bottom, Distribution profile of GUS activity in
a population of independently transformed tobacco: 19 UBQ-GUS and 16 GUS plants were analyzed. A fluorogenic assay determined GUS activity
by measuring the conversion of the substrate 4-methylumbelliferyl
-D-glucuronide to the product 4-methylumbelliferone
(MU).
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Expression of the UBQ-GUS and GUS vectors resulted in the accumulation
of active GUS protein. The GUS vector directed the synthesis of a 74-kD
protein (Fig. 3B), in agreement with previous SDS-PAGE determinations
of GUS (Jefferson et al., 1987). A protein of indistinguishable size
was also synthesized from the UBQ-GUS vector, despite the additional
sequence encoding UBQ (Fig. 3B). In fact, the products from the GUS and
UBQ-GUS vectors comigrated as a single species during SDS-PAGE. The
protein from UBQ-GUS was not recognized by UBQ antibodies, indicating
that most, if not all, of the UBQ moiety was absent from the mature
protein (Fig. 7A). Even after overloading the SDS-PAGE gels with
protein and overdeveloping the blots, we were unable to detect the
initial translation product of UBQ-GUS, suggesting that the UBQ moiety was rapidly and efficiently removed after synthesis (data not shown).
It is possible that internal initiation of the UBQ-GUS mRNA generated a
polypeptide lacking the UBQ sequence. However, subsequent analysis of
noncleavable UBQ-GUS fusions demonstrated that the UBQ coding region
was translated (see below).
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| Figure 7.
Expression and ubiquitination of UBQ-GUS fusions
containing mutations surrounding the Ubp cleavage site. Mutant versions
of UBQ-GUS that contained the G-76-to-A substitution in UBQ (lanes
UBQ[A]-GUS) or the M-1-to-P substitution in GUS (UBQ-[P]GUS), with
or without K-48-to-A substitution in UBQ (UBQR48) were
created (see Fig. 2). The proteins were expressed in tobacco and
extracted from young leaves. Samples were subjected to SDS-PAGE and
immunoblot analysis with UBQ preimmune serum (PI), GUS antibodies, or
UBQ antibodies. A, Immunoblot analysis of the various GUS proteins
purified from leaves by saccharo-1,4-lactone agarose-affinity
chromatography. B, Immunoblot analysis of total leaf protein.
Arrowhead indicates the migration position of nonprocessed UBQ-GUS.
Dots show the migration positions of UBQ conjugates of UBQ-GUS formed
in vivo. C, Immunoblot analysis of UBQ-GUS immunoprecipitated from
total soluble protein with GUS antibodies and then subjected to
SDS-PAGE and immunoblot analysis with UBQ antibodies. Arrowheads show
unmodified UBQ-GUS and the heavy chain of the GUS IgG. The bracket
indicates the UBQ conjugates of UBQ-GUS.
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To determine the exact cleavage site(s) in the UBQ-GUS protein, we
purified the GUS protein from tobacco expressing UBQ-GUS and GUS and
subjected both preparations to N-terminal amino acid sequence analysis.
Initial yields of phenylthiohydantoin amino acids were close to the
expected values, indicating that each protein probably contained a
free, nonacetylated N terminus (data not shown). The N-terminal
sequence of the protein purified from the GUS-expressing plants was
MLRPVETPTREIKKL for the first 15 cycles, which was identical to that
predicted from the nucleotide sequence (Jefferson et al., 1987). The
same sequence was obtained for UBQ-GUS. In the first cycle, Met was the
only residue in significant quantity (>95% of total residues), with
no evidence of other amino acids that would have resulted from
imprecise cleavage at the UBQ/GUS junction (i.e. Gly, Leu, or Arg
[Fig. 2]).
From a collection of independently transformed tobacco plants, we
compared the amount of GUS protein generated from the GUS and UBQ-GUS
vectors (Fig. 3C). All transgenic plants (19 plants for UBQ-GUS and 16 plants for GUS) that contained one or more nonrearranged copies of the
appropriate gene (as determined by DNA gel-blot analysis) were
included. As is commonly observed for transgene expression in plants
(e.g. Martin et al., 1992; Cherry et al., 1993), the levels of GUS
(determined either immunologically or enzymatically) varied widely
among the independent transformants. However, when the expression
levels were collectively assessed, significantly greater amounts of
GUS protein were generated on average from the UBQ-GUS vector than from
the GUS vector (Fig. 3C). By enzymatic assay, 4.1 times more GUS
activity was synthesized in the UBQ-GUS plants (Table
I). Furthermore, the percentage of the
transgenic plants that accumulated GUS to high levels (>200 nmol
4-methylumbelliferone min
1 mg1 leaf
protein) was 10-fold greater in the UBQ-GUS population (Fig. 3C).
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Table I.
Accumulation of GUS and LUC expressed alone or as a
UBQ fusion
Protein content was assayed in young, mature-green leaves of
independently transformed T0 plants. GUS levels were
determined by fluorogenic assay that measured the conversion of the
substrate 4-methylumbelliferyl -D-glucuronide to the
product 4-methylumbelliferone. LUC levels were determined by
chemiluminescence assay using the activity of the purified LUC as the
standard. n, Number of independent transformants analyzed
for each vector.
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LUC Expression
In a manner similar to the procedure for GUS, we examined the
potential benefits of UBQ fusion for expressing LUC. As can be seen in
Figure 4, a collection of independently
transformed tobacco was generated that expressed LUC or UBQ-LUC. For
the LUC vector, an mRNA of the appropriate size (2.0 kb) was
transcribed and translated into an active LUC protein of 62 kD (Fig. 4,
A and B), in agreement with the apparent molecular mass of the LUC protein reported previously (de Wet et al., 1987). For the UBQ-LUC vector, a slightly larger mRNA of 2.3 kb that contained both LUC- and
UBQ-coding sequences was evident (Fig. 4A). This mRNA generated a
protein indistinguishable in size to that expressed from the LUC vector
(Fig. 4B). Similar to our observations with UBQ-GUS, the UBQ-LUC
protein was easily recognized by LUC antibodies but not by UBQ
antibodies, indicating that the UBQ moiety was removed posttranslationally. Moreover, the 62-kD LUC protein was the only species detected even when the SDS-PAGE gels were overloaded with protein and the blots were overdeveloped (data not shown), suggesting that cleavage was rapid and quantitative.
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| Figure 4.
Expression and processing of UBQ-LUC fusions in
transgenic tobacco. A, RNA gel-blot analysis of 10 µg of total
RNA isolated from young leaves expressing LUC alone or as a UBQ fusion
(lanes UBQ-LUC). Left, RNA blot hybridized with a
LUC-specific probe. Right , RNA blot hybridized
with a UBQ-specific probe. The arrowhead indicates
migration position of the UBQ-LUC transcript. RNAs of 1.6 and 0.8 kb
(*) that also hybridize with the UBQ probe
correspond in size to transcripts derived from endogenous tobacco
UBQ genes. B, Immunoblot analysis of plants
expressing LUC or UBQ-LUC. Soluble protein (10 µg) from young leaves
was subjected to SDS-PAGE and LUC protein was visualized by
immunoblotting with LUC antibodies. Lane UBQ-LUC + LUC, Equal amounts
of leaf extract from plants expressing UBQ-LUC and LUC mixed prior to
electrophoresis. Lane NT, Sample from a nontransformed plant. C,
Accumulation of LUC protein in plants transformed with either the LUC
or the UBQ-LUC vector. Top and middle, Immunoblot analysis with
LUC antibodies of soluble leaf protein extracted from randomly selected
T0 plants independently transformed with either the UBQ-LUC
or the LUC vector. Arrowheads indicate the position of mature LUC.
Bottom, Distribution profile of LUC activity in a population of
independently transformed tobacco: 64 UBQ-LUC and 43 LUC plants were
analyzed. LUC activity was determined by chemiluminescence assay using
the activity of the purified protein as a standard.
|
|
From enzymatic and immunoblot analyses of a collection of transgenic
plants harboring either the LUC or the UBQ-LUC vectors (43 and 64 independent transformants, respectively), we found that the addition of
the UBQ moiety also increased the level of LUC protein (Fig. 4C). Using
a chemiluminescence assay, an average of 2.2 times more active LUC
protein accumulated (Table I). Furthermore, the percentage of the
transgenic plants that had high levels of LUC protein (>160 pg
mg1 leaf protein) was 8-fold greater in the
population expressing UBQ-LUC (Fig. 4C).
Expression of Compartmentalized Proteins
Although it is apparent that cytoplasmic proteins can be readily
synthesized as UBQ fusions (Garbino et al., 1995; Worley et al., 1998;
the present study), this approach may not be successful for many
compartmentalized proteins in which the UBQ moiety could interfere with
proper localization. This is especially true for chloroplastic and
ER/secretory system-resident proteins because the UBQ moiety would
immediately precede the requisite N-terminal transport sequences. The
added UBQ moiety may be less intrusive for chloroplast proteins because
their import occurs posttranslationally (Cline and Henry, 1996), thus
allowing time for UBQ removal by Ubps. However, ER-directed protein is
imported cotranslationally (Walter and Johnson, 1994). Johnson and
Varshavsky (1995) showed previously that inserting the UBQ moiety
following the signal sequence did not impair ER localization in yeast.
However, if UBQ preceded the signal sequence, it was unclear whether
removal of the UBQ moiety would be sufficiently rapid to allow import.
As an example of a chloroplast-localized protein, we tested spinach
ACPII bearing its own TS (Schmid and Ohlrogge, 1990). ACP was modified
at the C terminus to contain the 10-amino acid c-Myc epitope (Kolodziej
and Young, 1991) to help discriminate the spinach protein from the
tobacco ACPs (see Fig. 5A for a
description). When TS-ACP was expressed in transgenic tobacco, the
tagged 17.5-kD ACP protein was easily detected in crude leaf extracts
(Fig. 5B). It was indistinguishable in size from mature ACP and was
substantially smaller than ACP bearing the TS (both expressed in
E. coli), indicating that proper removal of the TS had
probably occurred in tobacco. We also detected a similarly sized
protein of 17.5 kD in plants synthesizing ACP as a UBQ fusion
(UBQ-TS-ACP) (Fig. 5B). The tobacco-expressed UBQ-TS-ACP was easily
recognized by the c-Myc antibody, but unlike an E. coli-derived version, it was not detected with UBQ antibodies (data not shown). Both the antigenicity and the size of the product from the UBQ-TS-ACP vector indicated that the UBQ moiety and the TS had
been removed in planta.
We addressed the proper localization of ACP from the analysis of
chloroplasts isolated from young tobacco leaves. Following the
isolation by Percoll gradient centrifugation, intact chloroplasts were
lysed to release the stromal fraction. Enrichment of the stromal
fraction and the lack of cytosolic contamination were confirmed by the
presence of the stromal enzyme CH-42 subunit of the
Mg2+ protoporphyrin chelatase (Guo et al., 1998)
and the absence of the cytosolic protein UBC1 (Sullivan et al., 1994)
in the final preparations, as determined by immunoblot analysis (Fig.
5C). As can be seen in Figure 5, B and C, the ACP protein expressed from either the TS-ACP or UBQ-TS-ACP vectors properly localized to
chloroplasts. Using transgenic plants expressing near-equivalent levels
of ACP, we found a similar level of enrichment of ACP protein in the
chloroplast stromal fraction.
As an example of an ER-targeted protein, we used an AMY from B. licheniformis (Pen et al., 1992), which was directed to the ER and
ultimately secreted to the apoplast by the N-terminal signal sequence
from the soybean VSP (Mason et al., 1988) (see Fig.
6A for a description). Previous studies
showed that the AMY protein expressed in tobacco by this strategy had
an apparent molecular mass of 62 kD after removal of the signal
sequence, and that this polypeptide had become glycosylated and
exported to the apoplast as a mixture of approximately 70-kD
enzymatically active proteins (Pen et al., 1992; D.E. Mathews,
unpublished data). We confirmed these observations with the AMY vector
described here. In total leaf homogenates from AMY plants, we detected
immunologically a mixture of four to five approximately 70-kD proteins
with molecular masses higher than the single 62-kD AMY protein
expressed in E. coli without the VSP signal sequence (Fig.
6B). These species were present in the apoplastic fraction.
A similar processing and localization was observed for AMY expressed as
a UBQ fusion. In total leaf homogenates from tobacco expressing
UBQ-AMY, a comparable mixture of approximately 70-kD AMY proteins was
detected that could be extracted with the apoplast fluid (Fig. 6B). In
fact, when AMY and UBQ-AMY plants expressing equivalent levels of
protein were compared, similar levels of AMY protein were present in
the apoplast fraction. The only notable difference between the proteins
expressed from the AMY and the UBQ-AMY vectors was an enrichment for
the forms of higher molecular mass in the UBQ-AMY plants (Fig. 6B).
These higher-mass forms could be created by various mechanisms,
including differences in glycosylation, improper cleavage of the signal
sequence, and/or retention of the N-terminal UBQ moiety. From
immunoblot analyses with UBQ antibodies, we eliminated the possibility
that much of the UBQ moiety remained attached. Whereas UBQ-AMY
expressed in E. coli was easily detected, an equivalent
amount of its counterpart collected from tobacco apoplastic fluid was
not (Fig. 6B).
Expression of Noncleavable UBQ Fusions
Processing of UBQ fusions by Ubps is highly sensitive to the amino
acid sequence immediately surrounding the cleavage site. In particular,
substituting other amino acids for the C-terminal Gly of UBQ or
introducing Pro as the first residue of the adjacent polypeptide can
effectively inhibit processing (Baker, 1996; Varshavsky, 1997). The
retained N-terminal UBQ moiety in turn can destabilize the fusion by
serving as an acceptor site for linking the multiubiquitin chain, which
then targets the proteins for breakdown by the 26S proteasome (see Fig.
1). In yeast this conjugation is accomplished by the UBQ-fusion
degradation subpathway and probably uses the free 
-amino group in
Lys-48 in the fused UBQ for binding additional UBQs (Johnson et al.,
1995). By various combinations of UBQ mutations at the Ubp cleavage
site and at Lys-48, noncleavable UBQ fusions can be created that either
destabilize or stabilize proteins in yeast. A noncleavable fusion with
wild-type UBQ (K-48) can be a target for further ubiquitination and
thus may confer a shorter half-life, whereas one bearing a UBQ mutant
containing R at position 48 (R-48) could be resistant to
multiubiquitination and consequently may confer a longer half-life
(Fig. 1). Worley et al. (1998) recently provided evidence that a
similar affect may occur in plants. From studies in which LUC was
transiently expressed in tobacco, they found that appending a truncated
and theoretically noncleavable version of UBQ (residues 1-72)
significantly reduced the levels of active LUC, suggesting that the
half-life of the modified LUC was substantially reduced.
To further investigate the use of noncleavable UBQ fusions in plants,
we generated two sequence variants of the UBQ-GUS vector (see Fig. 1)
that substituted either A for the C-terminal G-76 of UBQ (UBQ[A]-GUS)
or P for the N-terminal M-1 of GUS (UBQ-[P]GUS). These mutations were
combined with either wild-type UBQ or the R mutant
(UBQR-48). Both the UBQ(A)-GUS and UBQ-(P)GUS
mutants were readily expressed in tobacco, producing protein that was enzymatically active and easily detected by GUS antibodies (Fig. 7A and data not shown). Consistent with
the retention of the UBQ moiety, each had an apparent molecular mass of
80 kD, which was approximately 6 kD larger than that of GUS (Fig. 7A).
The presence of the UBQ moiety was confirmed subsequently by the
recognition of both proteins with UBQ antibodies (Fig. 7A). In a
similar fashion, we expressed in tobacco a LUC mutant, bearing a P for
M-1 substitution (UBQ-[P]LUC), and found that this variant also
retained the UBQ moiety (data not shown).
From immunoblot analysis with UBQ antibodies, it became apparent that
the retained UBQ moiety in UBQ(A)-GUS and UBQ-(P)GUS stimulated further
ubiquitination of the fusion, provided wild-type UBQ (K-48) was used.
When total tobacco leaf homogenates were subjected to immunoblot
analysis with UBQ antibodies, we observed a heterogeneous smear of UBQ
conjugates (Fig. 7B) that represented a wide range of polypeptides
modified with multiple UBQs in vivo (van Nocker et al., 1993). In
addition to these, the 80-kD, noncleaved UBQ fusion was evident in
plants expressing UBQ(A)-GUS or UBQ-(P)GUS (Fig. 7B and data not
shown). In fact, these species were the most abundant UBQ conjugates in
the extracts. Above the 80-kD UBQ-GUS fusion, we detected additional
UBQ-immunoreactive proteins in the UBQ(A)-GUS and UBQ-(P)GUS plants but
not in plants expressing UBQR-48(A)-GUS or
UBQR-48-(P)GUS. Their size increments were
consistent with the posttranslational addition of one or more UBQs,
suggesting that the UBQ(A)-GUS and UBQ-(P)GUS proteins became targets
for further ubiquitination in a process that required K-48.
To confirm the identity of the UBQ conjugates, GUS protein was
immunoprecipitated from the extracts with GUS antibodies and the
immunoprecipitates were then subjected to immunoblot analysis with UBQ
antibodies. As can be seen in Figure 7C, we detected a heterogeneous
array of multiubiquitinated GUS proteins from the noncleavable UBQ-GUS
proteins containing wild-type UBQ (i.e. K-48 (UBQ[A]-GUS
or UBQ-[P]GUS). This array was substantially reduced in plants
expressing the noncleavable fusions bearing the R-48 UBQ mutation
(UBQR-48[A]-GUS or
UBQR-48-[P]GUS). Given the possibility that
multiubiquitination of UBQ-(P)GUS destabilizes the protein, we expected
that the half-life of UBQ-(P)GUS would be substantially shorter.
However, using pulse-chase analysis of leaf discs, we found that the
half-lives of UBQ-(P)GUS and UBQR-48-(P)GUS, and
GUS were indistinguishable (about 70 h [data not shown]).
| |
DISCUSSION |
In both yeast and E. coli, UBQ fusions offer a
versatile method to manipulate protein expression (Baker, 1996;
Varshavsky, 1997). By appropriate combinations of amino acids within or
adjacent to the UBQ moiety, accumulation of protein can be dramatically enhanced or, alternatively, the stability of the protein can be substantially reduced (Fig. 1). Here we show that UBQ fusions may
provide similar benefits in plants. We found using transgenic tobacco
that: (a) protein expression as a UBQ fusion can augment protein
accumulation; (b) the N-terminal UBQ moiety does not interfere with
proper localization of chloroplast- or ER-targeted proteins; (c)
through the use of wild-type or modified forms of UBQ, the UBQ moiety
can be rapidly released or remain stably attached to the protein; and
(d) that noncleavable variants of UBQ-protein fusions can become
substrates for further ubiquitination. In yeast multiubiquitinated
forms of these noncleavable fusions are rapidly degraded (Varshavksy,
1997). We observed in all cases that UBQ fusion expression did not
detectably alter function of the gene or activity of the fused protein.
In addition to GUS, LUC, ACP, and AMY, synthesis of active oat
phytochrome A, B. thuringiensis 
endotoxin, and
Aequoria victoria green fluorescent protein was possible
(data not shown).
For all proteins tested, we found that the UBQ moiety was rapidly
removed from the initial translation product to release the fused
protein in an unmodified form. In fact, this processing was so
efficient that we were unable to detect the unprocessed form of any of
the four proteins tested. This processing is not restricted to tobacco;
in transgenic potato and rice, the initial translation product of
UBQ-GUS was also rapidly cleaved, releasing quantitatively the 74-kD
GUS protein (Garbino et al., 1995; P. Christou, D. Hondred, and R.D.
Vierstra, unpublished data). It is remotely possible that internal
initiation ignores the UBQ coding sequence in the fusion-vector mRNAs.
However, results from noncleavable UBQ fusions bearing amino acid
substitutions 76 or 77 codons downstream of the UBQ initiation codon
strongly suggest that the UBQ sequence was translated and removed
posttranslationally.
Although the plant enzyme(s) involved in UBQ processing have not yet
been identified, the nature of the cleavage site (between G-76 of UBQ
and M-1 of GUS) and the sensitivity of cleavage to amino acid
substitutions at this site (G-76 to A or M-1 to P) are consistent with
the action of Ubps (Varshavsky, 1997; Wilkinson, 1997). Plants contain
a number of distinct Ubps that are possible candidates (16 Ubps
identified so far in Arabidopsis), some of which can process UBQ
fusions in vitro and/or in vivo (Sullivan et al., 1990; Chandler et
al., 1997; N. Yan, T. Falbel, and R.D. Vierstra, unpublished data). In
yeast, processing of UBQ fusions by Ubps appears to be cotranslational
(Johnson and Varshavsky, 1995; Varshavsky, 1997). A similar timing is
likely in plants given our observations that an N-terminal UBQ moiety
does not block protein import into the ER. Such import would probably
require removal of the UBQ moiety before docking of the signal sequence with the ER transport machinery.
As is observed in microorganisms, synthesis of proteins as UBQ fusions
can significantly augment accumulation in plants. From analysis of a
population of transgenic tobacco, we found that the presence of the
N-terminal UBQ moiety increased the enzymatically detectable levels of
GUS and LUC. A similar benefit was suggested by Garbino et al. (1995)
from expression of a UBQ-GUS fusion in transgenic potato. However,
because their GUS and UBQ-GUS vectors differed at the Kozak sequence
and because their UBQ-GUS vector also contained an intron within the
5-UTR, it was not possible to differentiate the effects caused by the
UBQ coding region from those caused by the intron or sequence
differences surrounding the translation initiation codon. The
enhancements observed here are more modest than some of those reported
using E. coli or yeast (Butt et al., 1989; Ecker et al.,
1989; Baker, 1996). However, we should note that both the GUS and LUC
proteins are easily expressed and stable in plants. As a result,
greater benefits may be possible for proteins more recalcitrant to
high-level expression. It remains to be determined whether the
UBQ-fusion approach can increase expression of compartmentalized
proteins. Although we showed that AMY and ACP can be synthesized as UBQ
fusions and correctly localized, an insufficient number of
transformants was available to compare expression levels with
confidence. Preliminary studies on a small group of transgenic plants
certainly indicated that the UBQ moiety was not detrimental to ACP and
AMY accumulation (data not shown).
The mechanism(s) whereby an N-terminal UBQ moiety can augment protein
accumulation remain unclear. One possibility is that the favorable
codon bias of UBQ enhances translation of the appended coding region.
However, this possibility is diminished by the fact that yeast UBQ
works in E. coli despite substantial differences in codon
bias (Butt et al., 1989). Our analysis of individual GUS and UBQ-GUS
plants showed that the amount of GUS protein synthesized per mRNA was
indistinguishable for the fusion and control vectors (J.M. Walker and
R.D. Vierstra, unpublished data). Still, it remains possible that
enhanced translation stabilizes the mRNA by association with polysomes,
so that the levels of the protein and the mRNA rise concomitantly.
Another possibility is that the UBQ moiety facilitates folding and/or
stability of the nascent polypeptide or shields the protein from
cotranslational degradation. This is supported by the observations that
UBQ is an extremely stable and proteolytically resistant protein that
folds rapidly (Briggs and Roder, 1992). Given the likelihood that the
UBQ sequence is removed quickly, its benefits to protein accumulation
would have to occur before translation is complete.
As shown previously in yeast, we were able to express noncleaved UBQ
fusions simply by substituting single amino acids at the cleavage site.
Changes in either residue 76 of UBQ or introduction of P as the first
residue of the adjacent fusion were effective. Whereas most Ubp are
unable to process UBQ fusions linked via a P residue, Gilchrist et al.
(1997) recently reported on two from mammalian cells that are able to
break this linkage. The stability of UBQ-P fusions in tobacco suggests
that homologs of these Ubps are not present in plants.
The UBQ fusion vector offers two simple ways to confer a shorter
half-life to transgenic proteins (Fig. 1). The use of cleavable UBQ
fusions allows the production of proteins with N-terminal residues
other than M, after processing of the fusions by Ubps (all residues are
possible except P). By exposing specific "destabilizing" amino
acids at the N terminus, the protein may be rapidly degraded by the
N-end rule pathway, a subpathway within the UBQ system (Varshavsky,
1997). Although the N-end rule pathway is not yet fully described in
plants, recent studies suggest that the same hierarchy of stabilizing
and destabilizing amino acids exists (Potuschak et al., 1998; Worley et
al., 1998).
Another way to confer a short half-life is to express the protein as a
noncleavable fusion with wild-type UBQ. As shown in yeast, these
fusions became targets for further ubiquitination through the K-48
residue, which enhanced their degradation by the UBQ pathway (Johnson
et al., 1995). A similar situation may exist in plants as well but it
appears to be target specific. Worley et al. (1998) reported
that the addition of a noncleavable version of UBQ to LUC dramatically
impaired LUC expression in tobacco, suggesting that the half-life of
the protein was decreased. However, that study and the present study
observed no such "destabilizing" effect for a similar fusion with
GUS. Here we found that a noncleavable version of UBQ-GUS became
extensively ubiquitinated in a reaction that required K-48. Taken
together, the data suggest that the presence of the noncleavable UBQ
and subsequent linkage of additional UBQs are insufficient for rapid
turnover and that other properties (e.g. structure, conformational
stability) of the protein may be involved as well.
Conversely, because noncleavable UBQ fusions bearing the R-48 mutation
appeared to be immune to further ubiquitination, they may be stable in
plants. If so, such fusions may provide a way to stabilize unstable
proteins and could be especially useful for short peptides or protein
subdomains that are typically degraded rapidly if expressed by
themselves.
In conclusion, we found that expressing proteins as UBQ fusions offers
a number of ways to manipulate protein expression in transgenic plants.
Most important is the observation that synthesis of a protein as a UBQ
fusion can augment accumulation. Conveniently, the UBQ moiety is
subsequently removed so that only the unmodified protein accumulates in
vivo. Of immediate interest is a new set of stronger GUS and LUC
reporter vectors that should help in expression analysis of weak
promoters (Gallagher, 1992). Given that the enhancement probably occurs
posttranscriptionally, the UBQ-fusion strategy can be combined with
enhancements in other aspects of transgene expression to elevate
protein accumulation. Clearly, additional examples (especially of those
proteins difficult to express) will be required to determine the extent
to which these vectors will benefit protein production in plants.
| |
FOOTNOTES |
1
This work was supported the U.S. Department of
Agriculture funded through the Consortium for Plant Biotechnology
Research (grant no. 92-34190-6941) and the North Central Biotechnology Initiative (grant no. 94-34190-1204), Pioneer Hi-Bred International, Proctor & Gamble, ICI Seeds, Rhône Poulenc S.A.,
Agrigenetics, Dow Elanco, Northrup King, the Graduate School of the
University of Wisconsin, and the Research Division of the University of
Wisconsin, College of Agriculture and Life Sciences, Madison.
2
These authors contributed equally to this
work.
3
Present address: Pioneer Hi-Bred International,
7300 NW 62nd Street, B.P.O. Box 1004, Johnston, IA 50131-1004.
4
Present address: Department of Plant Biology, 46 College Road, University of New Hampshire, Durham, NH 03824.
*
Corresponding author; e-mail vierstra{at}facstaff.wisc.edu; fax:
1-608-262-4743.
Received September 11, 1998;
accepted November 8, 1998.
| |
ABBREVIATIONS |
Abbreviations:
ACP, acyl carrier protein.
AMV, alfalfa mosaic
virus.
AMY, 
-amylase.
CaMV, cauliflower mosaic virus.
LUC, luciferase.
NOS, nopaline synthase.
TS, transit sequence.
Ubp, UBQ-specific protease.
UBQ, ubiquitin.
UTR, untranslated region VSP,
vegetative storage protein.
| |
ACKNOWLEDGMENTS |
We thank Agracetus, Mycogen, E.I. DuPont Nemours, and Proctor & Gamble for supplying the oligonucleotides used to assemble the various
constructions. We are grateful to Dr. Steve Kay for providing LUC
antibodies; to Drs. David Russell and Mike Miller at Agracetus for the
plasmid pCMC1100 (GUS); to Drs. Anthony Cashmore and William B. Terzaghi for plasmid pAB14016LBS (LUC); to Clara Kielkopf for technical
assistance; and to Dr. Jane Wallent (University of Wisconsin Biotech
Center) for help with protein sequence analysis.
| |
LITERATURE CITED |
Baker RT
(1996)
Protein expression using ubiquitin fusion and cleavage.
Curr Opin Biotechnol
7:
541-546
[CrossRef][Medline]
Barton KA,
Whiteley HR,
Yang N-S
(1987)
Bacillus thuringiensis delta-endotoxin expressed in transgenic Nicotiana tabacum provides resistance to Lepidopteran insects.
Plant Physiol
85:
1103-1109
[Abstract/Free Full Text]
Bevan M
(1984)
Binary Agrobacterium vectors for plant transformation.
Nucleic Acids Res
12:
8711-8721
[Abstract/Free Full Text]
Bevan M,
Barnes WM,
Chilton M-D
(1983)
Structure and transcription of the nopaline synthase gene region of T-DNA.
Nucleic Acid Res
11:
369-385
[Abstract/Free Full Text]
Bradford HM
(1976)
A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal Biochem
72:
248-254
[CrossRef][ISI][Medline]
Briggs MS,
Roder H
(1992)
Early hydrogen-bonding events in the folding reaction of ubiquitin.
Proc Natl Acad Sci USA
89:
2017-2021
[Abstract/Free Full Text]
Butt TR,
Jannalagadda S,
Monia B,
Sternberg E,
Marsh JA,
Stadel JM,
Ecker DJ,
Crooke ST
(1989)
Ubiquitin fusion augments the yield of cloned gene products in Escherichia coli.
Proc Natl Acad Sci USA
86:
2540-2544
[Abstract/Free Full Text]
Callis JA,
Carpenter TB,
Sun CW,
Vierstra RD
(1995)
Structure and evolution of genes encoding polyubiquitin and ubiquitin-like proteins in Arabidopsis thaliana ecotype Columbia.
Genetics
139:
921-939
[Abstract]
Chandler JS,
McArdle B,
Callis J
(1997)
AtUBP3 and AtUBP4 are two closely related Arabidopsis thaliana ubiquitin-specific proteases present in the nucleus.
Mol Gen Genet
255:
302-310
[CrossRef][Medline]
Cherry JR,
Hondred D,
Walker JM,
Keller JM,
Hershey HP,
Vierstra RD
(1993)
Carboxy-terminal deletion analysis of oat phytochrome A reveals the presence of separate domains required for structure and biological activity.
Plant Cell
5:
565-575
[Abstract]
Cline K,
Henry R
(1996)
Import and routing of nucleus-encoded chloroplast proteins.
Annu Rev Cell Dev Biol
12:
1-26
[CrossRef][ISI][Medline]
de Wet JR,
Wood KV,
DeLuca M,
Helinski DR,
Subramani S
(1987)
Firefly luciferase gene: structure and expression in mammalian cells.
Mol Cell Biol
7:
725-737
[Abstract/Free Full Text]
Doyle JJ,
Doyle JL
(1987)
A rapid RNA isolation procedure for small amounts of fresh leaf tissue.
Phytochem Bull
19:
11-15
Ecker DJ,
Stadel JM,
Butt TR,
Marsh JA,
Monia BP,
Powers DA,
Gorman JA,
Clark PE,
Warren F,
Shatzman A,
and others
(1989)
Increasing gene expression in yeast by fusion to ubiquitin.
J Biol Chem
264:
7715-7719
[Abstract/Free Full Text]
Falbel TG,
Staehelin LA
(1994)
Characterization of a family of chlorophyll-deficient wheat (Triticum) and barley (Hordeum vulgare) mutants with defects in the magnesium-insertion step of chlorophyll biosynthesis.
Plant Physiol
104:
639-648
[Abstract]
Gallagher SR
(1992)
Quantitation of GUS activity by fluorometry.
In
SR Gallagher,
eds, GUS Protocols: Using the GUS Gene as a Reporter of Gene Expression.
Academic Press, Boston, pp 47-59
Gallie DR
(1993)
Posttranslational regulation of gene expression.
Annu Rev Plant Physiol Plant Mol Biol
44:
77-105
[CrossRef][ISI]
Garbino JE,
Oosumi T,
Belknap WR
(1995)
Isolation of a polyubiquitin promoter and its expression in transgenic potato plants.
Plant Physiol
109:
1371-1378
[Abstract]
Gehrke L,
Auron PE,
Quigley GJ,
Rich A,
Sonenberg N
(1983)
5-Conformation of capped alfalfa mosaic virus ribonucleic acid 4 may reflect its independence of the cap structure or of cap-binding protein for efficient translation.
Biochemistry
22:
5157-5164
[CrossRef][Medline]
Gilchrist CA,
Gray DA,
Baker RT
(1997)
A ubiquitin-specific protease that efficiently cleaves the ubiquitin-proline bond.
J Biol Chem
272:
32280-32285
[Abstract/Free Full Text]
Goddijn OJM,
Pen J
(1995)
Plants as bioreactors.
Trends Biotechnol
13:
379-387
[CrossRef]
Gould SJ,
Keller GA,
Schneider M,
Howell SH,
Garrard LJ,
Goodman JM,
Distel B,
Tabak H,
Subramani S
(1990)
Peroxisomal protein import is conserved between yeast, plants, insects, and mammals.
EMBO J
9:
85-90
[ISI][Medline]
Guo R,
Luo M,
Weinstein JD
(1998)
Magnesium-chelatase from developing pea leaves.
Plant Physiol
116:
605-615
[Abstract/Free Full Text]
Haq TA,
Mason HS,
Clements JD,
Arntzen CJ
(1995)
Oral immunization with a recombinant bacterial antigen produced in transgenic plants.
Science
268:
714-719
[Abstract/Free Full Text]
Harris RG,
Rowe JJM,
Stewart PS,
Williams DC
(1973)
Affinity chromatography of -glucuronidase.
FEBS Lett
29:
189-192
[CrossRef][ISI][Medline]
Hondred D,
Vierstra RD
(1992)
Novel applications of the ubiquitin-dependent proteolytic pathway in plant genetic engineering.
Curr Opin Biotechnol
3:
147-151
[Medline]
Jefferson R,
Kavanagh T,
Bevans M
(1987)
GUS fusions: -glucuronidase as a sensitive and versatile gene fusion marker in higher plants.
EMBO J
6:
3901-3907
[ISI][Medline]
Johnson ES,
Ma PC,
Ota IM,
Varshavsky A
(1995)
A proteolytic pathway that recognizes ubiquitin as a degradation signal.
J Biol Chem
270:
17442-17456
[Abstract/Free Full Text]
Johnson N,
Varshavsky A
(1995)
Ubiquitin-assisted dissection of protein transport across membranes.
EMBO J
13:
2686-2698
[ISI][Medline]
Kolodziej P,
Young RA
(1991)
Epitope tagging and protein surveillance.
Methods Enzymol
194:
508-519
[ISI][Medline]
Kozak M
(1986)
Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes.
Cell
44:
283-292
[CrossRef][ISI][Medline]
LaValle ER,
McCoy JM
(1995)
Gene fusion expression systems in Escherichia coli.
Curr Opin Biotechnol
6:
501-506
[CrossRef][ISI][Medline]
Martin T,
Wohner R-V,
Hummel S,
Willmitzer L,
Frommer WB
(1992)
The GUS reporter system as a tool for the study of plant gene expression.
In
SR Gallagher,
eds, Gus Protocols: Using the GUS Gene as a Reporter of Gene Expression.
Academic Press, San Diego, CA, pp 23-43
Mason HS,
Guerrero FD,
Boyer JS,
Mullet JE
(1988)
Proteins homologous to leaf glycoproteins are abundant in stems of dark-grown soybean seedlings: analysis of proteins and cDNAs.
Plant Mol Biol
11:
845-856
[CrossRef][ISI]
McCabe DE,
Swain WF,
Martinell BJ,
Christou P
(1988)
Stable transformation of soybean (Glycine max) by particle acceleration.
Biotechnology
6:
923-926
[CrossRef]
Millar AJ,
Short SR,
Chua N-H,
Kay S
(1992)
A novel circadian phenotype based on firefly luciferase expression in transgenic plants.
Plant Cell
4:
1075-1087
[Abstract/Free Full Text]
Odell JT,
Caimi PG,
Yadav NS,
Mauvais CJ
(1990)
Comparison of increased expression of wild-type and herbicide-resistant acetolactate synthase genes in transgenic plants, and implications of posttranscriptional limitations on enzyme activity.
Plant Physiol
94:
1647-1654
[Abstract/Free Full Text]
Odell JT,
Nagy F,
Chua N-H
(1985)
Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter.
Nature
313:
810-812
[CrossRef][Medline]
Ohtani T,
Galili G,
Wallace JC,
Thompson GS,
Larkins B
(1991)
Normal and lysine-containing zeins are unstable in transgenic tobacco.
Plant Mol Biol
16:
117-128
[Medline]
Pen J,
Molendijk L,
Quax WJ,
Sijmons PC,
van Ooyen AJJ,
van den Elzen PJM,
Rietveld K,
Hoekema A
(1992)
Production of active Bacillus licheniformis alpha-amylase in tobacco and its application in starch liquefaction.
Biotechnology
10:
292-296
[Medline]
Potuschak T,
Stary S,
Schlögelhofer P,
Becker F,
Nejinskaia V,
Bachmair A
(1998)
PRT1 of Arabidopsis thaliana encodes a component of the plant N-end rule pathway.
Proc Natl Acad Sci USA
95:
7904-7908
[Abstract/Free Full Text]
Schmid KM,
Ohlrogge JB
(1990)
A root acyl carrier protein-II from spinach is also expressed in leaves and seeds.
Plant Mol Biol
15:
765-778
[CrossRef][ISI][Medline]
Shah DM
(1997)
Genetic engineering for fungal and bacterial diseases.
Curr Opin Biotechnol
8:
208-214
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Abstract
Introduction