
. 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 deubiquitinating enzymes), a family of novel, sequencespecific 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 Ecker et al., 1989; Baker, 1996 ) (see Fig. 1 ). This approach was especially beneficial for short peptides and gene products that were expressed poorly, caleb39@example.org. 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) .

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.
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 5Ј end. The promoter sequence was followed by the 39-bp 5Ј-UTR from AMV (Gehrke et al., 1983) . Polyadenylation signals were obtained from the 3Ј 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.
Construction of Chimeric UBQ Fusion Vectors
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 3Ј 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 5Ј 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 3Ј 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 5Ј 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 3Ј 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 Na 4 EDTA, 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 g) was subjected to N-terminal amino acid sequence analysis using an Applied Biosystems model 470A protein sequencer.
Enzyme Extraction and Analysis
We homogenized leaf tissue expressing LUC in 25 mm Tris-phosphate (pH 7.8), 2 mm DTT, 2 mm 1,2-diaminocyclohexane-N,N,NЈ,NЈ-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).
Enzyme Extraction and Analysis
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 MgCl 2 , and 3 mm DTT. Afterward, the strips were blotted dry, rolled between strips of Parafilm, and centrifuged at 4°C for 10 caleb39@example.org. We isolated chloroplasts from 15-d-old plants by Percoll density-gradient centrifugation (Falbel and Staehelin, 1994) , with the inclusion of 1 mm MgCl 2 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).
Immunoblot Analysis
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 32 P-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 formaldehydecontaining agarose gels, transferred onto a Zetaprobe membrane (Bio-Rad), and subsequently hybridized with 32 P-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 LUCspecific 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.
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 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.
Construction of the UBQ Fusion Vector
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 5Ј-regulatory elements, 3Ј-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 5Ј-UTR, and the NOS 3Ј-UTR. The nucleotide sequence immediately 5Ј 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 5Ј to the initiation codon to avoid any differences in translation efficiency not associated with the presence of the UBQcoding 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 UBQspecific 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) .
GUS Expression
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 agree- 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 5Ј-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 5Ј-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. Expression and processing of UBQ-GUS fusions in transgenic tobacco. A, RNA gel-blot analysis of 10 g 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 g) from young leaves was subjected to SDS-PAGE, and GUS protein was visualized by immunoblotting with GUS antibodies. Lane UBQ-GUS ϩ 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 T 0 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). ment 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).
GUS Expression
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 (Ͼ95% 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]) .
GUS Expression
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 (Ͼ200 nmol 4-methylumbelliferone min Ϫ1 mg Ϫ1 leaf protein) was 10-fold greater in the UBQ-GUS population (Fig. 3C) .
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.
LUC Expression
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 (Ͼ160 pg mg Ϫ1 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 Table I . Accumulation of GUS and LUC expressed alone or as a UBQ fusion
Expression of Compartmentalized Proteins
Protein content was assayed in young, mature-green leaves of independently transformed T 0 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. intrusive for chloroplast proteins because their import occurs posttranslationally (Cline and Henry, 1996) , thus allowing time for UBQ removal by Ubps. However, ERdirected protein is imported cotranslationally (Walter and Johnson, 1994) . 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.
Expression of Compartmentalized Proteins
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.
Expression of Compartmentalized Proteins
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 Mg 2ϩ 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.
Expression of Compartmentalized Proteins
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 polypep- tide 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.
Expression of Compartmentalized Proteins
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 degrada- tion subpathway and probably uses the free ⑀-amino group in Lys-48 in the fused UBQ for binding additional UBQs . 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.
Expression of Noncleavable UBQ Fusions
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 (UBQ R-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).
Expression of Noncleavable UBQ Fusions
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 UBQimmunoreactive proteins in the UBQ(A)-GUS and UBQ-(P)GUS plants but not in plants expressing UBQ R-48 (A)-GUS or UBQ R-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.
Expression of Noncleavable UBQ Fusions
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 
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 (UBQ R-48 [A]-GUS or UBQ R-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 UBQ R-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).
DISCUSSION
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.
DISCUSSION
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 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.
DISCUSSION
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 5Ј-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 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 UBQfusion 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).
DISCUSSION
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 . 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.
DISCUSSION
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.
DISCUSSION
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) .
DISCUSSION
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 . 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.
DISCUSSION
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.
DISCUSSION
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.

{'paper_id': '12706923', 'title': 'Use of Ubiquitin Fusions to Augment Protein Expression in Transgenic Plants', 's2_url': 'https://api.semanticscholar.org/CorpusID:12706923', 'source': 'S2ORC'}