natural plant DNA sequences - PNAS

Proc. Nati. Acad. Sci. USA Vol. 82, pp. 334-338, January 1985 Biochemistry

Expression of phaseolin cDNA genes in yeast under control of natural plant DNA sequences (plant genes/Saccharomyces cerevisiae/post-translational processing)

JANE HARRIS CRAMER, KRISTI LEA, AND JERRY L. SLIGHTOM Agrigenetics Advanced Research Division, 5649 East Buckeye Road, Madison, WI 53716

Communicated by Masayasu Nomura, September 17, 1984

tures of the (3-type phaseolin gene in XPvPh177.4 are illustrated in Fig. 1. The Bgl II/BamHI restriction fragment shown includes approximately 800 base pairs (bp) of DNA adjacent to the 5' end and 1200 bp adjacent to the 3' end of the phaseolin transcribed region. The transcript for this gene contains 80 bases of 5' untranslated RNA, a coding region of 1260 bases interrupted by five introns representing a total of 515 bases, and 135 bases of 3' untranslated RNA. The amino acid sequence deduced from the DNA sequence includes a

We have constructed a strain of SaccharomyABSTRACT ces cerevisiae that expresses two different members of the multigene family encoding phaseolin, the major seed storage glycoprotein from the French bean, Phaseolus vulgaris. Yeast vector plasmids have been engineered to include a Phaseolus DNA segment that contains the natural 5' and 3' plant genomic regulatory sequences flanking a cDNA copy of the proteinencoding region. Characterization of phaseolin transcripts isolated from transformed yeast cells revealed the presence of two classes of polyadenylylated RNA, approximately 1400 and 1800 bases, which initiate and terminate in plant DNA sequences. Protein extracts from transformants contain phaseolin-immunoreactive proteins similar in size to those isolated from plant tissue. These polypeptides are glycosylated in yeast and their molecular weights are consistent with the possibility that the phaseolin signal peptide has been cleaved to form the mature protein.

hydrophobic amino-terminal region typical of signal peptide sequences and two regions that represent characteristic attachment sites for N-glycosylation (11). Sequences encoding the larger a polypeptides differ from the .3 types mainly by the inclusion of two small repeated sequences, adding a total of 14 amino acids (10). Phaseolin polypeptides are synthesized on rough endoplasmic reticulum, modified, and transported to membranedelimited protein bodies (storage vacuoles) (12). The yeast system offers the advantage of having well-characterized vacuolar and secretory protein modification and transport pathways (13). By expressing phaseolin in this microbial eukaryote, we can examine polypeptides synthesized from individual members of this multigene family and compare cotranslational and post-translational modifications in plant and yeast systems. MATERIALS AND METHODS Yeast Strains and Culture Conditions. JHC8-24C (MATa ura3-52 his3-11 his3-15 leu2-3 leu2-112 inol-13 ino4-8) was used as the yeast host in all experiments. Yeast transformants were grown under selection for leucine independence in YNB (Difco) minimal medium and 2% glucose with appropriate supplements. Construction of Recombinant Plasmids. Plasmids composed of pBR322 plus Phaseolus genomic or cDNA sequences have been described (11). YEp13 (14) was a gift from J. Broach. Plasmid DNA was purified according to Cramer et al. (15). Restriction enzymes (Promega Biotec, Madison, WI, and Bethesda Research Laboratories) and T4 DNA ligase (Collaborative Research) were used as recommended by the supplier. Restriction fragments were purified for molecular cloning as described previously (16). Bacterial and Yeast Transformations. Bacterial transformations were done according to Lederberg and Cohen (17). Yeast cells were transformed by the lithium acetate method of Ito et al. (18).

The availability of transformation procedures for introducing expgenous DNA into Saccharomyces cerevisiae and the development of a variety of plasmid vehicles for cloning and expressing foreign genes have permitted studies on transcription and translation of several higher eukaryotic genes in yeast. Most of these studies have involved DNA from higher animals or animal viruses (1-5); however, recent experiments have shown that products encoded by plant genes can also be synthesized and processed in yeast (6, 7). In this study, we report the expression in yeast of the genes for phaseolin, the major seed storage glycoprotein of French bean (Phaseolus vulgaris Linnaeus, cultivar Tendergreen). The term "phaseolin" applies to a group of polypeptides that are synthesized rapidly during a specific growth period in developing seed cotyledons and eventually represent about 50% of the total protein in mature seeds. One-dimensional NaDodSO4/polyacrylamide gel electrophoresis of these proteins resolves three molecular mass classes of glycopeptides, 51 kilodaltons (kDa), 48 kDa, and 45.5 kDa (8). After chemical deglycosylation, only two polypeptides, apprpximately 47.5 kDa and 45 kDa, are observed (H. Paaren, personal communication). In addition, phaseolin mRNA isolated from bean cotyledons directs the synthesis of only two molecular mass variants in cell-free systems devoid of membranes (9). Thus, the complexity of the in vivo protein fraction apparently stems from various degrees of glycosylation of two basic polypeptide types. Phaseolin is encoded by a family of 7 to 14 related genes (10). The complete nucleotide sequences of one genomic clone XPvPh177.4, and a number of cDNA copies have been reported (10, 11). On the basis of polypeptide molecular weights deduced from DNA sequences, the genes can be classified into two types, a and ,B, each containing a limited numqber of base substitutions and deletions (10). Several fea-

RNA Isolation and Analysis. Whole cell yeast RNA was prepared by a modification of the method of Elder et al. (19). Poly(A)+ RNA was purified by poly(U)-Sepharose chromatography. RNA was fractionated on agarose/formaldehyde gels, transferred to nitrocellulose, and hybridized to 32P-labeled DNA as described (20). The 5' termini of phaseolin transcripts from yeast and bean were determined by S1 nuclease mapping (21). Yeast poly(A)+ RNA or total bean cot-

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Abbreviations: bp, base pair(s); kb, kilobase(s).

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Proc. NatL. Acad. Sci. USA 82 (1985) A

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yledon RNA was annealed with constant amounts of a 5'labeled Nco I/EcoRI phaseolin restriction fragment under conditions of DNA excess exactly as described (20). After reassociation, samples were diluted, treated with S1 nuclease (22), and analyzed on acrylamide/urea sequencing gels (23). Identical results were obtained over concentration ranges of 3 ng to 5 ,4g for bean and 2 to 10 ,ug for yeast RNA, or using 10 to 200 units of S1 nuclease per ,ug of total DNA (unpublished data; M. Murray, personal communication). Protein Extract Preparation and Phaseolin Detection. Yeast cultures were grown to an OD650 of 0.5 and divided, and one half was treated with tunicamycin (Sigma) at a final concentration of 5 gg/ml for 4 hr. Protein extracts from cells disrupted by Vortex mixing with glass beads in buffer containing 10 mM NaH2PO4 at pH 7.5, 1 mM phenylmethylsulfonyl fluoride, and 0. 1% Triton X-100 were clarified by centrifugation, quantitated by the Bradford protein assay (24), and analyzed by immunodetection techniques. ELISA analysis was performed by the double antibody sandwich technique and antigen was quantitated by using the alkaline phosphatase assay (25). Phaseolin in yeast extracts was quantitated on serial dilutions such that the amount of phaseolin fell within the linear range of our standard curve (0.05 to 1.2 ng). Extracellular phaseolin in yeast transformant cultures was determined by ELISA after concentrating the growth medium by lyophilization and dialyzing exhaustively vs. 20 mM sodium phosphate, pH 7.5. For immunoblot analysis, protein extracts were fractionated on 13% acrylamide gels (acrylamide to bisacrylamide ratio, 30:0.15) containing NaDodSO4 (26), then transferred to nitrocellulose by electroelution (27). Filters were incubated with polyclonal rabbit antiserum to phaseolin followed by 125I-labeled Staphylococcus aureus protein A (New England Nuclear), and antigen-antibody complexes were visualized by autoradiography (28).

RESULTS Yeast Plasmid Construction. To determine whether the plant DNA regulatory sequences responsible for phaseolin expression in Phaseolus might function in a yeast host, we constructed yeast plasmids containing the genomic Bgl II/ BamHI restriction fragment shown in Fig. 1. Preliminary experiments showed that phaseolin genomic DNA was transcribed in yeast (Fig. 3, lane A), but the major transcripts of approximately 950 and 1150 bases were too small to include the entire coding region, and no phaseolin polypeptides were detected. Recent evidence (29, 30) has shown that for introns in yeast mRNAs to be excised correctly, they must include the sequence 5'-T-A-C-T-A-A-C-3' upstream from the 3' splice site. None of the five phaseolin introns contain this sequence (11). To circumvent transcription problems owing to the presence of introns, we have replaced the protein coding region of the genomic DNA sequence with either an iden

3.0

FIG. 1. Phaseolin storage protein gene. A 3.8-kilobase (kb) genomic Bgl II/BamHI restriction fragment encompassing the phaseolin gene is shown (11). Solid lines flank the gene, thin bars represent 5' and 3' transcribed but untranslated regions, thick bars show phaseolin exons, and open bars show introns. DNA regions designated A, B, and C were used as radioactive probes in hybridization experiments. The DNA sequence surrounding the methionine initiator codon (ATG) is shown on an expanded scale.

tical /-type cDNA copy or an a-type cDNA (Fig. 2). The resulting Phaseolus DNA retains the natural 5' and 3' flanking sequences (approximately 800 and 1200 bp, respectively) for a 3-type gene. Therefore, the sequence containing /3 cDNA is identical to that in the plant genome except for the absence of introns. The sequence containing a cDNA is a fusion between two types of phaseolin genes and may not have a direct counterpart in the plant genome, as the gene flanking regions for a phaseolins may not be equivalent to ,3 phaseolins. A HindIII/BamHI restriction endonuclease fragment spanning the Phaseolus DNA plus a small segment of pBR322 in AG-pPvPh3.8/cDNAa or -,8 was then inserted into the yeast vector YEp13. The resulting plasmids, AGYEpl3 PvPh3.8ca or -,3 (referred to as YE Pha or YE-Ph,3), contain the yeast 2-aim plasmid origin of replication, enabling them to be maintained as extrachromosomal highcopy-number vectors in yeast cells, plus the yeast LEU2 gene, which permits selection for the plasmids in leu2 cells growing in medium lacking leucine. Phaseolin Transcription in Yeast. Analysis of total cellular RNA from yeast transformants containing YE-Phf3 or YEW Pha (Fig. 3, lanes B and C) or poly(A)+ RNA from cultures containing YE-Ph,/ (lane E) reveals two polyadenylylated species, approximately 1400 and 1800 bases, which hybridize to 32P-labeled phaseolin cDNA spanning region A in Fig. 1. Transcripts from YE-Pha and YE.Ph,3 are indistinguishable in these experiments. Although neither transcript is similar in size to the 1650-base phaseolin mRNA in bean cotyledons (lane F), both are large enough to include the phaseolin coding region. To determine if the yeast phaseolin transcripts extend into adjacent vector sequences, RNA from transformants containing YE&Phj was also hybridized to 32plabeled pBR322 (Fig. 3, lane D). Homology was observed to transcripts of about 1700 and 2700 bases, but not to the 1400or 1800-base RNAs. Furthermore, none of the RNA species from transformed cells hybridized to either the 32P-labeled Bgl II/IinclI or the Hinfl/BamHI restriction fragments designated regions B and C in Fig. 1, indicating that the two yeast phaseolin transcripts most likely initiate and terminate in the phaseolin sequence bordered by the HincII and Hinfl restriction sites. The 5' termini of yeast phaseolin transcripts have been determined more accurately by S1 nuclease protection experiments. Nuclease-resistant hybrids were formed between an Nco IlEcoRI restriction fragment that includes approximately 500 bp of DNA upstream from the phaseolin initiation codon plus 36 bp of the coding region (see Fig. 1) and either poly(A)+ RNA from yeast transformants containing YE-Ph,8 or, for comparison, total bean cotyledon RNA (Fig. 4). Both transformed yeast and bean cotyledon RNA protect multiple DNA fragments, whereas no protected fragments were observed with untransformed yeast RNA under identical experimental conditions (not shown). Reproducible results

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FIG. 3. Transcription of the phaseolin gene in yeast. Yeast total RNA (5-10 ,g) or poly(A)+ RNA (0.2 pg) purified by poly(U)-Sepharose chromatography was resolved on 1% agarose/formaldehyde gels, transferred to nitrocellulose, and hybridized to a 32P-labeled phaseolin cDNA restriction fragment spanning region A in Fig. 1 (lanes A, B, C, E, and F) or to 32P-labeled pBR322 (lane D). Lanes A-D, whole cell RNA from yeast transformants containing the genomic 3.8-kb 3phaseolin restriction fragment (lane A), YE-Phf3 (lane B) or YE-Pha (lane C). The filter used for lane B was incubated in 1 mM EDTA at 650C to discharge the phaseolin cDNA and then its RNA was rehybridized to pBR322 (lane D). Lane E, poly(A)+ RNA purified from transformants containing YE.Ph(3; lane F, 0.2 /.g of bean cotyledon RNA. Size standards (bromegrass mosaic virus RNA and yeast ribosomal RNA) are indicated.

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FIG. 2. Construction of yeast plasmids for phaseolin expression. AG-pPvPh3.8, which is pBR322 plus the Bgl II/BamHI restriction fragment in Fig. 1, was digested partially with EcoRI and to completion with Sac I. The 6378-bp fragment containing pBR322 plus sequences 5' and 3' to the phaseolin protein encoding region was ligated to a fragment obtained by complete EcoRI and partial Sac I digestion of AG-pPvPh3l or AG-pPvPh39, which contain cDNA copies of P- or a-type phaseolin genes, respectively. The resulting plasmids, AG-pPvPh3.8/cDNAP and -a, yielded HindIII/BamHI fragments subsequently inserted into YEp13 cleaved with HindIII and BamHI. Closed bars represent Phaseolus DNA, cross-hatched regions represent pBR322, and open bars indicate yeast LEU2 and 2-,um DNA.

have been obtained over a wide range of experimental conditions (see Materials and Methods). Therefore, the complex pattern of protected fragments does not appear to be an artifact of specific S1 nuclease/DNA or RNA/DNA ratios, and we surmise that most of the fragments represent 5' transcript termini. We have grouped the yeast termini into two clusters, each lying 20-30 bp downstream from a "TATA" element in the phaseolin 5' flanking DNA (ref. 11; unpublished data) and separated by a 50-bp region containing no termini (see Fig. 4). Region I extends 35 to 100 bp upstream from the methionine initiator codon and region II extends 150 to 185 bp upstream. No termini are found closer to the translation initiation site or in the first 36 bp of the coding region. Use of the Nco I/EcoRI fragment in these experiments does not allow detection of termini in the remainder of the phaseolin coding region. Notably, many of the same fragments are protected by RNA from both yeast and bean (note asterisks in Fig. 4). However, the major bean 5' termini, 70 to 80 bp upstream from the ATG codon (ref. 20; also Fig. 4), do not appear to be represented in yeast. The S1 nuclease mapping data are consistent with the RNA hybridization experiments, which show that yeast phaseolin transcripts do not extend farther upstream from the phaseolin coding region than the HincHI restriction site (region B in Fig. 1). Synthesis of Phaseolin Polypeptides in Yeast. Expression of phaseolin genes at the translational level in yeast cells has been demonstrated by immunodetection techniques. Quantitative ELISA analysis (see Materials and Methods) of soluble protein extracts from transformed cells containing either YE.Ph,8 or YE-Pha showed that 0.01-0.03% of the total cellular protein was phaseolin-immunoreactive material. Phaseolin polypeptides are not exported from yeast cells in appreciable amounts. Only about 0.3% of the total phaseolin in a culture is in the extracellular medium. This small amount is probably due to cell leakage or autolysis during culture growth. When protein preparations from yeast transformants were fractionated on polyacrylamide gels (Fig. 5), several prominent phaseolin polypeptides not evident in extracts of untransformed cells (lane E) were present. The amounts of phaseolin-immunoreactive material estimated from the gels are consistent with the ELISA data. The (3type phaseolin gene directs the synthesis of three molecular mass variants of 49, 47, and 44.5 kDa (lane C), slightly smaller than the

*a

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Biochemistry: Cramer et aL A

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FIG. 4. Si nuclease mapping of yeast phaseolin poly(A)+ RNA 5' termini. SI nuclease-resistant hybrids were formed be* _ tween the Nco I/EcoRI Phaseoii lus restriction fragment shown in Fig. 1 and 5 pg or 1 ug of two different poly(A)+ RNA preparations from yeast transformed with YE.Ph(3 (lanes A and B, re-> a spectively). Lane C shows Si nuclease-resistant hybrids be=t *tween the Nco I/EcoRI frag-> * a* ment and 10 ng of bean cotyle.^ don RNA (containing about 100 pg of phaseolin mRNA). Pro*= ^ tected DNA was electropho*~ resed on 6% acrylamide/7 M *- tIm urea gels and compared to the . -* sequence of the same fragment (lane D) prepared according to Now the method of Maxam and Gilbert (31). Arrows on the left and ;ROM right indicate clusters of DNA fragments protected by yeast and bean cotyledon phaseolin *transcripts, respectively. Frag-]a_: protected by both are ~~~ments noted by asterisks.

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bean phaseolin polypeptides. Two smaller components of approximately 25-26 kDa are also present, probably resulting from limited proteolytic cleavage of the larger polypeptides. In contrast, the a-type gene directs the synthesis of 52-, 50-, and 47.5-kDa polypeptides (lane F). Again, two smaller polypeptides, 22 and 35 kDa, are detectable. The mobility of native phaseolin polypeptides is not altered in the presence of yeast extracts (lane B), therefore the sizes of phaseolin polypeptides synthesized from single genes in yeast are distinctly different from the sizes in the native phaseolin mixture. The 44.5- and 47.5-kDa polypeptides in transformants containing the (3-type and a-type phaseolin genes, respectively, are the sizes expected for the proteins encoded by these two genes after removal of the signal peptide (10, 11), and these species appear to migrate with chemically deglycosylated phaseolin from beans (lane H). To determine if the two larger polypeptides in each type of transformant are glycosylated forms of the 44.5- or the 47.5-kDa protein, we examined extracts from logarithmically growing yeast cultures treated with tunicamycin, an antibiotic that blocks the synthesis of N-linked oligosaccharides (32). Under these conditions, the proportion of the 44.5- or the 47.5-kDa polypeptide in the two different cultures increased dramatically (lanes D and G). A new 45-kDa polypeptide, presumably an unglycosylated variant, appears in transformants containing the a phaseolin gene after tunicamycin treatment. The small amount of the larger polypeptides still visible probably represents molecules that are glycosylated before drug treatment. Alterations that occur in polypeptide pattern during growth in tunicamycin suggest that phaseolin is glycosylated in yeast and that the two larger polypeptides arise from various amounts of oligosaccharide added to the unglycosylated 44.5-kDa /3type or the 47.5-kDa a-type phaseolin.

DISCUSSION We have used S. cerevisiae as an expression system for examining the biosynthesis, modification, and processing of

21.5 -

FIG. 5. Immunological detection of phaseolin polypeptides synthesized in yeast. Yeast protein extracts (200 pg of total protein) were fractionated and phaseolin was detected as described in Materials and Methods. Lane A, 80 ng of purified bean phaseolin; lane B, purified phaseolin incubated with untransformed yeast cells during extraction; lanes C and D, extracts from yeast transformants containing YE.Php grown with or without tunicamycin at 5 pg/ml, respectively; lane E, untransformed yeast extract; lanes F and G, extracts from yeast transformants containing YE-Pha grown with or without tunicamycin at 5 ug/ml, respectively; lane H, purified bean phaseolin polypeptides after chemical deglycosylation. Molecular masses are shown for bean phaseolin polypeptides, their deglycosylated derivatives, and markers bovine serum albumin (66.2 kDa), carbonic anhydrase (31 kDa), and soybean trypsin inhibitor (21.5 kDa), included in the same gel.

the plant protein phaseolin. Although phaseolin genomic sequences are transcribed in yeast, no proteins are made, presumably because the yeast transcription or splicing mechanisms cannot produce translatable mRNA when the introns are present. When the gene coding region is replaced by cDNA copies for either a- or (3-type phaseolin, phaseolin polypeptides are synthesized. Two size classes of polyadenylylated phaseolin mRNAs, constituting about 1% of the yeast mRNA population (unpublished data), are transcribed from a- or 3-phaseolin cDNA-containing plasmids in yeast. Such a high level of transcription undoubtedly results in part from the presence of the gene on the multicopy YEp13 vector, but it also suggests that the plant DNA supplies reasonably efficient promoter activity in the yeast host. S1 nuclease protection experiments show that the array of fragments protected by yeast phaseolin transcripts is similar, but not identical, to the array protected by bean mRNA. The reproducibility of these patterns suggests that most of the fragments represent bona fide 5' RNA termini, arising from transcription initiation or some type of processing event. For the bean RNA, this assumption is reinforced by sequence data for eight independent phaseolin cDNA clones (10, 11). Six have termini that lie 70-80 bp upstream from the ATG codon and are represented by the highest concentration of protected fragments (Fig. 4; ref. 20). Two other sets of protected fragments (top and bottom arrows on the right side of Fig. 4) are also equivalent to termini found among the clones (10). However, it is possible that some of the fragments protected by the bean RNA arise because of sequence divergence between the ,& phaseolin DNA fragment used in this experiment and transcripts from other members of the gene family. Although the major 5' termini in bean are not present in yeast, many of the yeast protected fragments correspond to minor bean termini.

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In comparing the relative amounts of different fragments protected by RNA from the two sources, it is important to note that yeast phaseolin transcripts arise from a single gene, while those in bean represent the entire multigene family. The contribution of the f3phaseolin gene used here to the total pool of bean phaseolin transcripts is unknown. We have grouped the multiple 5' termini of the yeast phaseolin transcripts into two clusters. The separation between these groups is not, however, sufficient to account for the difference between the 1400- and 1800-base size classes of phaseolin poly(A)+ RNAs, suggesting that the 3' termini are also heterogenous or that the RNAs have been polyadenylylated to quite different degrees. There are several sequences distributed throughout the 3' phaseolin flanking region (refs. 10 and 11; unpublished data) which are similar but not identical to the consensus signals T-T-T-T-T-A-T-A (33) and T-AG-...-T-A-G-T-...-T-T-T or T-A-G-...-T-A-T-G-T-...T-T-T (34) thought to be important in yeast transcription termination. Levels of phaseolin polypeptides synthesized in the yeast transformants approach 0.03% of the total protein, lower than expected for the amount of mRNA. These results suggest either that a limited class of the phaseolin transcripts from the YEp13 constructions serve as functional messages or the population as a whole is translated with low efficiency. Highly expressed yeast genes contain no G residues in the 15-20 bases immediately preceding the initiation codon (35). Additional characteristics include an A residue at the -3 position (with the A of the methionine ATG equal to + 1), a purine at +4, and a T at +6 (35, 36). The 5' end of the phaseolin coding sequence has two consecutive ATG triplets, either of which might serve as the initiation codon (ref. 10; see Fig. 1). The first ATG has a favorable environment for yeast, as it has no G residues preceding it until position -59 and conforms to the rest of the yeast consensus except for a G at +6. The second ATG, with a G at -1, would presumably be less desirable. One of the most interesting and useful aspects of phaseolin expression in this system is modification of the protein after translation. Phaseolin contains two Asn-X-Thr/Ser sequences (11), which are known from in vitro studies to be required for N-glycosylation in yeast (37). We presume phaseolin glycosylation in yeast occurs at one or both of these sites; however, additional studies are required to define the nature and location of the carbohydrate moiety. The molecular weights of ,3- and a-type unglycosylated components in yeast are consistent with sizes expected after removal of the 22- to 26-amino acid amino-terminal signal peptide (ref. 11; H. Paaren, personal communication), suggesting that S. cerevisiae may recognize the plant protein processing signal. This observation is consistent with results showing that plant proteins preprothaumitin (7) and possibly wheat a-amylase (6) undergo signal peptide cleavage in yeast. We have determined that phaseolin synthesized in yeast is not secreted, but its cellular location is not known. Core oligosaccharides are added in the endoplasmic reticulum and extended in the Golgi region (13); therefore, our glycosylation results suggest that phaseolin might be directed to these intracellular sites in yeast. We thank W. Kennard and R. Klassy for isotopically labeled DNA, M. Murray for assistance with S1 nuclease protection experiments, L. Hoffman for bean cotyledon RNA, H. Paaren for deglycosylated phaseolin, N. Reichert for purified bean phaseolin and instruction in ELISAs, D. Talbot and M. Yeazel for phaseolin antisera, and C. Sengupta-Gopalan and J. Pitas for helpful suggestions on methods and critical evaluation of results. We particularly appreciate the enthusiastic support of Tim Hall and John Kemp during the course of this work. This is Agrigenetics Advanced Research Division manuscript no. 29.

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