Proc. Nati. Acad. Sci. USA Vol. 88, pp. 10941-10945, December 1991 Plant Biology

Molecular cloning of a putative plant endomembrane protein resembling vertebrate protein disulfide-isomerase and a phosphatidylinositol-specific phospholipase C (endoplasmic reticulum/Medicago sadva/plant cell culture/tunicamycin)

BASIL S. SHORROSH AND RICHARD A. DIXON* Plant Biology Division, The Samuel Roberts Noble Foundation, P.O. Box 2180, Ardmore, OK 73402

Communicated by J. E. Varner, September 16, 1991 (received for review November 9, 1990)

ABSTRACT cDNA clones containing sequence similarity to the multifunctional vertebrate protein disulfide-isomerase (PDI, EC 5.3.4.1) were isolated from an alfalfa (Medicago saiva L.) cDNA library by screening with a cDNA sequence encoding human PDI. The polypeptide encoded by a clone designated B2 consisted of 512 amino acids and was characterized by a 24-amino acid hydrophobic leader sequence, two regions with absolute identity to the vertebrate PDI active site (Ala-Pro-Trp-Cys-Gly-His-Cys-Lys), and a C-terminal endoplasmic reticulum retention signal (Lys-Asp-Glu-Leu). The overall identity of the B2 sequence to that of human PDI was 35% at the amino acid level (79% when conservative substitutions were included) and 39% at the nucleotide level; this included homology between B2 and the region of human PDI believed to be involved in binding estrogens. The deduced amino acid sequence of B2 was also 35% identical to that of a rat form I phosphatidylinositol-specific phospholipase C. Lysates from Escherichia coli cells harboring an expression plasmid bearing the B2 sequence contained significantly elevated levels of PDI activity. Southern analysis indicated the presence of a small PDI-related gene family in alfalfa, of which B2 appeared to correspond to a single gene. An -2-kilobase B2 transcript was expressed in all alfalfa organs tested. In alfalfa cell suspension cultures, B2 transcripts were strongly induced by tunicamycin but not by exposure to fungal elicitor.

mains of the human estrogen receptor (12). Recently, a GSBP/PDI clone was isolated from yeast (13). Apart from its suggested endomembrane location (3), little is known about plant PDI. The enzyme has been purified from the unicellular green alga Chlamydomonas reinhardtii (14), from which it appears to be a dimer of a 60-kDa subunit antigenically unrelated to the vertebrate enzyme. It has been reported that human PDI sequences do not detect complementary sequences in a C. reinhardtii cDNA library (14). Antibody crossreactivity studies have provided conflicting evidence of a role for PDI in the Chlamydomonas PH (14, 15). PI-PLCs have been partially purified from plants (16), but their amino acid sequences, and therefore possible relationships to PDI, have yet to be determined. We here report the isolation of an alfalfa cDNA clonet whose deduced amino acid sequence exhibits extensive similarity to vertebrate PDI and PI-PLC and which exhibits PDI activity on expression in Escherichia coli. We describe the genomic organization of alfalfa PDI-related sequences, the levels of PDI-related transcripts in tissues of mature alfalfa plants, and changes in PDI-related transcript levels in alfalfa cell suspension cultures following treatment with tunicamycin or fungal elicitor. MATERIALS AND METHODS Library Screening. A 546-base-pair (bp) internal Xho I fragment from the human PDI (PH 1 subunit) cDNA (6) was used to screen a AZAPII (Stratagene) cDNA expression library constructed from poly(A)+ RNA isolated from an alfalfa cell suspension culture at 2, 3, and 4 hr after exposure to fungal elicitor. GeneScreenPlus nylon membranes containing plaque lifts were air-dried for 5-10 min, autoclaved at 120°C for 3 min, and then washed at 42°C for 2 hr in 0.1% (wt/vol) SDS/1 M NaCl/1 mM EDTA/50 mM Tris-HCI, pH 8.0. Prehybridization in the presence of 50% (vol/vol) formamide was performed, according to standard procedures (17), for 6 hr at 37°C. The probe [labeled by random oligonucleotide priming (18)] was then added and the membranes were incubated overnight at 37°C. The membranes were washed at 60°C in 2x standard saline citrate (SSC)/1% SDS and then at room temperature in 0.lx SSC prior to autoradiography. Positive plaques were removed and phage were eluted and replated twice, according to standard procedures (17). Template Isolation and DNA Sequence Analysis. The pBluescript vector was in vivo excised, in the presence of the helper phage R408 (Stratagene protocol), from AZAPII to yield a

Protein disulfide-isomerase (PDI, EC 5.3.4.1) catalyzes formation of the disulfide bonds that stabilize the tertiary and quaternary structures of many extracellular proteins (1, 2). PDI is localized in the endoplasmic reticulum of plant (3) and vertebrate (4) cells, where it appears to be loosely associated with the lumenal surface. The vertebrate enzyme is a dimer consisting of two identical subunits of -57 kDa, for which cDNA and genomic sequences have been obtained (5-7). It contains two nearly identical, putative active-site regions containing the consensus sequence Ala-Pro-Trp-Cys-GlyHis-Cys-Lys, a sequence closely related to the active site of the thioredoxins (5). PDI is identical to a number of other endomembrane proteins (or components of them): the j8 subunit of prolyl 4-hydroxylase (PH) (6, 7), the thyroidhormone-binding protein (8), and the lower molecular weight subunit of the microsomal triacylglycerol transfer protein complex (9). Its amino acid sequence is very similar to that of the glycosylation site-binding protein (GSPB) of the oligosaccharyltransferase complex (10). In addition, vertebrate PDI shares substantial similarity in its amino acid sequence (including the two active sites) with rat phosphatidylinositolspecific phospholipase C (PI-PLC) (11) and contains two regions with sequence similarity to hormone-binding do-

Abbreviations: IPTG, isopropyl P-D-thiogalactopyranoside; PDI, protein disulfide-isomerase; PI-PLC, phosphatidylinositol-specific phospholipase C; PH, prolyl 4-hydroxylase; GSBP, glycosylation

site-binding protein. *To whom reprint requests should be addressed. tThe sequence reported in this paper has been deposited in the GenBank data base (accession no. M72335).

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Plant Biology: Shorrosh and Dixon

phagemid containing the cloned insert. After propagation in E. coli XL1-Blue cells, insert cDNA was subjected to doublestranded DNA sequencing using dideoxynucleotide chain termination (19) with T7 DNA polymerase and pBluescript universal primers. Subsequent sequencing reactions were primed with synthetic oligonucleotides. End deletions of the cDNA insert for sequencing were generated by mung bean exonuclease (Promega). Deduced amino acid sequences were aligned by the CLUSTAL program (20). A score of 8 or greater in a Dayhoff PAM matrix of amino acid similarity was used as a cutoff to indicate amino acids with similar physicochemical properties or encoded by codons having one base exchange. Growth and Elicitation of Cell Cultures. Cell suspension cultures of alfalfa (Medicago sativa L.) cv. Apollo were initiated and maintained in a modified Schenk and Hildebrandt medium as described (21). Cells were treated with elicitor from the cell walls of Colletotrichum lindemuthianum (60 ug of glucose equivalents per ml of culture) (21) or with tunicamycin (10 pug/ml final concentration), frozen in liquid N2, and stored at -70'C. RNA Isolation and Northern Blot Analysis. Total RNA (15 ,ug) was isolated from alfalfa cell suspension cultures or plant tissues by the method of Logemann et al. (22), fractionated in 1% agarose gels containing 2.2 M formaldehyde, and blotted onto nitrocellulose membranes. Blots were prehybridized for 6 hr at 420C in the presence of 50% formamide. Labeled probe was added and blots were incubated overnight at 420C. Blots were washed at 650C in 0.2x SSC/0.1% SDS and then in 0.2x SSC at room temperature prior to autoradiography. Southern Blot Analysis. High molecular weight genomic DNA (17) from alfalfa leaves was digested with restriction endonucleases, electrophoresed in a 0.7% agarose gel, and blotted onto a GeneScreenPlus nylon membrane. Prehybridization and hybridization conditions were as described above for library screening, except that hybridization was at 42°C. The blot was washed at 65°C in 2 x SSC/1% SDS and then in 0.1 x SSC at 65°C prior to autoradiography. Measurement of Transcript Levels by RNase Protection. A subclone containing the first 342 bp of the 5' end of the B2 cDNA insert in pBluescript was digested with Nhe I at position 208 and then transcribed from the T7 promoter in the presence of [a-[35S]thio]UTP. The radiolabeled antisense RNA (134 bases) was used to detect B2-related transcripts in alfalfa by RNase protection (23). Expression of B2 in E. coli. The B2 cDNA insert was amplified by PCR in 10 mM Tris HCl, pH 8.3/50 mM KCl/3 mM MgCl2/0.01% gelatin/100 ,uM each dNTP containing 0.1 nmol of each primer (I, 5'-CGCGCCCATGGAGGAATCATCAACTGACGC-3'; II, KS primer in pBluescript), "500 ng of template DNA, and 2.5 units of Amplitaq DNA polymerase (Perkin-Elmer/Cetus) in a final volume of 100 ,u. Amplification (30 cycles) was at 94°C for 1.5 min, 55°C for 2 min, and 72°C for 2 min, with the final 72°C incubation for 10 min. Amplified DNA was isolated by electrophoresis onto Whatman 3MM paper, digested with Nco I/HindlI, and subcloned into the Nco I/HindIII sites of expression vector pSE380 (24). The construct was used to transform E. coli strain SB221 (25), and the cells were grown overnight at 37°C in 5 ml M9 minimal medium supplemented with 0.8% (wt/vol) glucose, 12 ,ug of thiamin per ml, 0.3 mM leucine, 0.2 mM tryptophan, 4 mM MgSO4, 0.2 mM CaCl2, and 50 ,ug of ampicillin per ml. After 3 hr growth in fresh medium (OD6N 0.3), cells were incubated for 3 hr in the presence of 5 mM isopropyl 3-D-thiogalactopyranoside (IPTG), collected by centrifugation at 40C, and lysed at room temperature for 10 min in 400 p.l of 0.1 M sodium phosphate, pH 7.5/5 mM EDTA/0.1% lysozyme. Cell debris was removed by centrif-

Proc. Natl. Acad. Sci. USA 88 (1991)

ugation at 40C and the protein concentration of the supernatant was determined by the Bradford assay (26). Assay of PDI Activity. PDI activity was assayed by a modification of the glutathione: insulin transhydrogenase assay (27). Each reaction mixture (200 14) contained 111.6 ILI (:600 tug of protein) of E. coli lysate, 0.1 M sodium phosphate buffer (pH 7.5), 5 mM EDTA, 1 mM reduced glutathione, 50 pug of insulin, and 0.1 p.Ci (37 kBq) of porcine insulin monoiodinated with 1251I at tyrosine A14 (371 .uCi/,ug; NEN). The mixture was incubated at 370C for 40 min, treated with 500 jul of 15% (wt/vol) trichloroacetic acid and 300 .ul of 2.5% (wt/vol) bovine serum albumin, incubated on ice for 15 min, and finally centrifuged at 14,000 x g for 30 min at 40C. Radioactivity in a 900-pl aliquot of the supernatant was determined by y counting.

RESULTS Three hundred thousand plaques from an alfalfa cDNA library were screened at moderate stringency with a labeled human PDI cDNA. Thirty positive clones were identified after two rounds of plaque purification, and three of these clones (B2, L1, and G1) were subjected to full DNA sequence analysis. The 1770-bp insert in clone B2 consists of a 1536-bp open reading frame, 6 bp of 5' untranslated region, and 228 bp of 3' untranslated region (Fig. 1). Five short sequence motifs are repeated within the open reading frame, and there are also two pairs of inverted repeats. Although the CCMCA

6

ATGGCGAAAAACGTTGCGATTTTCGGCTTATTG=---CTCTTCTTGTGTTGGTrCCTTCTCAGATCTTCG

76

CTGAGGMTCATCAACTGACGCTAAGGMTTTGTTCTTACATTGGATAACACTAA-TTCCATGACACTGT

146

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TAAGAAGCACGAtTTCATCGTCGTTGMTTCTAC | ACC

AAGCTAGCCC

214

IR1

CAGAGTATGAGAAGGCTGCTTCTATCTTGAGCACTCACGAGCCACCAGTTGTTTTGGCTAAAGTTGATGC

284

AGGAATGGTGGAAAGAATATTCAAGAATACAAAGGTCCCCGTGAAGCTGAAGGTATTGTTGAGTATTTGA

424

354

IR2

AAAAACAAAGTGGCCCTGCATCCACAGAAATTAAATCTGCTGATGATGCGACCGCTTTTGTTGGTGACAA

494

SL

1R2

CAAAGTTGTTATTGTCGGAGTTTTCCCTAATTTTCTGGTGAGGAGTACGATAACTTCATTGCATTAGCA

564

GAGAAGTTGCGTTCTGACTATGACTTTGCTCACACTTTGAATGCCAAACACCTTCCAAAGGGAGACTCAT

634 704

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TGTAGAAGCTCTAGAGAAATTCATTGAAGAATCCAGTACCCCAATTGTGACTGTCTTCAACAATGAGCCT

774

IRV' IRV' AGCAATCACCCTTTTGTTGTCAAATTCTTTACTCTCCCAACGCAAAGGCTATGTGTCATCAACTTA

844

R4

CTACCGAAGGTGCTGAATCTTTCAAAACAAAATACCATGAAGTTGCTGAGCAATACAAACAACAGGGAGT

914

TAGCTTCTTGTTGGAGATGTTGAGTCTAGTCAAGGTGCCTTCCAGTATTTTGGACTGAAGGAAGAACAA

984

GTACCTCTAATTATTATTCAGCATAATGATGGCAAGAAGTTTTTCAAACCCAATTTGGAACTTGATCAAC

1054

TCCCAACTTGGTTGAAGGCATACAAGGATGGCAAGGTTGAACCATTTGTCAAGTCTGAACCTATTCCTGA

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AACTAACAACGAGCCTGTTAAAGTGGTGGTTGGGCAAACTCTTGAGGACGTTGTTTTCAAGTCTGCGMG Rl'

1194

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AATGTTTTGATAGAGTTTTAT GCTCCTTGGTGTGGTCACTGCAAG CAGTTGGCTCCAATCTTGGATGA

1262

R2'

1332

CCAAGGAGACA 1 1T1GAT1GT1CCAAGCT1AT1CCAACCT1TGT1ACT1T1CABGGTGA@GAAGTGAAAACT1AT1CAC R3'

1402

AATACGACGGTGGTAGGACAAAGGAAGACATCATAGAATTCATTGAAAAGAACAAGGATAAAACTGGTGC R5 R5' TGCTCAACAAGAAGTAGAACAACCAAAAGCTGCTGCTCAGCCAGAAGCAGAACAACCAAAAGATGAGCTT

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TGAAAAGTTCCGCTTGGAGGATATCGGCACACAGTCATCTGCGGGCTTTACAACTCTTTTGTATCTCAGA

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TAATGTATTACTGAATAATGTGAGTTTTGGCGGAGTTTAGTACTGGAACTTTTGTTTCTGTAAAATTTAA

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1770

FIG. 1. Nucleotide sequence of the alfalfa B2 cDNA insert. Two active-site regions (boxed), repeats (R), inverted repeats (IR), potential stem-loop regions (SL), and putative polyadenylylation sites (PA) are indicated. The 5' and 3' untranslated regions are underlined.

Plant Biology: Shorrosh and Dixon

Proc. Natl. Acad. Sci. USA 88 (1991)

A.

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82

KNI-QEYKGPREAEGIVEYLKKQSGPASTEIKSADDATAFVGDNKVVIVGVFPKFSGEEY 179

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2-PH KQFLQAAEAI -DDIPFGITSNSDVFSKYQLDKDGVV- -LFKKFDE--- GRNNFEGEVTKE 223

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B2 cDNA insert does not contain a poly(A) tail, two putative polyadenylylation sites are located in the 3' untranslated region. Clone Li appears to encode an allelic variant of B2. The deduced amino acid sequence of B2 exhibits an overall identity of 35% to that of the human PH f8-subunit/PDI multifunctional polypeptide (Fig. 2A). Particularly striking is the sequence identity of B2, vertebrate PDIs, chicken GSPB, rat PI-PLC, and the endomembrane protein ERp72 (28, 29) in the two putative active-site regions (Fig. 2B). A single active-site region of close similarity to B2 is found in the lower molecular weight thioredoxins (Fig. 2B), but the alfalfa B2 sequences are clearly more characteristic of PDI than of thioredoxin. Outside the active-site regions, alfalfa B2 and human PDI share less sequence identity, although both contain hydrophobic N-terminal signal peptides predicted by the Von Heijne method (30), and terminate in the Lys-AspGlu-Leu endoplasmic reticulum retention sequence (31). The sequence Leu-Phe-Ser-Leu-Leu-Val-Leu-Val in the predicted B2 signal peptide is also found in the signal peptides of potato endochitinase (32) and rat pancreatic RNase (33). Alfalfa B2 contains a potential N-glycosylation site (AsnPhe-Thr) at amino acid 278. Rat PI-PLC exhibits strong sequence similarity to human PDI, especially with respect to the two active-site regions (11), and the deduced alfalfa B2 polypeptide shows an overall amino acid identity of 35% to the phospholipase (full sequence comparisons not shown). Interestingly, the B2 sequence contains a region of 9 amino acids (starting at position 365) which is identical to a correspondingly placed sequence in rat PI-PLC but which is not conserved in human, bovine, chicken, or rat PDIs (Fig. 2C). Attention has been drawn to the similarity of two regions within human PDI to the hormone-binding domains of the human estrogen receptor (12). The amino acid sequence similarity in these regions is shown in Fig. 2D. Although the corresponding regions in alfalfa B2 are less similar to the estrogen receptor sequences than is human PDI, alfalfa B2 nevertheless exhibits regions of identity to the human PDI throughout these regions. The deduced amino acid sequence of the alfalfa cDNA clone G1 also contains the two characteristic Ala-Pro-TrpCys-Gly-His-Cys-Lys active-site regions (Fig. 2B), and a hydrophobic N-terminal signal peptide. However, G1 resembles the mammalian endomembrane protein ERp72 (28, 29) more closely than it does PDI. Details of the sequence and expression of G1 will be presented elsewhere. Expression of the B2 sequence in the vector pSE380 in E. coli cells resulted in increased PDI activity. This activity was inducible by IPTOG, and was similar to the IPTG-induced activity in E. coli cells harboring an expression vector containing a mouse PDI sequence. In the absence of IPTG, leaky expression of B2 led to an increase of 18,200 cpm in

D.

NSLALSLTADQMVSA--LLDAEPPI--LYSEYDP---TRP-FSEASHMGLLTNI DDIIPFGITSNSDVFSKYQLDKDGVV- -LFKKFDE - - GRNNFEGEVTKENLLDEF

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350-392 ADRELVHMINWAKRVPGFVDLTLHDQVH--- LLECAWLEILMIGLV

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128-171 GPREAEGIVEYLICKQSGPASTEIKSADDATAFVGDN- -KVVIVGVF

FIG. 2. Amino acid similarities between alfalfa B2 and G1 polypeptides and related eukaryotic sequences. (A) Alignment of the amino acid sequence deduced from the B2 cDNA insert with that of human PDI [PH ,( subunit (3-PH)]. Identical amino acids in the two sequences are starred. Dots indicate pairs of amino acid residues that have a similarity score of .8 in a Dayhoff PAM matrix of amino

acid similarity. Conserved amino acids in human, bovine, chicken, and rat PDIs are underlined on the human (3-subunit sequence. A potential glycosylation site in the alfalfa B2 polypeptide is marked with a plus sign. Arrows mark the proposed sites of cleavage of the signal peptides. Gaps are indicated by dashed lines. An unidentified amino acid in the human sequence is represented by X. (B) Alignment of the proposed active-site regions (a and a') of alfalfa B2 and G1 polypeptides with related eukaryotic sequences. The sequences are the PDI consensus [human, bovine, chicken, and rat (PDI/C)], the ( subunit of human PH (p-PH), chicken GSBP, human ERp72 (initially described as deoxycytidine kinase; ref. 28) (dCK), rat form I PI-PLC, and spinach thioredoxin (STD). (C) Sequence similarity of a region of alfalfa B2, rat PI-PLC, and the PDI consensus (PDI/C). Invariant amino acids in human, bovine, chicken, and rat PDIs are underlined. (D) Sequence similarity of regions of alfalfa B2, human PDI (PDI/H), and the hormone-binding domain of the human estrogen receptor (ER/H).

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Plant Biology: Shorrosh and Dixon E kb 8.1

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Proc. Natl. Acad. Sci. USA 88 (1991) X

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Molecular cloning of a putative plant endomembrane protein resembling vertebrate protein disulfide-isomerase and a phosphatidylinositol-specific phospholipase C.

cDNA clones containing sequence similarity to the multifunctional vertebrate protein disulfide-isomerase (PDI, EC 5.3.4.1) were isolated from an alfal...
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