Nucleotide sequence and developmental expression of duplicated ...

Plant Physiol. (1994) 106: 1705-1 706

Plant Gene Register

Nucleotide Sequence and Developmental Expression of Duplicated Genes Encoding Protein Disulfide lsomerase in Barley (Hordeum vurgare 1.)' Fuqiang Chen* and Patrick M. Hayes

Department of Crop and Soil Science, Oregon State University, Corvallis, Oregon 97331-3002

PDI (EC 5.3.4.1), an ER luminal protein that catalyzes the formation of disulfide bonds of nascent polypeptide chains in the lumen of ER (Freedman, 1989), is a multifunctional protein that also functions as the p subunit of prolyl 4hydroxylase (Pihlajaniemi et al., 1987), a thyroid hormonebinding protein (Yamauchi et al., 1987), and a component of the triglyceride transfer protein complex (Wetterau et al., 1990). In addition, the amino acid sequence of PDI is similar to a number of enzymes (Bennett et al., 1988; Geetha-Habib et al., 1988; Tsibris et al., 1989). PDI contains two copies of an active site closely related to thioredoxin. A cDNA sequence for plant PDI has been reported in alfalfa (Medicago sativa) (Shorrosh and Dixon, 1991, 1992); however, here we report three different cDNA clones encoding PDI in barley (Hor-

Table 1. Characteristics of cDNA clones encodinn PDI in barlev

Orga n i sm : Barley (Hordeum vulgare L. cv Morex). Cenome Location: Two independent loci were detected. One of these loci was mapped to t h e plus arm of barley chromosome 1. Cene Function: Encodes PDI, which catalyzes the formation of disulfide bonds. Source: cDNA of poly(A)+ RNA from 3-d-old pollinated barley

ovaries. Sequencing Technique: Dideoxy chain termination method. Method of Identification: Sequence comparison with CenBank, EMBL, and SwissProt data bases. Expression Characteristics: The mRNA levels were elevated after pollination and peaked at d 8 after pollination. Features of Predicted Amino Acid Sequence: An open reading frame encodes a 51 3-amino acid protein that includes an N-terminal signal peptide of 23 amino acids and a C-terminal ER retention signal sequence. The mature protein contains 490 amino acids with a predicted M, of 54,142 and an isoelectric point of 4.8. Anti bodies:

deum vulgare). A cDNA library in Uni-ZAP XR was constructed from poly(A)+ RNA of barley ovaries 3 d after pollination. Three different cDNA clones (PDIl, PDI2, and PDI3) were identified in the course of differential screening to characterize fertilization-specific gene expression. The DNA sequence of each clone was determined by the dideoxy method of Sanger with an autosequencer (Applied Biosystems, Inc. [Foster City, CAI model 373A, version 1.2.0). PDI2 was used as a probe for restriction fragment-length polymorphism mapping in two barley doubled-haploid populations. PDIl (1816 bp) and PDI2 (1772 bp) contained a complete coding sequence, but PD13 (1191 bp) was truncated at the 5' region. The three cDNA clones differed at the 3' noncoding region before the poly(A) tail and were likely transcribed from different PDI loci. Two independent PDI loci were detected in 150 doubled-haploid progeny of Harrington X Morex. One of these loci was mapped to the plus arm of barley chromosome 1, which is 2.7 centimorgans from the restriction fragment-length polymorphism marker ABC154a in the barley genome mapping population of Steptoe X Morex (Kleinhofs et al., 1993). Additional PDI loci are likely since multiple bands were detected in DNA genomic blots. Multiple cDNA sequences and PDI loci indicate the presence of a small PDI gene family in barley.

None. PDIl and PD12 had an open reading frame that encodes a 513-amino acid protein. The deduced amino acid sequence was characterized by a 23-amino acid N-tenninal signal sequence, two copies of a domain identical to the vertebrate and a CPDI active site (Ala-Pro-Trp-Cys-Gly-His-Cys-Lys), terminal ER retention signal sequence (Lys-Asp-Glu-Leu). The sequence identity between barley PDI and alfalfa PDI was 67% at the nucleotide level and 63% at the amino acid level. Characteristics pertaining to the barley PDI clones are described in Table I. PDI gene expression was elevated in ovaries after pollination. The expression level peaked at d 8 after pollination with a 5-fold increase over unpollinated ovaries. The elevation pattern, however, was different from that of calreticulin, an ER luminal Ca2+-binding protein, which was also elevated

'This is Oregon Agricultura1 Experiment Station Joumal No. 10507. * Corresponding author; fax 1-503-737-1589.

Abbreviation: PDI, protein disulfide isomerase. 1705

Chen and Hayes

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after pollination but peaked a t a n earlier stage (d 2-3) after pollination (Chen et al., 1994). Received June 2, 1994; accepted June 30,1994. Copyright Clearance Center: 0032-0889/94/106/1705/02. The GenBank accession numbers for the sequences reported in this article are L33250, L33251, and L33252 for PDI1, PDI2, and PD13, respectively. LITERATURE ClTED

Bennett CF, Balcarek JM, Varrichio A, Crooke ST (1988)Molecular cloning and complete amino-acid sequence of form-I phosphoinositide-specificphospholipase C. Nature 334 268-270 Chen FQ, Hayes PM, Mulrooney DM, Pan AH (1994)Identification and characterization of cDNA clones encoding plant calreticulin in barley. Plant Cell 6: 835-843 Freedman RB (1989) Multiple role in modification of nascent secretory proteins. Cell57: 1069-1072 Geetha-Habib M, Noiva R, Kaplan HA, Lennarz WJ (1988) Glycosylation site binding protein, a component of oligosaccharyl transferase, is highly similar to three other 57 kd luminal proteins of the ER. Cell54: 1053-1060 Kleinhofs A, Kilian A, Saghai Maroof MA, Biyashev RM, Hayes P, Chen FQ, Lapitan N, Fenwick A, Blake TK, Kanazin V, Ananiev E, Dahleen L, Kudrna D, Bollinger J, Knapp SJ, Liu B,

Plant Physiol. Vol. 106, 1994

Sorrells M, Heun M, Franckowiak JD,Hoffman D, Skadsen R, Steffenson BJ (1993) A molecular, isozyme and morphological map of the barley (Hordeum vulgare) genome. TheoI Appl Genet 8 6 705-712 Pihlajaniemi T, Helaakoski T, Tasanen K, Myllyla R, Huhtala M-L, Koivu J, Kivirikko KI (1987) Molecular cloning of the 8subunit of human prolyl 4-hydroxylase. This subunil and protein disulfide isomerase are products of the same gene EMBO J 6: 643-649 Shorrosh BS, Dixon RA (1991) Molecular cloning of a plant putative endomembrane protein resembling vertebrate protein disulfideisomerase and a phosphoinositide-specific phospholipase C. Proc Natl Acad Sci USA 88: 10941-10945 Shorrosh BS, Dixon RA (1992) Sequence analysis and developmental expression of an alfalfa protein disulfide isonierase. Plant Mo1 Bioll9 319-321 Tsibris JCM, Hunt LT, Ballejo G,Barker WC, Toney LJ, Spellacy WN (1989) Selective inhibition of protein disulfide isomerase by estrogens. J Biol Chem 264: 13967-13970 Wetterau JR, Combs KA, Spinner SN, Joiner BJ (1'990) Protein disulfide isomerase is a component of the miaosomal triglyceride transfer protein complex. J Biol Chem 265 9800-980:' Yamauchi K, Yamamoto T, Hayashi H, Koya S, Takikawa H, Toyoshima K, Horiuchi R (1987) Sequence of membrane-associated thyroid hormone binding protein from bovine li ver: its identity with protein disulfide isomerase. Biochem Biophys Res Commun 146 1485-1492