Eur. J . Biochem. 243, 350-357 (1997) 0 FEBS 1997

The wheat poly(A)-binding protein functionally complements pabl in yeast Hanh LE, Su-Chih CHANG, Robert L. TANGUAY and Daniel R. GALLIE

Department of Biochemistry, University of California, Riverside CA, USA

(Received 20 August18 November 1996) - EJB 96 1243/2

Poly(A)-binding protein (PAB) binds to the poly(A) tail of most eukaryotic mRNAs and influences its translational efficiency as well as its stability. Although the primary structure of PAB is well conserved in eukaryotes, its functional conservation across species has not been extensively investigated. In order to determine whether PAB from a monocot plant species could function in yeast, a protein characterized as having PAB activity was purified from wheat and a cDNA encoding for PAB was isolated from a wheat seedling expression library. Wheat PAB (72 kDa as estimated by SDSPAGE and a theoretical mass of 70823 Da as determined from the cDNA) was present in multiple isoforms and exhibited binding characteristics similar to that determined for yeast PAB. Comparison of the wheat PAB protein sequence with PABs from yeast and other species revealed that wheat PAB contained the characteristic features of all PABs, including four RNA binding domains each of which contained the conserved RNPI and RNP2 sequence motifs. The wheat PAB cDNA functionally complemented a pub1 mutant in yeast suggesting that, although the amino acid sequence of wheat PAB is only 47% conserved from that of yeast PAB, this monocot protein can function in yeast.

Keywords: poly(A)-binding protein; wheat; mRNA:

Most eukaryotic mRNAs terminate in a poly(A) tail of 50- 150 adenylate residues in length which serves as the binding site for poly(A)-binding protein (PAB). PAB is an abundant cellular protein, present at 4 pM in actively growing HeLa cells, and has a high affinity for poly(A) of 25 bases or longer (Sachs et al., 1987; Gorlach et a]., 1994; Yang and Hunt, 1992). Two-dimen- sional electrophoresis analysis of PAB from sea urchin and yeast revealed that the protein is present as several isoforms in these species (Drawbridge et al., 1990). PAB is an essential protein in yeast (Adam et al., 1986; Sachs et al., 1986) and appears to be involved in mRNA stability (reviewed by Bernstein and Ross, 1989) and translation (reviewed by Sachs and Wahle, 1993; Ja- cobson, 1996; Jackson and Standart 1990). PAB facilitates translation in vitro (Grossi de Sa et al., 1988) and, as a regulator of translation initiation, the poly(A) tail is dependent on the presence of the 5’ cap structure (Gallie, 1991). Inactivation of PAB using a temperature-sensitive mutant in yeast resulted in the accumulation of monoribosomes and increased the average poly(A) tail length, observations implicating PAB in promoting translation initiation and poly(A) tail shortening (Sachs and Davis, 1989). The observations that suppressor mutations within a 60s ribosomal protein or involving an RNA helicase required for 25s rRNA maturation in yeast rescued viability in the ab- sence of functional PAB and that PAB promotes 40s ribosomal subunit binding to an mRNA, suggest that PAB is involved dur- ing translation initiation (Sachs and Davis, 1989, 1990; Tarun and Sachs, 1995). Recent evidence has suggested that PAB may

Correspondence ro D. R. Gallie, Department of Biochemistry, Uni-

Fcwt tl 909 787 3590. Abbreviations. elF, eukaryotic initiation factor; PAB, poly(A)-bind-

ing protein : RBD. RNA-binding domain; TMV, tobacco mosaic virus. Note. The novel amino acid sequence data published here have been

deposited with the GenBank sequence data bank and are available under accession number U81318.

versity of California, Riverside. CA 92521-0129, USA

translation; yeast.

interact with ribosomes even in the absence of poly(A) (Prowel- ler and Butler, 1996).

PAB also controls mRNA degradation. Once an mRNA is transported to the cytoplasm, the poly(A) tail is progressively shortened in a processive manner until a tail length of approxi- mately 10-25 adenosine residues is reached (Lowell et al., 1992; Caponigro and Parker, 1995). Following the shortening process, the 5’ cap structure is removed by the decapping en- zyme (Beelman et al., 1996) and the body of the message quickly degraded by 5‘-3’ exonuclease attack (Decker and Par- ker, 1993), although there may be exceptions to this particular sequence of degradatory events (Decker and Parker, 1993). The presence of PAB bound to the poly(A) tail prevents this decap- ping event (Caponigro and Parker, 1995). Thus, the binding of PAB to the poly(A) tail maybe an integral step for translation and for maintaining the integrity of an mRNA.

Characterization of PAB from yeast, human, mouse, Xeno- pus leuvis, Drosophila melanogaster, and Arubidopsis tlzaliana revealed that it is conserved both in its primary sequence and in gene structure in eukaryotes (Sachs et al., 1986; Grange et al., 1987; Lefrere et al., 1990; Nietfeld et al., 1990; Belostotsky and Meagher, 1993). PAB exhibits a modular structure composed of four, tandemly arranged RNA-binding domains (RBDs) of 90- 100 amino acid residues in length that comprises the N-terminal half of the protein and a C-terminal accessory domain that is poorly conserved between plants, animals, and yeast. PAB is but one member of a large family of RBD-containing RNA-binding proteins (reviewed by Dreyfuss et al., 1993 ; Burd and Dreyfuss 1994; Kenan et al., 1991). Each RBD contains two short con- served sequence elements known as RNPI (8 amino acids resi- dues) and RNP2 (6 amino acids residues). Although the structure of PAB has not been elucidated, the structure of the RBD do- main from other members of this family has been solved and all share a common tertiary fold. The RBD contains a PI -a1 -P2-/l3- a2$4 structure in which the four /l strands forms an antiparallel

Le et al. ( E m J. Biochem. 243) 351

p sheet against which the two a helices lie (reviewed by Drey- fuss et al., 1993; Kenan et a]., 1991). The RNPl and RNP2 motifs lie within the adjacent antiparallel /l strands 3 and 1, re- spectively.

In contrast to the extensive knowledge concerning the con- servation of PAB primary structure in eukaryotes, less is known about the functional conservation of PAB between species. In order to determine whether a plant PAB could function in an- other species, we purified PAB from wheat and isolated a PAB cDNA from a wheat seedling expression library. The wheat cDNA was then used to complement the pablp null mutation in yeast. These results demonstrate the functional complementation of a PAB from a monocot plant species in yeast.

MATERIALS AND METHODS

Purification of PAB protein. For all steps in the purifica- tion, fractions were screened for activity by assaying for binding activity with radiolabeled (A)50 in a gel shift assay (described below). The purification procedure was essentially as described by Ymg and Hunt (1992) except that KOAc was used in place of KCI and a Mono Q chromatography step was added following Affi-blue gel chromatography. The 25 - 60 % saturated ammo- nium sulfate fraction of extract from 200 g wheat germ (pre- pared as described by Gallie and Tanguay, 1994) was loaded onto an Affi-blue gel column. The column was washed with 4 M NaCl in buffer B (100 mM KOAc, 1 mM CaCl,, 1 mM MgOAc, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride and 1 mM dithiothreitol) and PAB eluted with 2 M guanidine . HCI in buffer B. The eluted protein was dialyzed against buffer C (buffer B containing 50 mM KOAc and 10% glycerol), loaded onto a Mono Q column, and the flow-through loaded onto a poly(A)-Sepharose 4B column. PAB was eluted with 1 M urea and 2 M LiCl in buffer B containing 10 % glycerol and dialyzed against buffer B.

Preparation and purification antibodies to wheat PAB. Purified wheat PAB was resolved on a preparative SDS/PAGE gel, a vertical section of the gel stained for protein, and the band corresponding to PAB excised from the remaining gel and used for injection in rabbits. Antibodies against wheat PAB were pre- pared from the antiserum as described (Von Boxberg et a]., 1994). Purified PAB was resolved on a SDS/PAGE gel, trans- ferred to nitrocellulose membrane using semi-dry electroblot- ting, and stained with Ponceau S. The section of the membrane containing PAB was cut, fixed for 10 min with 4% formalin, blocked with 5% non-fat dried milk for 30 min and incubated with anti-PAB sera. Bound antibodies to PAB were eluted with 3 M potassium thiocyanate and dialyzed against phosphate-buf- fered saline (1.4 mM KH,PO,, 3 mM Na,HPO,i, 150 mM NaCI).

Western analysis. Plant tissue was ground in 10% trichloro- acetic acid in acetone on solid CO,, precipitated 1 h, and centri- fuged at 12000Xg for 15 min. The precipitate was washed twice with acetone, air dried, and the protein resolubilized in 9.5 M urea. The protein concentration was determined as described by Bradford (1976). Protein was displayed on a 10% SDS/PAGE gel and visualized either by staining with Coomassie brilliant blue G-250 dye or transferred to nitrocellulose membrane for western analysis.

For two-dimensional gel/western analysis, the protein was resolved in the first dimension using isoelectric focusing with 25 % pH 3 - 10 and 75 % pH 5 - 8 ampholytes and in the second dimension using SDS/PAGE, followed by Western blotting analysis essentially as described (Burnette, 1981). Protein was transferred to 0.2-pm nitrocellulose using semi-dry electroblot- ting. The membrane was then blocked in 5 % non-fat dried milk,

incubated with the appropriate dilution of antibodies to wheat PAB, rinsed and incubated with goat anti-rabbit antibodies con- jugated with horseradish peroxidase. The signal was visualized using chemiluminescence detection.

Gel retardation assay. The pT7-A5, plasmid, containing a continuous tract of 50 adenosine residues downstream of the T7 RNA polymerase promoter, was linearized with NdeI, which linearizes the DNA template immediately downstream of the po- ly(A) sequence. In vitro transcription was performed as de- scribed (Yisraeli et al., 1989) using 40 mM Tris pH 7.5, 6 mM MgCI,, 100 pg/ml BSA, 0.3 U/pl RNAsin (Promega), 10 mM dithiothreitol, 0.5 Up1 T7 RNA polymerase (New England Bio- labs), 55 pM ATP, 0.5 mM each of GTP, UTP. CTP, and 80 pCi [a-’2P]ATP. Full-length transcript was purified by electroelution following resolution on native PAGE gels.

Purified PAB or plant extracts were incubated with 1 ng la- beled (A)50 in a 15-pI reaction for 15 min at 0°C in a buffer containing 25 mM Tris pH 7.5, 100 mM KCI, 10% glycerol, 1 mM dithiothreitol, and 0.5 mg/ml of total yeast RNA. Heparin was added to a final concentration of 1 mg/ml followed by 2 p1 80% glycerol and the reaction incubated for an additional 10 min. The RNA-protein complexes were resolved on native 4 % PAGE mini-gels containing 0.5 X Tris/borate/EDTA buffer. Dried gels were analyzed by autoradiography. Quantitation of RNA-protein complexes was determined using phosphorimaging (Molecular Dynamics Inc).

Isolation of wheat PAB cDNA clones. PAB antibodies were used to screen a A ZAP I1 wheat seedling cDNA expression library (provided by Dr Karen Browning, University of Texas) according to the method described by Allen et al. (1992). 25 000-30000 plaque-forming-units in 8 ml Luria-Bertani me- dium containing 0.2% maltose top agar were used for each 150- mm plate. Following a 4-h incubation at 42OC, the plate was overlaid with an air-dried nitrocellulose filter saturated with 10 mM isopropyl thio-p-D-galactoside and was incubated at 37°C for 6 h. The filter was washed four times with 10 mM Hepes/KOH pH 7.6, 150 mM NaC1, 0.2% BSA, and 0.05% Tween 20 (buffer D) and incubated overnight at 4°C with wheat PAB antibodies diluted in buffer D. The filter was washed in buffer D, incubated with goat anti-rabbit IgG conjugated with horseradish peroxidase for 4 h. The filter was washed in buffer D and developed using diaminobenzidine as the substrate (De Blas and Cherwinski, 1983); 53 putative positive plaques were rescreened and plaque purified. R408 helper phage was used to excise the cDNA-containing pBluescript from the /z ZAP I1 prior to sequencing.

Yeast strains and growth conditions. Escherichia coli strain XLI-blue (Stratagene) was used for ZAP 11 phage ma- nipulation. The yeast strains, YAS100 and YAS352, contain the PABl gene under the control of its own promoter or the galac- tose-inducible GAL1 promoter, respectively (provided by Alan Sachs, University of California-Berkeley). YAS120 @abI), con- tains a genomic deletion of PABI and a truncated PABl gene is expressed from a centromeric plasmid (pAS86) that produces a single yeast PABl RBD domain in which Phe has been substi- tuted for Leu364 (provided by Alan Sachs). Yeast strains were grown in synthetic glucose medium with the required additives (Sherman, 1991).

A BarnHI -XhoI 2.2-kbp cDNA fragment containing the wheat PAB coding region was introduced into the galactose- inducible yeast expression vector, pYES2 (URA3) (Stratagene) to result in pwPAB which was then introduced into YASl20. YAS120(pwPAB) that had lost the resident plasmid, i.e. pAS86 (TRPI) , were screened for tryptophan auxotrophy following growth in tryptophan-supplemented synthetic glucose medium in which galactose was substituted for the glucose (in order to

352 Le et al. (Eur: J. Biochem. 243)

200 - 98 - 66 - 45 -

I 2 3 4 5

- PAB

Competitor: -- Molarratio: 0 0 I 5 10 50 1 5 10 50

31 - Free RNA -

Fig. 1. Purification of PAR from wheat germ. The protein profile for each step in the purification procedure (see Materials and Methods) was resolved on a 10% SDSlPAGE gel followed by staining with Coomassie. 20 pg protein from each fraction (lanes 1-4) and 2 pg purified PAB protein (lane 5) were loaded on the gel. Quantitation of the amount and purity of PAB protein at each step during purification is shown in Table 1. Molecular mass markers in kDa are indicated to the left of the gel.

Table 1. Purification of PAB from wheat germ. One unit (U) is defined as the amount of PAB catalyying the gel shift of 1 pg (A)5c, under stan- dard conditions.

Step ~~~~~~~

Protein Total Specific Purifica- activity activity tion

mg kU kU/mg -fold

Crude extract 250 000 200 000 0.800 -

Affi-blue gel 150 10000 66 83 20-60% (NH,)2S0, 150000 180000 1.2 1 .5

Mono Q 100 6200 62 I 8 Poly(A) affinity 0.4 300 750 938

A50 Capped A50

Competitor: Poly c R RNA -- Molarratio: 0 0 10 100 500 1000 1 5 10 50

Free RNA - 1 2 3 4 5 6 7 8 9 1 0

PAB: - + + + + + + + + +

Competitor: Poly G

Molar ratio: 0 0 ‘ 1 10 50 500 ’

induce wheat PAB expression). The presence of pwPAB (which contains the URA3 gene) in YAS120 was confirmed by the fail- ure of YASl20(pwPAB) to grow on 5-fluoroorotic acid which is toxic to URA3’ yeast. Generation times of yeast strains in standard glucose medium (with required additives) were deter- mined from the absorbance at 600 nm.

RESULTS

Characterization of PAB protein from wheat. PAB was puri- fied to near homogeneity from wheat germ using a four-step purification procedure (Fig. 1) during which PAB activity was followed using binding to radiolabeled (A)io in a gel shift assay. The procedure was optimized for purity by selecting only the peak PAB-containing fraction at each step during purification which explains the low yield of PAB (see Table 1, Fig. 1). Elu- tion from the poly(A)-Sepharose 4B column consistently yielded a doublet of 72 kDa and 79 kDa (Fig. 1, lane 5) . N-ter- minal sequencing of the purified protein failed, suggesting that both protein forms were N-terminally blocked and that the 72- kDa protein is not an N-terminal degradation product of the 79- kDa protein. Digestion of PAB with N-glycosidase F, which cleaves N-linked glycosyl moieties from glycoproteins, did not alter the migration of the two proteins (data not shown). How- ever, antibodies raised against the 72-kDa protein also recog- nized the 79-kDa protein, suggesting that both are antigeneti- cally related (data not shown).

To examine the binding specificity of wheat PAB, competi- tion RNA gel shift assays were performed in which purified PAB was added to reactions containing iri-vitro-synthesized ra- diolabeled (A)so and unlabeled competitor RNA; 1 mg/ml hepa- rin was added to the binding reactions to eliminate non-specific

Free RNA - 1 2 3 4 5 6

PAB: - + + + + + Fig.2. Analysis of the specificity of PAR binding to poly(A). The specificity of PAB binding was determined using competition binding/ gel shift assays between radiolabeled (A)5o and unlabeled competitor RNAs. The effect of a cap (m’GpppG) on PAB binding was determined using 5 ng purified protein and unlabeled competitor (A)so synthesized as uncapped or capped RNA (top). Specificity of binding to non-poly(A) RNAs was examined using unlabeled poly(C) and R RNAs (middle) or poly(G) (bottom) as the competitor RNAs. 9 is the 68-base 5’ leader from TMV RNA which is composed of 50% adenosine. The molar ex- cess of the competitor RNAs to the radiolabeled present in the competition reactions is indicated above each lane.

binding. Two prominent complexes were observed when 2.5 ng PAB was added to the binding reaction (lane 2, top panel, Fig. 2). A third complex that ran just above the free form of the RNA was also observed. A fourth complex could also be ob- served under some conditions. Given that PAB has a packing density of approximately 25 adenosines residues (Sachs et al., 1987) the (A)scl used in this study should bind a maximum of only two molecules of PAB. However, binding of PAB to po- ly(A) which exceeds the theoretical maximum for a given length of poly(A) has been previously observed (Kuhn and Pieler, 1996). Moreover, the C-terminal domain of PAB has been impli- cated in protein-protein contacts during the cooperative binding of PAB to poly(A) (Kuhn and Pieler, 1996). These findings may explain the appearance of more than the expected two PAB/(A),,, complexes observed in Fig. 2.

Unlabeled (A)50 acted as an efficient competitor when it was added to the binding reaction even at an equal molar ratio (lanes

Le et al. (ELM J. Biochern. 243) 353

3-6, top panel, Fig. 2). As the cap-associated initiation factors, eIF4F and eIF4B, also bind poly(A) (Gallie and Tanguay, 1994), we examined whether the reverse may also be true, that is, whether PAB can bind to the cap structure. This was measured by determining whether capped (A)50 could act as a better com- petitor than uncapped (A)50. Capped (A)s0 competed as effec- tively (lanes 7-10, top panel, Fig. 2) but not more than the un- capped (A)sc, (lanes 3-6, top panel). Poly(G) was less effective as a competitor than (A)s0 in that PAB binding to (A)5(, was still detected even when poIy(G) was present at a 10-fold molar ex- cess (lanes 3-6, bottom panel, Fig. 2). Poly(G), however, was a better competitor than poly(C) which required a 1000-fold ex- cess to achieve detectable competition with (A)50 (lanes 3 -6, middle panel, Fig. 2). Addition of poly(U) to the binding reac- tion resulted in a smear (data not shown) which was probably due to hybridization with the labeled (A)su. The relative affinity of wheat PAB for the homopolymers is similar to that observed for PAB isolated from Xenopus, pea, and Amhidopsis (Nietfeld et al., 1990; Yang and Hunt, 1992; Belostotsky and Meagher,

In addition to (A)s,,, PAB appears to bind other sequences in vivo which include the 5‘ leader sequence of the human PAB gene (de Melo Net0 et al., 1995; Bag and Wu, 1996). Short stretches of adenosine residues (a maximum of 8 in the human 5’ leader) are characteristic of the 5’ leader of PAB genes (de Melo Net0 et al., 1995) and may serve to autoregulate PAB ex- pression (de Melo Neto et al., 1995; Bag and Wu, 1996). The 68-base 5‘ leader (a) from tobacco mosaic virus (TMV) is also an A-rich RNA composed of SO% adenosine and functions as a translational enhancer in eukaryotes (Gallie et al., 1987). To determine whether PAB could bind this A-rich RNA, SZ RNA was tested in the competition assay. Competition by R was not detected until a SO-fold excess was tested in the PAB/(A),,, bind- ing assay which was significantly less than that observed for (A)so or even poly(G) (lanes 7-10, middle panel, Fig. 2) sug- gesting that PAB binding can discriminate between poly(A) and an A-rich RNA. Although R RNA is 50% adenosine, the longest stretch of adenosine residues is only three. These observations suggest that, regardless of the adenosine composition of an RNA, PAB may require a minimum length of poly(A) in order to bind. PAB activity exhibited a broad pH optimum of 5.5- 9.5, did not require salt or Mg” for activity, and was thermo- labile (data not shown). These observations suggest that the pro- tein purified from wheat is similar in size and binding character- istics to that in other species.

Wheat PAB protein is present in multiple isoforms. Multiple isoforms of PAB were observed in yeast and sea urchin (Draw- bridge et al., 1990) whereas PAB in human and Drosuphila cells appears to exist in a single isoform (Gorlach et al., 1994; Lefrere and Duncan, 1994) suggesting that PAB is extensively post- translationally modified in some species but not others. To deter- mine whether the isoform distribution of PAB in wheat is similar to that of yeast and sea urchin or more similar to that in humans and Drosophilu, the protein was resolved on a two-dimensional gel, transferred to nitrocellulose membrane, and subsequently probed with antibodies raised against purified wheat PAB. West- ern analysis revealed that at least seven isoforms of PAB (72 kDa in mass) with isoelectric points (PI) ranging over 6.0- 7.8 were present, with the basic isoforms being more abundant (Fig. 3). A fainter array of acidic isoforms (PI range 6.0-6.8) with a mass of 79 kDa was observed above the 72-kDa isoforms. The 79-kDa isoforms correspond in size and abundance to the upper band of the purified PAB observed i n the one-dimensional SDS/PAGE analysis (lane 5 , Fig. 1). As the PAB antibodies were raised and purified against the 72-kDa form but also recognize

1993).

SDS-PAGE

PAB - - 112 - 84 - 63 - 52

I I I I PI 5.2 6.3 7.6 8.8

Fig. 3. Two-dimensional geUwestern analysis of PAB. 500 ng purified PAB protein from wheat germ was resolved in the first dimension using isoelectric focusing and in the second using SDS/PAGE. Following transfer to nitrocellulose, the membrane was probed with PAB antibodies and the signal detected using chemiluminescence. Molecular mass mark- ers in kDa are indicated to the right of the western blot.

the 79-kDa form, these observations suggest that the 72-kDa and 79-kDa proteins are antigenically similar and may represent the modified and unmodified forms of PAB. Interestingly, a similar set of two arrays of isoforms was also observed in yeast and sea urchin (Drawbridge et al., 1990), suggesting that the distribution of the PAB isoforms has been conserved between wheat and these species.

Isolation of a wheat PAB cDNA. To obtain a wheat PAB cDNA for the complementation studies in yeast, a wheat seedling cDNA expression library was screened using the antibodies to wheat PAB; 48 positive clones were obtained of which 9 were larger than 2 kbp. One cDNA (2244 bp in length) was sequenced in its entirety and contained a complete open reading frame that would code for a 651-amino-acid protein of 70823 kDa in mass (Fig. 4). The deduced protein sequence contains a hexapeptide sequence (residues 201 -206) that was obtained from sequenc- ing of a peptide fragment isolated from purified wheat PAB which suggests that the protein isolated from wheat was PAB. Wheat PAB is conserved with PAB from other species: 55.5% identity with Arubidopsis PAB (Belostotsky and Meagher, 1993 ; Hilson et al., 1993), approximately 48% identity with PAB from animal species (Grange et al., 1987; Wang et al., 1992; Nietfeld et al., 1990) and 47% identity with yeast PAB (Burd et al., 1991 ; Sachs et al., 1986). Wheat PAB contains the characteristic do- mains and features of PAB, including four RNA binding do- mains (RBD), each of which contains the conserved RNPl and RNP2 sequence elements. The carboxyl-terminal domain of PAB is less well conserved between species than is the N-termi- nal domain, but within the C-terminal domain, wheat PAB con- tains a 37-amino-acid region close to the C-terminus that is con- served among all known PAB (Hilson et al., 1993). Like its yeast homolog, plant PAB have an N-terminal extension compared to PAB in animals. Interestingly, wheat PAB is as divergent from Auahidopsis PAB (72.6% similarity and 55.5% identity) as are two tissue-specifically expressed Arubidopsis PAB from each other (PAB2 and PAB5: 72.2% similarity and 56.3% identity) (Belostotsky and Meagher, 1993; Hilson et al., 1993).

The wheat PAB cDNA complements a yeast pabl null mu- tant. Because the level of PAB identity between plants and yeast is significant but not substantial (47% between wheat and yeast PAB), we examined whether PAB from wheat could function in yeast. To test this, the wheat PAB cDNA was introduced into a pub1 null mutant strain of yeast and tested for functional com- plementation. The wheat PAB cDNA was placed under the con-

354 Le et al. (Eur J. Biochem. 243)

Fig. 4. Comparison of the wheat PAB cDNA sequence with others. Sequences were from wheat (Pifiificum uesfivum, T. a. ; this study), Arahidopsis tlzaliancr (A. t . : Belostotsky and Meagher, 1993), Homo supien.r (H. s.; Grange et al., 1987), Mus musculus (M. m.; Wang et al.. 1992), Xenopus Itrevis (X. 1 . ; Nietfeld et al., 1990), Dro,coplzilci nzelanoguster (D. m.; Lefrere et al., 1990), Schizosaccl?uromyces poinnbe (S. p.; Burd et al., 1991) and Schi7osac,c/?a,-ornvces cereliyiae (S. c.; Sachs et al., 1986). The initiation codon for the wheat PAB open reading frame is the presumed start site. Amino acid sequences are shown as a one-letter code. Absolute amino acid conservation between the wheat PAB protein and the other PAB proteins is indicated by shading. The four RNA binding domains (RBD) are indicated by the boxes and within each RBD, the RNP consensus sequences are marked by asterisks above the sequence.

Le et al. (ELK J. Bioclzem. 243) 355

G: m 9 *

97 - 66 -

45 - Glucose: -

Galactose: +

! a

- - - + + +

Table 2. Wheat PAB functionally complements the yeast PABl gene.

Strain Promoter Type of PAB present Gener- ation time

- W P A B

YAS 100 YAS352 YAS120 (pAS86) YAS120 (pAS86

+ pYES2)

YAS120 (pAS86 + pwPAB)

YAS120 (pwPAB)

I1

native wild-type yeast PABl 3.3 GAL1 wild-type yeast PABf 3.4 native truncated yeast PABf a 10.4

native (pAS86) truncated yeast PABf 11.0 GALl (pYES2)

native (pAS86) truncated yeast PABI 5.3 GALl (pwPAB) wheat PAB GALl wheat PAB 4.9

Fig.5. Detection of wheat PAB (wPAB) protein expression in yeast by western analysis. The wheat PAB cDNA was introduced into the expression vector, pYES2, under the control of the galactose-inducible GAL1 promoter. YAS352 (pAS137) contains the wild-type yeast PABl under the control of the CALI promoter. YASl00 (pAS77) contains the wild-type yeast PABf gene. YAS120 (pAS86) expresses a single yeast RBD domain in a pabf background. YAS120 (pAS86 + pYES2) con- tains the expression pYES2 vector. YAS120 (pAS86 + pwPAB) contains the wheat PAB cDNA in the pYES2 expression vector under the control of the GAL1 promoter. YAS120 (pwPAB), cured of the pAS86 vector, contains only the wheat PAB/pYES2 (pwPAB) construct. YAS120 (pAS86 + pwPAB) was grown in standard glucose medium containing either glucose to repress expression of the wheat PAB or galactose as the sole carbon source to induce wheat PAB expression. All other strains were cultivated in standard glucose medium containing galactose. Puri- fied wheat PAB protein was included as a positive control. Molecular mass markers in kDa are indicated to the left of the western blot.

trol of the galactose-inducible GALl promoter in the yeast ex- pression vector, pYES2, resulting in pwPAB. This construct was then introduced into the pub1 mutant strain, YAS120, which ex- presses a single yeast RBD domain from the plasmid expression vector pAS86 (Sachs and Davis, 1989). Both pwPAB and pAS86 can be maintained in YAS120 as they contain the URA3 and TRPI selectable marker genes, respectively. Following a plas- mid shuffle in galactose-containing medium, YASl20 containing only pwPAB that expressed just the wheat PAB was obtained. Failure of YAS12O containing pwPAB, which contains the URA3 gene, to grow on 5-fluoroorotic acid confirmed that all the cells contained pwPAB. Expression of wheat PAB was observed using western analysis when the yeast host was grown in galac- tose but not in glucose as would be expected for a gene under the control of the GALl promoter (compare lanes 6 to 5 , Fig. 5) . Moreover, wheat PAB expression was detected in YAS120 when pAS86 was present (lane 6, Fig. 5) or absent (lane 7, Fig. 5) . The protein product of the yeast expressing the wheat PAB was approximately 72 kDa, although additional, smaller, protein bands were observed which were specific only to those yeast strains harboring the pwPAB construct; these may represent de- gradation products of wheat PAB. The wheat PAB antibodies did not cross-react with wild-type yeast PABl expressed either from its own promoter or from the GALl promoter (lanes 1 and 2, respectively, Fig. 5) . These data demonstrate that yeast retains viability in the absence of its own PABl if expressing the wheat PAB .

" Contains the fourth RBD of the yeast PABf in which Phe has been substituted for Leu364 and the C-terminal domain (Sachs et al., 1987).

The growth rate of YAS120, in which the pwPAB was either present or absent, was compared to yeast with the identical back- ground containing the wild-type yeast PABl gene (YAS100) or the yeast PABl coding region under the control of the GALl promoter (YAS352). When cultured in minimal medium, the generation time of YASIOO was 3 h whereas that for YAS352 was 3.5 h (Table 2). The doubling time for YAS120 (containing pAS86 but not pwPAB) was more than three times as long as that for YASIOO which had been previously observed for this strain (Sachs and Davis, 1989). This slower growth rate is a result of the expression of the truncated PABI containing only a single PABl RBD plus the PABl C-terminal domain. Introduc- tion ofthe expression vector, pYES2, into YAS120 did not sub- stantially alter the generation time. In contrast, when wheat PAB was expressed in YAS120 (pwPAB), the generation time was 5.3 h or 4.9 h, depending on whether the pAS86 construct (con- taining the truncated yeast PABI gene) was present or absent, respectively. These data suggest that wheat PAB complements yeast PABI function which agrees well with the functional com- plementation of the Amhidopsis PABS gene in yeast (Belostot- sky and Meagher, 1996). As the growth rate of the yeast that expresses only wheat PAB was faster than the yeast expressing the truncated yeast PABl but was slower than the yeast express- ing the wild-type yeast PABl, the wheat PAB was functionally better than the truncated yeast PABl but may not fully comple- ment yeast PABl function.

DISCUSSION

Although PAB is conserved in its protein sequence and gene structure in eukaryotes, its functional conservation between spe- cies has not been extensively examined. Because of its potential involvement in the translation and mRNA turnover, both of which are multi-component pathways, the functional comple- mentation of PAB between higher and lower eukaryotes might also require a significant degree of functional conservation of these pathways with regard to those interactions involving PAB. To establish whether PAB function is conserved between a mo- nocot plant species and yeast, we first characterized PAB from wheat and isolated a cDNA encoding it. Wheat PAB contains the conserved features of PAB from other species, including four RBDs and a less well conserved carboxyl-terminal domain. Puri- fied wheat PAB bound poly(A) specifically. Poor binding to the TMV 5' leader, an A-rich RNA (50% adenosine residues), was

356 Le et al. (Eul: J. Biochem. 243)

observed. However, like PAB from yeast and pea (Burd et al., 1991; Yang and Hunt, 1992), moderate binding to poly(G) was observed. The failure of PAB to bind the TMV 5’ leader may be a consequence of the fact that no stretch of adenosine residues longer than three is present in this sequence. Therefore, PAB protein may require a minimum length of adenosine residues in order to bind a non-poly(A)-containing RNA. Wheat PAB is pre- sent in multiple isoforms, ranging in pI over 6.0-7.8 and is similar to the isoforms observed for PAB from yeast and sea urchin (Drawbridge et al., 1990). PAB in human and Drosoplzila differs considerably in this respect in that only a single isoform is present in these species (Gorlach et a]., 1994; Lefrere and Duncan, 1994). The nature and role of the modification of PAB in any of these species remains unknown. The presence of multiple isoforms was also observed in wheat embryos, leaves, and roots (H. Le and D. Gallie, unpublished observations) sug- gesting that the distribution of isoforms is not regulated in a tissue-specific manner, at least in these tissues.

Wheat PAB functionally complemented a pub1 mutant in yeast suggesting that PAB from a monocot plant species can function in yeast. This is particularly interesting as the level of conservation between the primary sequence of PAB from wheat and yeast is only 47%. However, the C-terminal domain of the yeast PABI is not required for viability at least in yeast (Sachs et a]., 1987) and most of the conservation between the wheat and yeast PAB proteins is localized to the N-terminal domain. Wheat PAB may not completely complement yeast PABI in that the growth rate of yeast expressing only wheat PAB was a little slower than that for yeast expressing wild-type yeast PABI. However, yeast expressing wheat PAB grew considerably faster than yeast expressing a truncated yeast PABf, suggesting that wheat PAB functions to a greater extent than the minimal essen- tial domain of yeast PABI. The observation that a plant PAB is functionally conserved in yeast provides the opportunity to iden- tify those conserved regions in eukaryotic PAB that are essential for its function in translation as well as in mRNA processing and turnover.

We thank Karen Browning for the wheat seedling cDNA expression library and Alan Sachs for the yeast strains YAS100, YAS120, and YAS352. This work was supported by a grant from the United States Department of Agriculture (NRICGP 95-37100-1618).

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