The Plant Cell, Vol. 6, 393-404, March 1994 O 1994 American Society of Plant Physiologists

A Higher Plant Extracellular Vitronectin-like Adhesion Protein 1s Related to the Translational Elongation Factor-I a

Jian-Kang Zhu,a9' Barbara Damsz,aib Andrzej K. Kononowicz,a9b Ray A. Bressan,a and Paul M. Hasegawaai2 a Center for Plant Environmental Stress Physiology, Purdue University, 1165 Horticulture Building, West Lafayette, Indiana 47907-1 165

Department of Plant Cytology and Cytochemistry, University of Lodz, 90-237 Lodz, Poland

Higher plant proteins immunologically related to the animal substrate adhesion molecule vitronectin have recently been observed and implicated in a variety of biological processes, such as plasma membrane-cell wall adhesion, pollen tube extension, and bacterium-plant interaction. We provide evidence that, similar to vitronectin, one of these proteins, PVNl @lant yitronectin-like lJ, isolated from 428 mM NaCI-adapted tobacco cells binds to glass surfaces and heparin. PVNl was isolated by glass bead affinity chromatography. lsolated PVNl has adhesive activity based on results from a baby hamster kidney cell-spreading assay. This plant adhesion protein was detected in all tissues examined but was most abundant in roots and salt-adapted cultured cells. lmmunogold labeling indicated that PVNl is localized in the cell wall of cortical and transmitting tissue cells of pollinated mature styles. A partia1 amino acid sequence of PVNl revealed no similarity with vitronectin but, instead, was nearly identical to the translational elongation factor-lu (EF-lu). A clone isolated by screening a tobacco cDNA expression library with anti-PVN1 encoded a protein with greater than 93% identity to sequences of EF-lu from plants of numerous species. lmmunological cross-reactivity between tobacco PVNl and EF- l u as well as the reaction between the EF-lu antibody and the 65- and 75-kD vitronectin-like proteins of a fucoidal alga supported the conclusion that the plant extracellular adhesion protein PVNl is related to EF-lu.

INTRODUCTION

Vitronectin is a glycoprotein that was originally identified in human sera as a cell attachment factor (Tomasini and Mosher, 1990; Preissner, 1991). It is also present in the extracellular matrix (ECM) of specific mammalian cells (Hayman et al., 1983). This multifunctional protein mediates cell adhesion, protects thrombin from inactivation by antithrombin 111, and protects by- stander cells from cytolysis by complementation (Tomasini and Mosher, 1990; Preissner, 1991). Vitronectin mediates cell-ECM adhesion because it binds both to the cell membrane and to the ECM. Vitronectin connects the ECM with the intracellular network through a subset of plasma membrane receptors known as integrins (Hynes, 1987). A tripeptide sequence Arg- GlyAsp (RGD) in the amino-terminal cell binding domain is involved in the ligand-receptor interaction with integrins (Suzuki et al., 1985). However, recent evidence indicates that a basic motif in the carboxyl-terminal portion of the protein also mediates binding to certain integrins (Vogel et al., 1993). Vitronectin has high affinity for the proteoglycan heparin sul- fate that anchors it to the ECM (Suzuki et al., 1984). The heparin

Current address: Laboratory of Plant Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10021-6399.

To whom correspondence should be addressed.

binding domain consists of a basic sequence motif near the carboxyl terminus.

Vitronectin-like proteins have recently been identified in the slime mold Physarum polycephalum (Miyazaki et al., 1992), the brown alga Fucus distichus (Quatrano et al., 1991; Wagner et al., 1992), and severa1 species of flowering plants (Sanders et al., 1991; Zhu et al., 1991, 1993b). Higher plant vitronectin- like proteins have been implicated in plasma membrane-cell wall adhesion (Zhu et al., 1993b), attachment of bacterial patho- gens to plant cells (Wagner and Mathysse, 1992), and pollen tube extension through the transmitting tissue (Sanders et al., 1991). These proteins were identified as a result of immunoreac- tivity with pqlyclonal antibodies raised against vertebrate vitronectin. However, none of these proteins has been isolated from higher plants and biochemically characterized. We have previously reported the enrichment of vitronectin-like proteins in NaCI-adapted tobacco cells (Zhu et al., 1991, 1993b). In this study, we describe the isolation of one of the vitronectin-like proteins PVN1 @lant yitronectin-like 1) from these tobacco cells and demonstrate that it can bind to glass and heparin. PVNl is an adhesion protein that is localized in the cell wall (the plant ECM) and can mediate cell attachment to the substratum in a heterologous system. Evidence is also presented indicating that PVNl is both structurally and immunologically related to the translational elongation factor-1 a (EF-1 a).

394 The Plant Cell

A. COOMASSIE

1 2 3 4

B. ANTI-HVN1 2 3 4

Figure 1. Isolation of PVN1 from 428 mM NaCI-Adapted Tobacco Cells.(A) Coomassie blue stained-SDS-polyacrylamide gel.(B) Immunoblot probed with anti-human vitronectin (anti-HVN).Lanes 1 contain total protein extract that was used for glass bead columnchromatography; lanes 2, proteins from the glass bead column elutedwith high salt and at high pH; lanes 3, SDS-PAGE-eluted protein fromthe samples in lanes 2; lanes 4, proteins that were washed off beforeelution. Arrows designate the position of PVN1 (~55 kD).

RESULTS

initially to purify human vitronectin (HVN). The tobaccovitronectin-like protein (~55 kD; Zhu et al., 1993b) had preferen-tial affinity for glass beads and was eluted with 0.6 MNaHCO3, 0.2 M NazCO3, pH 9.5 (Figure 1, lanes 2). Prolongedwashing with 0.6 M NaHCO3 also eluted the protein from theglass beads (data not shown). In contrast, animal vitronectinsand the Fucus vitronectin-like protein can only be eluted fromglass bead columns under more stringent conditions (0.6 MKHCO3, 0.2 M K2CO3, pH 9.7; Hayman et al., 1983; Ruoslahtiet al., 1987; Wagner et al., 1992).

We have designated the tobacco vitronectin-like protein thatbinds to glass surfaces PVN1. Immunoblotting of two-dimensional gels of tobacco cellular proteins with anti-humanvitronectin (anti-HVN) revealed the presence of at least sixvitronectin cross-reacting proteins with molecular masses of~55 kD (Figure 2). The most acidic group of proteins reactedmost strongly with anti-HVN (Figure 2B). Anti-PVN1 reactedonly with a protein on the basic side of the gel; this proteinis extremely heterogeneous in charge and also reacted withanti-HVN (Figures 2B and 2C). Two-dimensional gel analysisof purified PVN1 indicated that it contained only the basic, het-erogeneously charged form of the family of vitronectin-likeproteins; it also reacted with anti-HVN (data not shown). PVN1can also bind to heparin (Figure 3). As with animal vitronec-tins (Yatohgo et al., 1988), this binding occurred only whenPVN1 was denatured with 8 M urea (Figure 3). The affinity forheparin was weak because PVN1 could be eluted from thematrix with only 0.1 M NaCI (data not shown).

Isolation of PVN1

Vitronectin, as the name indicates, is a glass binding protein(Hayman et al., 1983), and this unique property was used

PVN1 Is an Adhesive Protein

Tobacco glass binding proteins (Figure 1, lanes 2) were sepa-rated by preparative SDS-PAGE, and PVN1 was isolated by

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Figure 2. Two-dimensional Gel Analysis of Tobacco Vitronectin-like Proteins.Total S26 protein (500 ng) was separated by two-dimensional gel electrophoresis.(A) Gel stained with Coomassie blue.(B) Protein blotted onto a nitrocellulose membrane and probed with anti-HVN (1:1000 dilution).(C) Protein blotted onto a nitrocellulose membrane and probed with anti-PVN1 (1:5000 dilution).IEF, isoelectric focusing.

Higher Plant Vitronectin-like Protein 395

ANTI-HVN

1 2 3

by the adhesion protein HVN but not by BSA (Figure 4). PVN1promoted substantial adhesion and spreading of BHK cells,although not nearly to the same extent as HVN. The concen-tration of PVN1 needed to support BHK cell spreading was~100 times higher than that of HVN.

Immunological Characterization of PVN1

Figure 3. PVN1 Binds to Heparin-Agarose under DenaturingConditions.An immunoblot probed with anti-HVN is shown. Lane 1 contains pro-teins eluted from a glass bead column and used for heparin-agaroseaffinity chromatography; lane 2, heparin-agarose column eluate ob-tained when chromatography was conducted in the presence of 8 Murea to denature PVN1; lane 3, column eluate of undenatured PVN1.Arrow designates the position of PVN1 (~55 kD).

Similar to anti-HVN, polyclonal anti-PVN1 reacted specificallyto a polypeptide of ~55 kD (Figure 5). Consistent with our previ-ous report (Zhu et al., 1993b), this protein is enriched inNaCI-adapted tobacco cells and is associated with plasmamembranes (Figure 5). PVN1 was detected in tissues andorgans throughout the tobacco plant, with substantial accumu-lation in the root, pith, epidermal peels, style, and flower bud(Figure 6). Anti-PVN1 also reacted specifically with a 55-kDprotein in a broad taxonomic range of plant species (Figure 7).

Anti-HVN reacted with PVN1 (Figure 1). Reciprocally, anti-PVN1 reacted preferentially with HVN but not with BSA or fibro-nectin (Figure 8). Together, these data established that thereis definitive immuno-cross-reactivity between the plant vitronec-tin-like protein and an animal vitronectin. The very weak reactivityof anti-PVN1 toward HVN was not surprising because anti-HVNaffinity purified from plant vitronectin-like proteins also showedvery weak reaction with HVN (Sanders et al., 1991).

Subcellular Localization of PVN1

electroelution from excised gel slices (Figure 1, lanes 3). A stan-dard bioassay, baby hamster kidney (BHK) cell spreading, wasemployed to assess the adhesion activity of PVN1. BHK celladhesion to and spreading on the plastic matrix was facilitated

Biochemical fractionation indicated that some PVN1 is presenton the plasma membrane of cultured tobacco cells (Figure 5).In pollinated mature tobacco styles, immunogold labeling local-ized PVN1 to the inner wall of transmitting tissue cells (Figure

PVN1 HVN BSA

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Microtiter wells were coated with PVN1 (100 ng/mL), or HVN (1 ng/mL), or BSA (100 ng/mL) as controls. BHK fibroblast cells were added to themicrotiter wells and incubated for 2 hr before microscopic observation.

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Figure 5. PVN1 Is Associated with Plasma Membranes of TobaccoCells.Immunoblots of SDS-PAGE separated proteins (20 ng per lane) fromtotal protein extracts, microsomes, or plasma membrane-enriched vesi-cle fractions of unadapted (So) or 428 mM NaCI-adapted (S25) tobaccocells.(A) Anti-HVN at a dilution of 1:1000 for 3 hr.(B) Anti-PVN1 at dilution of 1:5000 for 2 hr.Arrows designate the position of PVN1 (~55 kD).

PVN1 antibody and anti-HVN reacted with ABP50 (Figures 12Aand 12B) and an EF-1a from carrot cells (data not shown).

To further study the relationship between vitronectin-like pro-teins and EF-1 a, we analyzed protein extracts from the fucoidalalga Pelvetia in which the vitronectin-like proteins (65 and 75kD) are of different sizes from that of EF-1 a (50 kD), thus avoid-ing the issue that PVN1 may have been contaminated with EF-1 a(Figure 13). Polyclonal antibodies that are specific to the Dic-tyostelium EF-1 a (Yang et al., 1990) reacted strongly with a50-kD polypeptide from Pelvetia (Figure 13, lane C). This 50-kDprotein is most likely the EF-1 a of the fucoidal alga. Consistentwith the finding that PVN1 is related to EF-1 a, anti-EF-1a alsoreacted with the fucoidal alga vitronectin-like proteins at 65and 75 kD, respectively (Figure 13, lane C). Anti-PVN1 reactedstrongly with the 65- and 75-kD vitronectin-like proteins, butit also cross-reacted weakly with the 50-kD EF-1 a (Figure 13,lane B). Anti-PVN1 also reacted with the 65-kD vitronectin-likeprotein in total protein extracts of Fucus embryos (B. Goodnerand R.S. Quatrano, personal communication).

Anti-PVN1 was used to screen a 428 mM NaCI-adaptedtobacco cell (825) cDNA expression library. Forty clones thatreacted with anti-PVN1 were purified, and the complete se-quence (Figure 11 A) was obtained for one of these clones(pZ8H7). The encoded protein (ZBH1) has very high sequenceidentity with higher plant EF-1 as (>93°/o) throughout the en-tire open reading frame (Figure 11B), including conservationof the GTP binding consensus residues and the region corre-sponding to the tRNA binding domain in EF-Tu (Axelos et al.,

9) and to the outer wall of cortical tissue cells within intercellu-lar spaces (Figure 10). A low level of labeling in the cytoplasmof transmitting cells was also observed (Figure 9). In contrast,in cells of immature styles and nonpollinated mature styles,labeling in the cytoplasm was substantially higher, whereasthere was very low or no labeling in the cell walls (data notshown). Preimmune antibody did not label either the cytoplasmor cell wall of stylar cells at any stage of development. Anothercontrol utilizing anti-tomato y-glutamyl phosphate reductaselocalized this enzyme, as expected, only to the cytoplasm oftobacco stylar cells (data not shown).

PVN1 Is Related to EF-1 a

The partial amino acid sequence of one CNBr fragment ofPVN1 was obtained, and a comparison with polypeptides inthe GenBank data base revealed identity with the translationalEF-1 a (Figure 11B). Polyclonal antibodies raised against a Dic-tyostelium EF-1 a, which is known as an actin binding proteinof 50 kD (ABP50), reacted with PVN1 (Figure 12C). Both anti-

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Figure 6. Organ and Tissue Distribution of PVN1 in Tobacco.Immunoblots of total protein extracts (20 ug per lane) separated bySDS-PAGE from roots of plants grown hydroponically (root hydroponic),organs, or tissues of plants grown in soil or 428 mM NaCI-adapted(S25) cells.(A) Anti-HVN at a dilution of 1:1000 for 3 hr.(B) Anti-PVN1 at a dilution of 1:5000 for 2 hr.Arrows designate the position of PVN1 (~55 kD).

A. COOMASSIE

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B. ANTI-PVN11 2 3 4 5 6

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Figure 7. PVN1 Is Detected in Different Plant Species.

Total leaf proteins (20 ng) were fractionated by SDS-PAGE, electro-blotted, and probed with anti-PVN1 (1:3000; 2 hr). Lanes 1 containtobacco; lanes 2, maize; lanes 3, wheat; lanes 4, soybean; lanes 5,barley; lanes 6, A. nummularia.(A) Coomassie blue-stained gel.(B) Anti-PVN1 antibody-probed immunoblot.Arrow designates the position of PVN1 (~55 kD).

1989). Recombinant ZBH1 produced from this clone alsoreacted strongly with anti-PVN1 (data not shown). The remain-ing clones appeared to be related structurally because theyall hybridized to the original ZBH1 cDNA. Partial sequencedata from several of the remaining clones indicated that, al-though not identical to ZBH1, the encoded proteins all had veryhigh sequence identity with ZBH1 and with EF-1a from otherplants (data not shown). It is, of course, not certain that ZBH1or any of the other cDNAs encodes PVN1. It is possible thatthe isolated cDNAs represent regular EF-1a family membersrather than PVN1. The fact that anti-PVN1 identified only EF-1os in the expression library screening could just be a reflec-tion of the abundance of these mRNAs relative to PVN1message. No apparent signal sequence was found in ZBH1.On the other hand, because no amino-terminal peptide se-quence is known for PVN1 or EF-1a proteins, it is difficult toidentify definitively any signal sequence even if it does existin ZBH1. In addition, not all precursors of secreted proteinscontain signal sequences. /

Higher Plant Vitronectin-like Protein 397

activity and affinity for glass or heparin were weak comparedto that of animal vitronectins, indicating that PVN1 may havediverged considerably from its animal counterparts during evo-lution. There remain other vitronectin cross-reacting proteinsin tobacco cells that have yet to be isolated and biochemicallyand physiologically characterized.

Animal vitronectins are anchored in the ECM partly throughinteractions with heparins. Tobacco PVN1 and vitronectin-likeproteins from Fucus and Physarum bind heparin to a certainextent (Miyazaki et al., 1992; Wagner et al., 1992); however,heparins are not present in the ECM of these organisms. InFucus, the sulfated fucoidan (Crayton et al., 1974), which is acomponent of the ECM, is most likely a homolog of heparinto which the vitronectin-like protein is linked. In higher plantcells, the association of PVN1 with the cell wall may also bemediated through its binding to a proteoglycan that may besimilar to heparin. Cell surface arabinogalactan proteins (AGPs),whose functions are unknown, are the best-characterized pro-teoglycans in plant cells (Komalavilas et al., 1991). The cellsurface localization of PVN1 showed a similar pattern to thatof AGPs (Pennell et al., 1989). Our data (not shown) indicatedthat PVN1 can bind to the algal fucoidan, but not to the secretedAGPs from Arabic gum. The binding of PVN1 to fucoidan wassimilar to its binding to heparin, i.e., weak and occurred onlywhen denatured with 8 M urea. It remains to be shown whetherPVN1 will interact with plasma membrane AGPs.

Experiments with different protein preparations indicated thatthe cell-spreading activity of PVN1 correlated with the extent

A. COOMASSIE

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B. ANTI-PVN1

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DISCUSSION

PVN1 Is a Vitronectin-like Protein

PVN1 is not only antigenically related to human vitronectin,it also possesses some biochemical properties in common withHVN, including affinity for glass and binding to heparin whendenatured with 8 M urea. PVN1 mediated cell attachment andspreading in the heterologous BHK cell spreading assay, im-plicating a function as an adhesion protein. PVN1 adhesive

Figure 8. Anti-PVN1 Antibody Reacts with HVN.Lane M contains molecular mass standards (97,66,45,31, and 22 kD);lanes 1, HVN (2 ng); lanes 2, human fibronectin (5 ng); lanes 3, BSA(5 ng); lanes 4, mixture of HVN (1 ng), fibronectin (2.5 ng), and BSA(4 ng). Proteins were separated by SDS-PAGE.(A) Coomassie blue-stained gel.(B) Immunoblot probed with anti-PVN1. Arrows designate the positionsof HVN on the blot (65 and 75 kD).

398 The Plant Cell

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Figure 9. Immunogold Localization of PVN1 in Transmitting Tissue Cells of Mature Tobacco Style 24 hr after Pollination.(A) and (B) Sections reacted with anti-PVN1.(C) Section reacted with preimmune serum.CW, cell wall; M, mitochondria; V, vacuole; P, plasma membrane; cell wall labeling, solid arrowheads; cytoplasmic labeling, open arrowheads.Bar = 1 urn.

Higher Plant Vitronectin-like Protein 399

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Figure 10. Immunogold Localization of PVN1 in Cortical Tissue Cells of a Pollinated Mature Tobacco Style.(A) Section reacted with anti-PVN1.(B) Section reacted with preimmune serum.C, chloroplast; CW, cell wall; IS, intercellular space; M, mitochondria; V, vacuole. Solid arrowheads point to cell wall labeling. Bar = 1 urn.

of enrichment for the protein (data not shown). These data to-gether with the cellular localization of PVN1 in the ECM andsurface of the plasma membrane implicate PVN1 involve-ment in mediating plasma membrane-cell wall adhesion in

NaCI-adapted cells. This activity has been attributed tovitronectin-like proteins (Zhu et al., 1993b). However, the adhe-sive property of PVN1 needs to be further characterized toascertain its physiological significance.

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Figure 11. cDNA and Translated Peptide Sequences of pZBH7 and Comparison of ZBHl Peptide Sequence with those of Plant EF-1a.s.

' \ Nucleotide and deduced amino acid sequences ofpZBH7 insert. These sequences have been submitted to GenBank as accession number U04632. .A) Comparison of ZBHl (ZBH) with EF-lu from tomato (Pokalsky et al., 1990; Genbank accession number X14449) and carrot (EMBL accession number X60302) indicating identities of 97.3 and 94.4%, respectively. The underlined sequence is from the partia1 amino acid sequence determi- nation of PVNI. Boldface letters indicate the RYD motif, and asterisks indicate positions of stop,codons.

Higher Plant Vitronectin-like Protein 401

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Figure 12. PVN1 Is Antigenically Related to EF-1a.

Lanes 1 contain prestained molecular mass standards (low range, Bio-Rad, catalog number 161-0304); lanes 2, total S0 cell protein extract; lanes3, total S25 cell protein extract; lanes 4, 0.5 ng of ABP50 (actin binding EF-1a [50 kD] from Dictyostelium); lanes 5, 1 |ig of ABP50; lane 6, 2 ngof ABP50; lanes 7, 2 ng of PVN1 eluted from a glass bead column (partially degraded).(A) Anti-PVN1-probed immunoblot.(B) Anti-HVN-probed immunoblot.(C) Anti-ABP50-probed immunoblot.

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-75-65-50

Figure 13. Analysis of the Brown Alga Pelvetia Vitronectin-like Proteins.Protein extracts (20 jig per lane) from two-celled Pelvetia embryos wereseparated by SDS-PAGE, and immunoblots were incubated with anti-HVN (lane A), anti-PVN1 (lane B), or anti-ABP50, i.e., anti-EF-1a (laneC). Approximate molecular masses are indicated at right in kilodaltons.

Immunogold localization with anti-PVN1 revealed intenselabeling in the walls of transmitting cells of pollinated maturetobacco styles. However, cytoplasmic labeling was also pres-ent, especially in certain cells of immature styles. Cytoplasmiclocalization was also observed for the Fucus vitronectin-likeprotein during early embryo development (Wagner et al., 1992).The observed cytoplasmic labeling in tobacco cells could bedue to immuno-cross-reactivity between PVN1 and an EF-1athat functions in the cytoplasm, or it could reflect the synthe-sis and secretion of PVN1 prior to deposition in the wall.

PVN1 Is Related to EF-1a

The fact that PVN1 reacted with antibodies against humanvitronectin and EF-1 a suggests that this higher plant vitronectin-like protein is related to EF-1 a. Furthermore, both anti-HVNand anti-PVN1 cross-reacted with Dictyostelium EF-1 a. A par-tial amino acid sequence obtained from a PVN1 CNBr fragmenthad high sequence identity with EF-1 a. Consistent with thisidea that PVN1 is related to EF-1 a, we showed that antibodiesmade specifically against EF-1 a (ABP50; Yang et al., 1990)can react with the 65- and 75-kD vitronectin-like proteins ofa brown alga, although less strongly than anti-PVN1 reactsto these proteins.

EF-1 a, a GTP binding subunit of the translation elongationcomplex, may have a diverse array of functions. Besides its

402 The Plant Cell

role in the elongation phase of protein synthesis, EF-la has been found to be an actin binding protein (Yang et al., 1990), an activator for the carrot phosphatidylinositol-4-kinase (Yang et al., 1993), and a malignant transformation determinant in animal cell cultures (Tatsuka et al., 1992). Our study illustrates that proteins structurally related to EF-1 a may function in the ECM by possibly interacting with membrane receptor proteins. The high homology between the partial sequence of WN1 and the sequence of EF-1 a indicates that the two proteins may be structurally very similar. It is likely that PVNl is a member of a superfamily of EF-1 a proteins. PVNl must somehow be dis- tinct from other EF-ia proteins because it is localized to the cell wall and most likely performs functions not related to translation.

Our finding that a cell surface protein is structurally and im- munologically related to a component of the translational machinery is not without precedent. An animal cell surface protein, the 67-kD high-affinity receptor for the ECM adhesion protein laminin (wewer et al., 1986), is immunologically related to a 33- to 37-kD polypeptide (Rao et al., 1989) of the transla- tional machinery (Auth and Brawerman, 1992). A partial amino acid sequence obtained from a CNBr fragment of the laminin receptor is identical to the sequence of the 33- to 37-kD pro- tein (Wewer et al., 1986). For both PVNl and the 67-kD laminin receptor, more sequence information is needed to ascertain the relationship between these proteins and the proteins of the translational machinery.

The partial sequence of PVNl contains a RYD motif. The RYD in the major surface glycoprotein (gp63) of Leishmania has been shown to mimic the RGD ligand attachment region of fibronec- tin in mediating parasite-macrophage interaction (Soteriadou et al., 1992). It would be interesting to determine whether the RYD sequence in PVNl is functional as a cell binding motif. In fact, knowledge from gp63 research may have more rele- vance to this study than just the common RYD motif. The Leishmania gp63 is considered to be afibronectin-like protein because of its immunological cross-reactivity and commonal- ity of certain cell binding characteristics with fibronectin (Rizvi et al., 1988). However, the primary sequence of gp63 has no identity with that of fibronectin (Button and McMaster, 1988). The RYD-containing region of gp63 has subsequently been determined to be responsible for the cross-reactivity with fibro- nectin (Soteriadou et al., 1992). It is interesting to note that not all of the currently isolated plant EF-lu type genes contain this RYD domain, and it is not present in the Eschefichia coli EFTu sequence. It is possible that plant vitronectin-like pro- teins have little or no primary sequence identity with animal vitronectin. After all, most of the plant cell wall proteins identi- fied to date have no sequence identity with any of the animal extracellular proteins. Even the plant cell wall enzyme non- specific lipid transferase contains no sequence similarity to the animal enzyme (Thoma et al., 1993). Therefore, despite recent emphasis on the similarities between the plant cell wall and the animal ECM (Roberts, 1989, 1990), we should also keep in mind the well-established differences between these two structures.

METHODS

Plant Material

Unadapted (So) and 428 mM NaCI-adapted (SZ5) tobacco (Nicotiana tabacum cv Wisconsin 38) cells were maintained as described previ- ously(Zhu et al., 1993b). Tobacco, maize, wheat, soybean, barley, and Atriplex nummulafia were grown in soil in a greenhouse. Two hours after fertilization, zygotes of brown alga Pelvetia fastigiata were ob- tained as described previously by Brawley and Robinson (1985).

lsolation of PVNl

A plant vitronectin homolog (PVN1) was isolated by glass-affinity chro- matography. Early stationary phase SZ5 cells were separated from medium by filtration, ground into fine powder in liquid nitrogen, and then incubated with a large volume of acetone overnight at -20°C. Cell powder was collected onto Whatman No. 1 filter paper, washed with cold acetone, lyophilized, and stored dry at -20°C. All subse- quent manipulations were conducted at O to 4°C with caution to avoid using glassware. To extract protein, 10 g of acetoneextracted cell powder was mixed with 200 mL of extraction buffer containing 50 mM Tris- HCI, pH 7.5,5 mM EDTA, 1% P-mercaptoethanol, 1 mM phenylmethyl- sulfonyl fluoride, and 0.01 mglmL leupeptin. The mixture was vortexed, sonicated briefly, and then shaken continuously for 1 hr. The mixture was then centrifuged at 10,OOOg for 20 min. The supernatant (150 mL) was loaded onto a column (14 cm2 x 30 cm) of acid washed glass beads (Sigma) equilibrated with 0.6 M NaH C03, pH 8.3. The column was washed with 600 mL of 0.6 M NaH C03, and then bound proteins were eluted with 1200 mL of 0.6 M NaH C03 plus 0.2 M Na2C03, pH 9.5. The eluate was dialyzed thoroughly against H20, lyophilized, and analyzed by SDS-PAGE and immunoblotting with anti-human vitronec- tin (anti-HVN).

Proteins eluted from the glass bead column were separated by preparative SDS-PAGE. A portion of the gel containing a band corre- sponding to the molecular mass of the tobacco vitronectin-like protein (Zhu et al., 199313) was excised, and the protein was electroeluted from the gel slice. This gel-eluted protein was designated PVNl and, after renaturation, was used in subsequent cell-spreading assays and amino acid sequence analyses.

Proteins eluted from the glass bead column were also loaded onto a heparin-agarose column (Sigma) either in the presence or absence of 8 M urea in a manner similar to that described by Yatohgo et al. (1988). Proteins bound to heparin-agarose were eluted with the load- ing buffer plus 0.5 M NaCI.

Partia1 Amino Acid Sequence Determination

The amino terminus of PVNl was blocked for sequence analysis. There- fore, polypeptide fragments of WNI were generated by CNBr cleavage. A partial amino acid sequence was obtained from one of the fragments by automated Edman degradation.

Cell-Spreading Assay

Cell attachment and spreading activities were determined using baby hamster kidney (BHK) fibroblast cells according to the procedure of

Higher Plant Vitronectinrlike Protein 403

Grinnell et al. (1977). Microtiter wells (96-well polystyrene tissue cul- turepIate)werecoatedwith50pLofPVNl, HVN,orBSA(attheindicated concentrations) in an adhesion medium (150 mM NaCI, 1 mM CaClz, 3 mM KCI, 0.5 mM MgCl2, 6 mM NqHPO4, and 1 mM KH2P04, pH 7.3) for 1.5 hr at 37%. BHK cells were incubated with O.!, mg/mL tryp- sin in adhesion medium for 20 min, washed with adhesion medium, and then treated with 0.5 mg/mL soybean trypsin inhibitor for 5 min. Approximately 4 x 103 BHK cells in 50 pL of adhesion medium were added to each well and incubated for 2 hr at 37%.

lmmunoblot Analyses

Cell fractionation, protein extraction, SDS-PAGE, and immunoblotting were conducted as described previously by Zhu et al. (1993b). Gels were stained with Coomassie Brilliant Blue R 250. Polyclonal chicken antibody (anti-WN1) was obtained after immunizing a hen with PVNl (Zhu et al., 1993a). Polyclonal rabbit anti-HVN antibodywas purchased from Telios (San Diego, CA). Dicfyostelium elongation factor-la (EF la) protein (actin binding protein of 50 kD; ABP50) and polyclonal an- tibody (anti-EM a) were generous gifts from J. Condeelis (Albert Einstein College of Medicine, New York) (Yang et al., 1990). Daucus carota EF- 1 a (PIK-A49) was provided by W.F. Boss (North Carolina State Univer- sity, Raleigh, NC). Twedimensional gel electrophoresis was performed as described by LaRosa et al. (1989).

lmmunocytochemlstry

Pollinated mature styles from tobacco (N. tabacum cv Wisconsin 38) flowers at developmental stage 12 (described by Koltunow et al., 1990) were fixed in 2% glutaraldehyde in 0.05 M potassium phosphate buffer, pH 6.8, washed with the buffer, and dehydrated in an ethanol dilution series. Tissue samples were embedded in London Resin White (hard grade; EM Sciences, Fort Washington, PA) according to the manufac- turer's directions. Ultrathin sections collected on gold grids were blocked in 1% BSA, 10% (v/v) rabbit normal serum (RNS) in TTBS (10 mM Tris-HCI, 150 mM sodium chloride, 0.05% Tween 20, pH 7.6) and reacted successively with a 1:lOO dilution of anti-PVN1 in antibody dilution buffer (Wo BSA, 1% RNS in TTBS) and a 1:30 dilution of gold-conjugated rabbit anti-chicken antibodies (Jackson lmmuno Research Laborato- ries, Inc., West Grove, PA) in 1% BSA in TTBS. Sections were stained in uranyl acetate and poststained in lead citrate. Sections were viewed and photographed with a transmission electron microscope (EM200; Philips Electronic Instruments, Mahwah, NJ).

lsolatlon and Characterizatlon of cDNA Clones

A tobacco Su cell hzapll (Stratagene) library was screened with anti- PVNl ata dilution of 1:5000 (v/v). Blocking, washing, and secondary antibody reaction conditions were the same as described for the im- munoblot analysis. Forty hzap clones were purified and converted to pBluescript SK- by coinfection with R408 helper phage. The com- plete sequence of both strands of the cDNA insert of clone pZ6H7 and partia1 sequences of 39 other clones were determined by the dideoxy nucleotide chain termination method using the Sequenase kit (U.S. Biochemicals).

ACKNOWLEDGMENTS

We thank Dr. Kenneth Robinson of Purdue University for providing klvetia embryos, Dr. John Condeelis of the Albert Einstein College of Medicine for the gift of EF-la protein (ABP50) and antibody (anti- ABP50), Dr. Wendy F. Boss of North Carolina State University for the gift of PIK-A49 protein, Xiaomu Niu for providing the computer analy- sis of DNA and peptide sequences, Jean Clithero for technical support, and the Electron Microscopy Center in Agriculture at Purdue Univer- sity for the use of the transmission electron microscope system. This work was supported by U.S. Department of Agriculture/National Re- search lnitiative Competitive Grants Program Grant No. 92-37100-7626 and by a McKnight Fellowship to J.-K.Z. This is journal paper number 14,029 of the Purdue University Agricultura1 Experiment Station.

Received November 29, 1993; accepted January 5, 1994.

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 This information is current as of January 13, 2009

 DOI: 10.1105/tpc.6.3.393

1994;6;393-404 PLANT CELLJ. K. Zhu, B. Damsz, A. K. Kononowicz, R. A. Bressan and P. M. Hasegawa

Elongation Factor-1[alpha]A Higher Plant Extracellular Vitronectin-like Adhesion Protein Is Related to the Translational

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