the tobacco luminal binding protein is encoded by a multigene family

The Plant Cell, Vol. 3, 1025-1035, September 1991 O 1991 American Society of Plant Physiologists

The Tobacco Luminal Binding Protein 1s Encoded by a Multigene Family

Jürgen Denecke,’,’ Maria Helena S. Goldman,b Jan Demolder,b Jef Seurinck,a and Johan Bottermana a Plant Genetic Systems N.V., J. Plateaustraat 22, 9000 Gent, Belgium

Laboratory of Genetics, K.L. Ledeganckstraat 35, 9000 Gent, Belgium

We have cloned cDNAs of the tobacco homolog of the luminal binding protein (BiP) that has been described in other higher eukaryotes. In contrast to the mammalian and yeast protein, tobacco BiP is encoded by a multigene family. The gene products of all the cloned members of this family contain a carboxy-terminal His-Asp-Glu-Leu peptide that may form the signal for retention in the endoplasmic reticulum. Analysis of expression patterns revealed that BiP transcripts are predominantly present in tissues with high rates of cell divisions, in secretory tissues, and in cells treated with tunicamycin. We also show that a chimeric gene containing the coding region of one of the tobacco BiP genes is able to complement a mutation in the Saccharomyces cerevisiae BiP gene.

INTRODUCTION

Severa1 proteins, commonly referred to as “polypeptide chain-binding (PCB) proteins” (Rothman, 1989) have been identified in various compartments of eukaryotic cells as part of a polypeptide folding machinery. These so-called “molecular chaperons” (Ellis, 1990) bind to nascent polypeptide chains and transiently stabilize the unfolded state until correct folding (Cheng et al., 1989; Goloubinoff et al., 1989) or transport to another cellular compartment (Chirico et al., 1988; Deshaies et al., 1988; Zimmermann et al., 1988; Phillips and Silhavy, 1990) is accomplished. A second role, that of preventing the aggregation of malfolded proteins and dissolving such aggregates both under normal or stress conditions, has also been proposed (Pelham, 1986).

Heat shock proteins of the hsp70 family and their related constitutive analogs (Lindquist and Craig, 1988) form a group of PCB proteins that are very conserved among different eukaryotes. They have a highly conserved N-terminal ATP binding domain and a more variable protein binding domain (Flaherty et al., 1990). The luminal binding protein (BiP), identified in various mammals (Munro and Pelham, 1986; Ting et al., 1987; Haas and Meo, 1988; Wooden et al., 1988) and yeasts (Normington et al., 1989; Rose et al., 1989), is a member of the hsp70 family that accomplishes its function in the lumen of the endoplasmic reticulum (ER). BiP differs from the other family members in the presence of an N-terminal signal sequence that is

’ Current address: Department of Molecular Genetics, Swedish University of Agricultura1 Sciences, S-750 07 Uppsala, Sweden. To whom correspondence should be addressed.

required for the cotranslational translocation of proteins through the ER membrane. Another specific feature is the C-terminal tetrapeptide Lys-Asp-Glu-Leu (KDEL) for mammals and His-Asp-Glu-Leu (HDEL) for yeast that serves as a general retention signal for soluble reticuloplasmins in the ER lumen (Munro and Pelham, 1987; Pelham et al., 1988). BiP was shown to bind to newly synthesized, incompletely assembled or malfolded proteins in the lumen of the ER (Haas and Wabl, 1983; Bole et al., 1986; Gething et al., 1986; Kassenbrock et al., 1988). Although consti- tutively present during normal growth, a sharp induction occurs upon conditions that cause the accumulation of malfolded proteins in the lumen of the ER (Lee, 1987; Kozutsumi et ai., 1988). BiP may prevent the secretion of proteins that have not yet acquired their mature confor- mation (Bole et al., 1986; Gething et al., 1986), promote protein folding and assembly (Gething et al., 1986), or dissolve protein aggregates that have been formed under normal or stress conditions in the ER (Munro and Pelham, 1986). The recent finding that loss of BiP function blocks translocation of secretory proteins in yeast (Vogel et al., 1990) suggests that the gene product plays a constitutive role both in protein import into the lumen of the ER and in the subsequent maturation steps in vivo. Continuous binding of BiP to proteins would only occur so long as proper folding or assembly cannot be achieved, because of either mutations in the transported proteins or externa1 stresses that impair maturation.

We recently demonstrated the presence of a default pathway for protein secretion in plants (Denecke et al., 1990). Three cytosolic proteins could be forced to enter

1026 The Plant Cell

the ER lumen by the addition of an N-terminal signalsequence and were slowly secreted from plant protoplasts.The rate of protein secretion was shown to be largelydependent on the nature of the transported protein. Fur-thermore, redirecting of cytosolic proteins into the endo-membrane system resulted in a drastic reduction of thesteady-state levels of the encoded gene products. As partof a more detailed analysis of the fate of proteins in foreigncompartments, we were interested in characterizing plantPCB proteins of the ER and to determine whether theycontribute to the lability of heterologous proteins in theER.

Here we present the molecular cloning and the functionalanalysis of the tobacco homolog of BiP. In contrast to themammalian or yeast counterpart, tobacco BiP is encodedby a multigene family. At least one of the encoded proteinsis able to complement a mutation in the yeast BiP genewhen synthesized under the control of a constitutive yeastpromoter.

RESULTS

Amplification of a Tobacco BiP Transcript

The cloning strategy was based on specific amplificationof transcripts by the polymerase chain reaction (PCR) toavoid cloning of the closely related hsp70 proteins. Basedon the sequences of mammalian BiP genes, a 5' p.'imerwas selected in the region encoding the carboxy terminus,which appeared to be conserved between BiP proteins butdiverged from that of hsp70 proteins. Reversed transcrip-tion of RNA and the subsequent amplification reactionswere done with a random primer. As starting material, totalRNA isolated from tobacco leaf protoplasts either culti-vated under standard conditions, treated with tunicamycin,or exposed to heat shock was used. Tunicamycin inhibitsW-linked glycosylation (Elbein, 1987), which results in theaccumulation of malfolded proteins in the ER and theinduction of elevated BiP mRNA levels in mammalian cells(Lee, 1987). In contrast, heat shock does not influenceexpression of mammalian BiP. We assumed that similarpatterns of gene expression would exist in plant cells.

Using RNA extracted from tunicamycin-treated proto-plasts, three amplification products were visualized onethidium bromide-stained agarose gels. After a secondround of amplification, similar fragments could be detectedusing the other RNA preparations as well. These resultssuggested that the amplification products originated fromtranscripts that are more abundant in cells treated withtunicamycin. Sequence analysis revealed that the threefragments were derived from the same transcript. All frag-ments contained an open reading frame extending into the5' primer. The deduced amino acid sequence showed highsimilarity with mammalian and yeast BiP sequences. In

addition, the open reading frame of the largest fragment(387 bp) ends with a sequence encoding the tetrapeptideHDEL, which is identical to the signal responsible for theretention of BiP and other soluble reticuloplasmins in yeast(Pelham et al., 1988).

RNA gel blot analysis using the largest amplified frag-ment as probe revealed a 2.5-kb transcript in plant tissues,which is similar to the size of known BiP transcripts, asshown in Figure 1A. The level of hybridizing transcripts ishigher in stigma tissue than in leaves of tobacco plants.Because the stigma contains a large proportion of secre-tory tissue (Kandasamy et al., 1990), it is expected tocontain elevated levels of BiP compared with leaf cells.

To localize the corresponding gene product in the plantcell, a polyclonal antiserum was raised against a fusionprotein consisting of phosphinothricin acetyl transferase(PAT) and the polypeptide encoded by the open readingframe on the amplified fragment. Tobacco leaf protoplastswere disrupted and separated into a microsomal fractionincluding ER-derived vesicles (lane M in Figure 1B) andinto a cytoplasmic fraction (lane C). The antibodies de-tected several proteins with an apparent molecular massranging from 75 to 80 kD on denaturing gels, whichconfirms the RNA gel blot analysis (Figure 1A). The factthat proteins with identical mobilities copurified with themicrosome fraction suggested that at least some, if notall, of the corresponding gene products are present in theendomembrane system. Proteins with similar mobilitieswere also detected in stem extracts from tobacco as wellas from tomato and potato, suggesting that the corre-sponding gene is highly conserved among solanaceousplants.

A L St B M C TO TM PO

-2.1.— 972

— 66.2

Figure 1. Analysis of Gene Products Corresponding to theAmplified DNA Fragment.

(A) RNA gel blot of 20 ^9 of total RNA prepared from tobaccoleaves (L) and stigmas and styles (St) using a multiprime probeprepared from the largest amplified DNA fragment. The sizemarker (given in kilobases) is indicated by an arrow.(B) Protein gel blot of microsomal fractions (M) and cytoplasm (C)of tobacco protoplasts and of total stem extracts from tobacco(TO), tomato (TM), and potato (PO). Size markers (given in kilo-daltons) are indicated by arrows.

Luminal Binding Protein 1027

Taken together, these data strongly indicated that the amplified DNA fragment originated from a transcript that encodes a BiP-like gene product.

cDNA Cloning and Structural Analysis

To isolate full-length clones of the corresponding gene, the amplified DNA fragment was used as a probe to screen a cDNA library prepared from poly(A)' RNA from styles and stigmas of tobacco plants. These tissues were chosen because elevated levels of transcripts hybridizing to the probe were found in RNA gel blot analysis (Figure IA). Eight different clones were isolated and sequenced (BLP1-8); six of these clones are clearly distinct from each other at the nucleotide sequence level.

The clones were grouped into three different classes based on the comparison between the 3' untranslated ends of the respective cDNA clones. Class I consists of BLPl (1117 bp), BLP2 (1092), and BLP3 (807). BLPl and 8LP2 hybridized most strongly with the probe, and 8LP2 contains a region whose sequence is identical to the amplified DNA fragment. Class II (BLP5, 2273 bp) and class 111 (BLP4, 2420 bp and BLP8, 1082 bp) hybridize only weakly. The nucleotide sequences of the different clones are available from the EMBL database with the Accession Nos. X60057 to X60062.

The sequences of a representative clone for each class are shown in Figure 2A. Different classes contain com- pletely unrelated sequences in the 3' and 5' untranslated regions. The coding regions appear to be more conserved between the three classes. The majority of the nucleotide substitutions do not alter the encoded amino acids, and most of the alterations do not change the charge or hydrophobicity of the residue (see legend of Figure 2). Within a class, significant homology between the 3' ends can be observed. Figure 2B shows an alignment of a region within the different clones of class I. Most sequence differences are present in the 3' untranslated end. BLP3 also contains a high degree of sequence divergence within its coding region compared with the other two clones. These data do not allow us to determine whether the different clones are allelic or not. However, the presence of at least six different mRNAs in tobacco demonstrates the existence of a gene family that cannot be explained by the amphidiploidy of this plant species.

The proteins encoded by BLP4 and BLP5 contain a typical signal peptide for translocation through the ER membrane (Von Heijne, 1986) and the C-terminal tetrapeptide HDEL, which may serve as a retention signal for soluble reticuloplasmins in tobacco. In Figure 3, a comparison of the amino acid sequences of one of the members of tobacco BiP (BLP4), a plant heat shock protein (petunia hsp70, Winter et al., 1988), hamster BiP (Ting et al., 1987), and yeast BiP (Normington et al., 1989; Rose et al., 1989) shows that tobacco BiP is most related to mammalian BiP.

The alignment of the amino acid sequences also illustrates that the 5' primer used for the amplification was chosen in a region that is highly conserved between tobacco and mammalian BiP, but diverges from plant hsp70. The signal sequence and the carboxy-terminal part preceding the HDEL signal are the most divergent portions of the protein sequence. Both the signal sequence and the HDEL tetrapeptide are lacking in the case of the petunia hsp70. The alignment also shows that the polypeptide encoded by the PCR fragment covered the most divergent portion of both BiP and hsp70 proteins. The antibodies raised against this polypeptide are, therefore, unlikely to show detectable affinity for other members of the hsp7O family.

Analysis of the amino acid sequences encoded by the open reading frames allowed us to predict the signal peptide processing sites for BLP4 and BLP5. For example, two putative processing sites for BLP4 were predicted and are indicated in Figure 3. Amino acid substitutions in the close environment of these sites, like the replacement of the threonine (position 30) residue in BLP4 by a lysine in BLP5, could cause a shift in the cleavage preference. It remains to be shown whether different processing sites contribute to the multiple bands detected in the protein gel blot analysis.

A remarkable difference between tobacco BiP and the mammalian or the yeast homolog is the presence of a consensus sequence for N-linked glycosylation in the C-terminal region of the protein. This site is also present at the same location in the putative Plasmodium falciparum BiP (Peterson et al., 1988). However, we could not observe binding of the tobacco protein to concanavalin A, and incubation of plant cells with tunicamycin did not lead to the accumulation of lower molecular weight forms (J. Denecke, unpublished results). The possibility that the multiple bands detected in Figure 1B correspond to either glycosylated or unglycosylated forms is, therefore, unlikely.

Figure 4 shows an alignment of the C-terminal amino acid sequences encoded by the isolated cDNA clones. A variable region containing small deletions and amino acid substitutions precedes the HDEL tetrapeptide. This inter- genic variation contrasts with the high degree of conservation found over the whole part of the protein sequence. Amino acid differences within this region could account for the minor variations in the electrophoretic mobility of the different BiP family members (Figure 1 B).

The size of the tobacco gene family encoding BiP-like proteins might also be reflected by the number of bands that hybridize on a genomic DNA gel blot. Unfortunately, the C-terminal part of the coding regions or the 3' untranslated ends do not cross-hybridize sufficiently to obtain detectable bands in genomic DNA gel analysis (see Figure 2). Probes that cross-hybridize with all the clones also cover regions that may be homologous to other members of the hsp70 family. With a fragment of BLP5 covering the region that encodes the signal peptide and part of the

1028 The Plant Cell

n GAAGAGA GGCGAAGGAGGAGTTGTTAGCCTGTGTTT AAAGAARGTTGATAAGTGGAACTGGTTCAGATCGTGGAGATCGTGGAGCAA~GC~~GTGCGTGGMTAGACGC~AT . . . . . . . . . . . . . . . Q . . . . . . O.. .

109 CCTTGATCGTCTTCOOCATCGTCTTGTTCGGGTCCTTGTTCGCAT~TCTATAGCCAC~AAGAAGCTACC~G~GGG~CAGTTATTGGAATAGATCTTGG~CAACTTACTCATG~TTGGT 115 . . . . . . . . . . T . . . . CA . . . . . . C........A......................T.M.................A..............T.................................

A TTCCA. . . . . . .AACGATTCAAC .AC . . . .ATATT. . .CAG. .GCA.. . . . .TGT. . . . . . A...GTCCT.GTG..C.TAC....T. Lil I

234 GmACAAGAATGGACATGTTGAAATTATATAGCGAATGACCAAGGTAATCGTATCACCCCTTCATGGGTTGCCTTCACTGATGGTGAGAGGCTGATTGGTGAGGCAGC~GAAT~AGCCGCTGT 2 4 0 . . C . . . . . . . . C..............C.....A..C...........C....................A................................A.....T..A...C...T..... 3 5 9 TAACCCAGAGAGGACCQTCTTCGATGTCAAARGACTTATTGGAAGAARG~TGATGACk4AGAAGT.~CAARGGGACA~CTTGTTCCGTAC~TTGTTAACAAGGATGGT~CCATATA 3 6 5 . . . T . . ......................................................................................................................

484 TCCAAGTTAAGATTAAAGATGGGGAGACCAAGA~TTCAGTCCTGAGGAGATCAGTGCAATGATCCTGACCAAGATGAAGGAARCAGCTGAAGCTTACCTCGG~G~TCAAGGATGCAGTT I

490 . . . . . . . . . . . . . C . . . . . . . . . . . . . . T...Q.........................T.....T..............A........A...........T........................ 609 GTCACTGTTCCAGCATACTTTAACGATGCCCAGAGGCAAGCCACTAAGGATGCAGGTGTAATCGCTGGACTAARTGTGGCCAGAATTATTAATGAACCAACTGCAGCAGC~TTGCCTATGGATT 615 . . . . . A . . . . . . . . . . . . . . C..T..............G..............C.....T..T.....TT.G..C.....A.....C.....C..............T..C..............

734 AGATAAGAAAGATCRGGTGGGTGGTG~GAACATCCTTGTCTTTTGACC~GGTGGTGGTACATTTGATGTTAGCATCCTCACCATTGATAATGGTGTTTTTGAAGTTCTT~CACk4ATGGAGACAC~ACC 7 4 0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T........T.....C..C...........G......Q................... T.

a 5 9 TGGGAGGAGAGGACT~TGACCAGAGGATTATGGAGTACTTGCTGAGCGT 865 .A . . . . . . . . . . . T...........A..........................A...........T........C...........T........T.....A.....C..G........C......

984 GCCAAGAGAGCTTTGAGCAGTCAACACCAAGTTAGGG~GAGATTGAATCTCTTTTCGATGGTGTGGATTTCTCTGAACCACTTACTCGGGCACGT7TTGAGGAGCT~CAATGA~ATTCAG 9 9 0 . . . . . . . . . . . AC . . . . . . . . . . G . . T.....C....................C...........A..........................T..............G.....C...........

1109 k4AGACAATGGGACCTGTTAGAAGGCTATGOA?\jATGCTGGTCTGGAG~CCAGAT~ATG~TTGTCCTTGTTGGTGGAAGTACCAGGATTCC~G~CAACAGCTTTTGAAGGATT 1115 G . . . . . . . . . . . T....................Q........A..A..A..G.h.........C........TT.G.......................T....................A.... BLPZ T . . . . . G........CC....... C. 1234 AmTGATGGCRAGGAGCCCAACAAGGGTGTCAACCCTGATGAAGCAGTTGC~ATGGTGCAGCCGTAC~GGAGGAATT~GAGTGGCGAGGGAGGTGATG~CC~GATATTCTTCTTCTG 1240 . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . ........................................................................................

27 . . . . . . . C . . G....................A..T........G..........T......T..T..T.....T..T..CC.T..C..A.....T..A........T..............C...

1 3 5 9 GATGTGGCTCCACT17ACTCTTGGTATTGAAACTGTTGTTGGTGGAGTGATGACTAAGCTGA~CCAAG~CACTGTTAT~CTACT~GTCTCAAGTCTTCACCACTTATCAGGATCAGCAGAC 1365 . . . . . T . . . . . . . . ............................................................................................................... 152 . . . . . T . . . . . T.....C....................G..T..C.....C..AT....T..T..G.....C..C..C..A..C..G.....C.................C..A..C........

1484 AACAGTGhCCI\RTCCAOGTCTTrCAAGGTGAACGCRGTG~CGCAGTCTCAC~GGACTGCAGACTGCTGGGGAAGT~GATTTACCGGk4TAGCTCCAGCTCC~GAGGAACTCCTC~TCGAGGTTACAT 1 4 9 0 . . . . . . C... . . Trr. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C.G . . A . . T.....A..C..C.....T...................................T..A.......

2 7 1 C . . . . . C T . C . . T... . . T............A.G.....T..T........T..G.....T..A.........C.G..A.....T..C..G........G.....C..A.....T.....C....

1 6 0 9 TTGAAGTTGATGCIL9ATGGTA~CTAAACGTCAAAGATCRGGTGGCTGAAGACAAR~C~~G~TCAGAGAAGATCAC~TCACCAACGACk4AGGTCGC~GAGTCAAGAAGAGATTGAGCGTATGGTC 1615 . . . . G . . . . . . . . C..C.....C..G..T..G...........T..............G..............C..T.....T.....G....................A.....A.........

. . . . . . . . . . . . . C . . . . . . . . . T.G . . T..T.....A........G.GAA....T.....C........T..C..T..A........G..C.....A..C....................... T

1734 AA0'3AGGCTGAGGAGTTTCAGAGGAGGATAAGk4AGTAAAAGk4AGAATTGATGCCAG~CAGCCTGGAGACATATGTGTAC~TATG~AACC~Tk4ATGACAGGACAAGCTTGCAGA 1740 ........ C . . . . . . . . . . . T........C.....G..G..............C...C................T..C..A.....C..............C..............A........

5 2 7 COT . . A..A..A........T.....A..C....I..G..G..G..G.........C.T.....T..T..A..C..C........C....M.....G..C..C..............A..T..

1859 CAAGCTAGAGTCTGATGAGkGAGAAGATTG~~CCGCGAC~GAAGCGCTTGAATGGTTGGA~GACACCAGAGTGC~AGAAGGAAGATTA~~GCTGk4AGAAGTGGAAGCTG

. . . . . . G . . . . . A........G..............A..A...........C..C......C.A...........A..C..C.....A..G.....C..A..G.....T.....G..A..G.. A.

4 0 2

1865 . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1984 TGTGCAACCC.~ATAATCACTGCGGTGTATCAG~TCTGGTGGAGCACCAGGAGGA GAA'ITMOTCCTAQCGAGAT CATGACGAGCTGTAGATGATTGCTAGTTTACAAGTT

652 n

1 9 9 0 . . . . . . . . . . . . . T . . . . . G..T..........GA...........C........?WCAOCOAI).MT.C.A.............rrA..............ATA.... TGTAGG.TAT . . . . 7 1 7 .C . . . . . . . . . . . T.....A..T..T.....A.OA..A........C.....T..?mlrr...Q.M.AnaAA..A..A..A...occ.....T........ A G

Y

2103 TTCTGTTATCACAAGTGATAGGATACACAGCCTTTTCT CATAT~~TTTTCCTGTTGACGGTGGCTATCAG~TAGATT~GATGA~AGG~~GACGTTT~~CTAAATTGATTAATTAAGGACGG 2114 . . . . . . . GC . . A.TAGACATAC.AWTTGAT.A . . . . . TTG-G . . . . . . . . CCA.A . . GAG.GTGG.ACG . . GA.CG.CTC . . . . . . AA.CGTGGGACG.... .T..C

aa2 C.A.TC.GAA.A.TC.CGATT . . C..TG.T.AAAAGA.G..AGA...GAGGA.A.A..ATAG.TTG~GT..GGGTT.C.GCA.ATTTTGTC..TTGT.AAAGC.CGAG.GAAG.GGTGTCC.TT

2227 CGAAAGAACCTTCCTCTCGTTCATTCTT GGAAGACATGTATAGAGACATTCATCAATTTAGAGTTGGAACTTTTGTGTTCTTTCAGATATGAGATTAAAAT 2 2 2 2 TCTGGT.T G T.......T.. TTCGT C G . A A 1007 . T T C G C T T A G . . T T . T . A . G . G . A G A . . T T C G T G G A A C A A T G T T T C C T T T C T T . G . T G T T G C . T A . P . . A G

. . . . . . . . . . . . . . . . . . . . . .

B BLp3 B L P l

BLPZ

120

1 2 0

126

CCcaATCAtcaCaGcTgtTtAtcaAagAtcaGGcGyagCctcaGGTGGTTCATC atcAtcAGAAGAAGAtGGaCATGATGAaCTGTAgGtTtaaCTtAAcAcT gCCATTGACACcaCT

MTTATCACAGCTGTTTATCAGATCAGGTGGAGCCCCAGGTGGTGGTTCGTCG GAAGAAG.4AGAAGAcGGCCATGATGAGCTGTMGCTATTCTG~TC~CATTGACACTGCT

AATTATCACAGCT'j~ATCAAAGATCRGGTGGATCAGGTGGAGCCCCAGGTGGTGGTTCATCGgaaga~GAAGAAGAAGAA~AtGGCCATGATGAGCTGTMGCTATTCTG~TCTCgATTGACACTGaT

/ / / I 1 1 1 1 1 I I I I I I l l l l l l l l l l l I I I I I I I I I I I I I I I I I I I I I I I I I I I / I I I I I l l l l l l l l l l I1

l l l l l l l I / i l l l / 1 1 l / l l l l l l i l l l l i l l ~ l i / l l l l l l l I / l l l l / l l l l l l i l l l l l l l l l l l l / l l / l l I / l I l l l l l I i l l l l l l l l l l l I / / l l l l l l l i l l l l l i l I

GAAAAGAgGCP.aaACATGAGGATAcAGgATAGaccaAGacGA GtTTAGTGtAAGTTgcaagttTTGTcAttGcttaAGTaCGa~g~a~gaggTATcc~TtcgctTgyTCttTtGAggtgAaaaT

GAAAAGATGCAcGAAATGAGGATATAGAATAGGTTGAGGTGAGGG~'AGTGcAAGTTTTGTcAATTGTT~GCACGAGTtCGCttAGtTtTtTATaggTgaaGaTtTTC GTgGA aCaAtGTT I I I I I I I I I I I l l l l l l l l l / I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I / I 1 I I I1 I I I I I

l l l l l l l l l l l / 1 I I I I / / / / l / I l l 1 I I I l I I / I l / I l 1 I I / 1 1 1 I I I I I I l l l l l l l l l l l l l l l l l l l l l l l l I I I I I I I I I I I I I 1 1 G ~ G A T G C A a G A A A T G A G G A T A T A G A A T A G G T G c A c G A G gGaagAGgTgTcccT tCT tCG CTT aGT tttCttAyGTg

Figure 2. DNA Sequence of a Representative Clone of Three Tobacco BiP Classes.

(A) An alignment of the nucleotide sequences of BLP2, BLP4, and BLP5 is shown. ldentical nucleotides are represented as dots. Nonconserved codons leading to amino acid transitions are indicated in boldface letters. The first and last ccdon are boxed. The arrows indicate the margins of the largest DNA fragment amplified by the PCR. The bars define the margins of the Aval1 fragment of BLP5 that was used as a probe for DNA gel blot analysis (see Figure 5). Note the sequence divergence in the 3' and 5' untranslated regions. (6) An alignment according to the Needleman-Wunsch algorithm (Intelligenetics, Mountainview, CA) of a region around the stop codon of class I clones, showing the high sequence divergence of the 3' untranslated ends. The stop codons are indicated in boldface letters.


PEBS M B i

v KGE.PA.. ........... .WQHDR.. . . . . . . . . .T.. .Y.G.. MAGGAWNRRTSLIVFGIVLFGCLFAFSIATEEATKLGTVIGIDLGTTYSCVGWKNGHVEI IANDQGNRITPSWVAFT

.T ..... D....QV.M..IN....A...... R . LZERLIGEAAKNLPAVNPERTVFDVKRLIGRKF .~__

IuBi MKFPMVAAAL LL.CAVRAEEEDKK.DV .. V . . . . . . . . . . . . . F...R...............Y....PE......D....QLTS...N....A... .. ,'Iw

S.PS .. S.1 . . W.F.VIPGP.DKPM1 .VTYK . . E.Q . A A . . . . S.V . . . . . . I......TT..N...........S................M..................ASSA....V.I...

125 N.RS..K.I.HL.F NV . . . . . . . AVEVSVK . . K.V.T . . . . . G...G...QI..D...T.V.H... .................................... 5DK.HQ.I.Y..

SCBI MFFNRLSAGKLLVPLSWLYALFW.LPLQNS.HSSNVLVRGADDVENY . . . . . . . . . . . A.M ...KT..L..E........Y.... .D .....D.... QV.A ..?N.I..I..... L.Y

82 112 DDKEVQRDMKLVPYKIVNKDKPYIQVKIK~~~ETKIFSPEEIS~ILTKMKETAEAYLGKKIKDA~PAYFNDAQRQATKDA~V~AG~~IINEPTAAAIAYGLDKK GGEKNILVFDL 104 ...........T......M................. R E . . . . . . . . . . N.PS . . Q.I.FL.F.V.E.KT . . . . . . D.GG.Q . . T.A ....... V . . . . . . . . . . . . . . . V.H . . .

207 ..TA .TTI . . D..YE.I..YSTI.........M...CE..E.CLR.. K 234 GGGTFDVSILTIDNGVFEVLSTNGDTHLGGEDFW2RIMEYFIKLIKKKHGKDISKDNRALGKLRREAER~RALSSQHQVRVEIESLF~V PLTRARFEELNNDLFRKTMGPvKKAMDDAG 226 T ....K.....M....S..K..Q.VL E.SD 246 T ....K.....L...K..LK..E.~ .S.

........ L ... EE.1 . . . KA.A . . . . . . . . . . . N.~H.V?EF.R.NK....GNP...RR.. TAC

. . . . . . . . L . . . . . . . . . . VA . . . . . . . . . . . . . . . V..H.... Y. ........ L.S,E . . . . . . QA.S . . . . . . . . . . . YK.VRQL..AF. .. VR . . . . . VQ . . . . . V.K . . . . . . . . . . A.I....F . E.E 1.V.DN.K . . A . . K....K........MST.I..D.FV.. I

332 359 351 371

... Q.F.N ...LC. SI . . . . . . . . . . . . . A A . . . . . . NEKVQ.L . . . . . T .. S..L..A..G..V......T.....E...S..S.N.PG.L...Y.. ..AR. LEKTQIDEIVLVGGSTRIPKVQQLLKDYFM;KEPNKGVNPDEAVAYGPAV~GILSGEGGDETKDILLLDVAPLTLGIETVGG~KLIPRNTVIPTKKSQVFTTYQDQQTTVTIQVFEGERSLT .K.SD . . . . . . . . . . . . . . . I...V.EF.N....SR.I.............A.V... ~ D . G . L V . . . . C ...................... V......I.S.AS.N.P....K.Y....P.. . . . KDV.D . . . . . . . . . . . . . . . . . . ES. . . . . KAS..I...... . . . . . . . A.V. . . EEGVE . . V . . . . NA . . . . . . . T.....P..K...A.......I.S.AV.N.P..M.K.Y.... AMS

457 . . N N . . . . . E.S . . P . . . . . V...T.C.Dl........S....TT.QKN............K.......Q...KYKS..EEL.KKVE.K.A..N.A..... TIKD,.INSQ.SRAD.KR..D.I

. . NH . . . T . . . . . . P.....V........I.V....R.T....GT.NKN.......QN...P.......ND..)(......;L.....T..E..S.A.SL....G..E..GG..S.ED..TM. K.V 494 . . N N . . . . . E. . . . P . . . . . V.......AL......K.S.T..GT....S...............D...E...K..S..ASI.AKVES..K..N.AHSL... VNGD.GE ... EED..TLLD.A 4 8 4 KDCRLLGKFDLTGIAPAPFGTPQIEVTFEVDANGILNVKAEDKASGKSEKITITNDKGRLSQEEIERENKEAEEFAEEDKKVKERIDARNSLETYWNMRNQINDKDK~DKLESDEKEKIETAT 474

581 609 KEALEWLDDN QSAEKEDYEEKLKEVEAVCNPIITAVYQ KSGGA PGGE SGASEDDDEDEL 599 E.KI...ESH .D.DI..FKA.K..L.EIVQ...SKL. GSA. PP.T. .ED T.. K.. . 617 NDV.... ... FET.IA..FD..FESLSK.AY..TSKL. G . . LZSGRADYDDEDEDDDGDYFE....

D..IK...N. .L . . ADEF.D.M . . L.SI . . . . . AKM . . GGA . . . TMDEDGPSV..SAGSQTGAGPK I.EVD

Figure 3. Comparison of Tobacco BiP with Related Proteins.

The figure shows an alignment of several proteins: PEHS, petunia HSP70; TOBI, tobacco BiP (BLP4); HABI, hamster BiP; SCBI, S. cerevisiae BiP. Amino acids that are identical to the tobacco BiP sequence are indicated by dots. The region that was used for priming in the DNA amplification reactions is underlined. The consensus site for N-linked glycosylation and the signal for retention in the ER are presented in boldface letters. The arrows refer to potential signal peptide cleavage sites as determined by computer analysis (Intelligenetics).

mature protein (indicated in Figure 2A), different cross- hybridizing bands were observed in Figure 5. The presence of the signal peptide encoding region is unique to BiP within the superfamily of hsp70. However, it cannot be excluded that some of the bands might correspond to other members of the hsp70 family. The exact number of BiP genes in tobacco, therefore, cannot be determined by this kind of analysis and remains to be elucidated.

Organ-Specific and Stress-Related Expression Patterns

To determine whether the tobacco BiP genes are expressed in a tissue-specific manner, we extracted RNA from different tissues covering several developmental stages of the plant. The 3' untranslated end of BLP2 was used as a probe in RNA gel blot analysis. Transcripts hybridizing to the probe were almost undetectable in roots, of low abundance in leaves and sepals, somewhat elevated in stems and petals and abundant in flower tissues and both immature and germinating seeds, as shown in Figure 6A. We have also observed that flower tissues and germinating seeds contain an elevated amount of ER membranes and reticuloplasmins compared with leaf tissue (J. Denecke, unpublished results). This suggests that the abundance of BiP transcripts is related to the amount of

ER in nonstressed cells. BiP mRNA levels appear thus to be more abundant in tissues containing large amounts of ER membranes. Germinating seeds 4 days (G4) after imbibition had the highest BiP mRNA levels of all tissues analyzed. This coincides with a developmental stage that is predominated by meristematic cells. The BiP mRNA levels decreased rapidly as root elongation proceeds (G5 and G6).

In mammalian cells, BiP mRNA accumulates upon treatment with the calcium ionophore A231 87 and tunicamycin. Calcium ionophores are believed to cause transient loss

BLP3 PIITAWQRSGGA sGGSS ssEEDGHDEL l l1 l l11111111 I I I I I I I I I I I I

BLPl PIITAVYQRSGGAPGGGSS EEEEDGHDEL I l l l l 1 1 1 1 1 l l 1 1 l l 1 1 1 I I I I I I I I I I

BLP2 PIITAVYQRSGGAPGGGSSEeEEEEDGHDEL I I I I l l 1 1 1 1 1 l l 1 1 1 I I I I I I I I

BLP5 PIITAWQRSGGAPGGaSeESnEDDDsHDEL I I I I I I I I I I I I I I I I I I I I I I I I I

BLPI PIITAVYQkSGGAPGGeSgaS EDDD HDEL

Figure 4. Comparison of Different Tobacco BiP C Termini.

An alignment of the C-terminal amino acid sequences of BLPl , BLP2, BLP3 (class I), BLP4 (class II), and BLP5 (class 111) according to the Needleman-Wunsch algorithm (Intelligenetics) is shown.

1030 The Plant Cell

Figure 5. DMA Gel Blot Analysis of Genomic Tobacco DMA.

Genomic tobacco DMA was cut with either Hindlll or EcoRI. Amultiprime probe prepared from a BLP5 fragment (indicated inFigure 2) was used for the hybridizations. Arrows indicate bandsthat hybridize above background.

of reticuloplasmins by perturbing a higher order structurein the ER (Booth and Koch, 1989), whereas tunicamycinwould cause the accumulation of malfolded proteins in theER (Lee, 1987). Both conditions would require enhancedBiP synthesis to compensate for the loss of free luminalBiP by either secretion or sequestration by malfoldedproteins. Similar observations were made in yeast, but incontrast to mammalian cells, yeast BiP is also inducedduring heat shock (Normington et al., 1989; Rose et al.,1989). In contrast, tobacco BiP mRNA levels were onlyelevated upon treatment of the tissues with tunicamycinand not by extreme temperatures or treatment with thecalcium ionophore A23187 (Figure 6B). Preliminary exper-iments using probes from 3' untranslated ends of the othercDNA clones showed expression patterns similar to thoseshown in Figure 6 (J. Denecke and M.H. Souza Goldman,unpublished results). However, it should be noted that thedifferent probes cannot be regarded as gene specific be-cause we do not know whether all the BiP genes arerepresented within the three cloned classes. A completeanalysis of gene-specific expression profiles can only beperformed when all the members of the tobacco BiP familyare cloned and sequenced.

Tobacco BiP Complements a Saccharomycescerevisiae BiP Mutation

To prove the functionality of the cloned tobacco BiP geneproducts, we determined whether the plant gene couldcomplement for the homologous gene (kar2) in the yeastS. cerevisiae. Mammalian BiP was shown to complementkar2 mutations in yeast, even when its original KDELretention signal was not replaced by the yeast signal(HDEL) (Normington et al., 1989; Rose et al., 1989). Thissuggests that even relatively small amounts of BiP canrestore the kar2 deficiency because KDEL was shown tobe an ineffective retention signal in yeast (Pelham et al.,1988). We used a temperature-sensitive kar2 mutant(Polaina, 1980) for the complementation experiments. Thisstrain is unable to grow at 37°C, whereas no aberrantgrowth phenotype is observed at the permissive temper-ature, 28°C. A chimeric gene containing the pyruvatedecarboxylase promoter of yeast (Kellermann et al., 1986)fused to the coding region of BLP4 was inserted into anintegrative plasmid (R. Contreras, unpublished results), asshown in Figure 7A, containing a dominant marker (Zhu etal., 1986) and aTy1 -derived sequence (Ciriacy and William-son, 1981). Tobacco BiP containing its own signal peptidewas synthesized and processed in the transformed yeaststrains, as shown by protein gel blot analysis (Figure 7B).Several bands with higher mobilities were detected aswell, which most likely represent degradation products.The presence of the tobacco gene product enabled themutant strain to grow at both 28°C and 37°C, albeit moreslowly at 37°C (Figure 7C). The latter can be explained bythe fact that tobacco BiP appeared to be degraded rapidlyin yeast grown at 37°C, resulting in almost undetectablelevels of the full-size gene product and increased amountsof degradation products (Figure 7D). We conclude thattobacco BiP is able to substitute for the temperature-sensitive kar2 gene product, but that complementation isimpaired by the instability of the tobacco protein in yeastat the nonpermissive temperature.

DISCUSSION

This paper describes the cDNA cloning and the functionalanalysis of tobacco BiP. Unlike the mammalian and yeasthomologs, tobacco BiP is encoded by a multigene family.At least one member of this gene family was able tocomplement a mutation in the yeast BiP gene. The differentmembers may represent isoforms that accomplish similarfunctions but contain marginal differences in substratespecificity or expression patterns. Some of the clonesmight be allelic, especially within the different classes. Thepresence of a gene family can be partially explained by theamphidiploidy of tobacco (Akehurst, 1981). However, the


RO SM LE 5E PE AN SI 0V SD Gi G5 G6

CO HS CS CA TUB

Figure 6. RNA Gel Blot Analysis.

(A) BiP mRNA levels in different tobacco tissues. Twenty micro-grams of total RNA was loaded in each lane and hybridized witha riboprobe prepared from the 3' untranslated region of BLP2.RO, roots; SM, stems; LE, leaves; SE, sepals; PE, petals; AN,anthers; ST, styles and stigmas; OV, ovaries; SD, seeds (allstages); G4, G5, G6, germinating seeds 4 to 6 days after imbibitionstart.(B) BiP mRNA levels in response to different stimuli in tobaccoseedlings (5 days after imbibition start). Hybridization conditionsare as in (A). CO, control; HS, heat shock (42°C for 3 hr); CS,cold shock (4°C for 16 hr); CA, treatment with calcium ionophoresA23187 (5 MM for 16 hr); TU, treatment with tunicamycin (20 ng/mLfor 16 hr).

occurrence of six distinct BiP-related messages demon-strates that at least one of the parental plants contains aBiP gene family. The detection of multiple bands on proteingel blots from extracts of other solanaceous plants sup-ports, but does not prove, this view. It will be interestingto analyze BiP genes in other plant species. Recently, thecloning and characterization of a partial cDNA clone en-coding an hsp70-related protein b-70 from maize wasreported (Fontes et al., 1991). b-70 is associated with theER and the protein bodies and is highly overproduced inthe endosperm of f!2 mutants that have reduced zeinsynthesis. The open reading frame encodes a C-terminalHDEL peptide, and the predicted amino acid sequence is93% identical to the corresponding region in BLP4. Twodifferent forms of b-70 can be separated (Boston et al.,1991; Fontes etal., 1991; Marocco et al., 1991). This mayalso reflect the presence of a BiP gene family in this plantspecies.

It has been proposed that BiP genes share a commonancestor that diverged from other hsp70 genes near thetime when eukaryotes first appeared (Nicholson et al.,1990). BiP genes might have evolved independently fromthe other members of the hsp70 family after duplication of

an ancestral hsp70 gene. Our alignment shows that to-bacco BiP is more closely related to mammalian BiP thanto S. cerew's/ae BiP, although it contains the carboxy-terminal sequence HDEL rather than KDEL. The highsimilarity of the different cloned members of the tobaccoBiP family suggests an identical function. However, thefunctionality of the other BiP members remains to bedemonstrated.

All the cloned members of the tobacco BiP gene familycontain the C-terminal tetrapeptide HDEL. This sequenceis preceded by a variable region, in contrast to the highsimilarity found over the remainder of the protein. TheHDEL sequence is shown to be responsible and mostlikely sufficient for the retention of soluble reticuloplasminsin yeast (Pelham et al., 1988). The presence of a variable

A pDC1

ORF4

Q 1 2 3 4 5 PL

B

28°C 37°CUT C T T PL

Figure 7. Complementation of a Mutation in the S. cerew's/ae BiPGene.

(A) Schematic representation of the chimeric gene and the vector.pDC1, pyruvate decarboxylase promoter from S. cerew's/ae;ORF4, open reading frame of BLP4; ORI, E. coli origin of replica-tion; Amp, ampicillin resistance; TY, transposable yeast element;met", chimeric dihydrofolate reductase gene conferring metho-trexate resistance; cycT, yeast cytochrome c terminator.(B) Protein gel blot analysis of total yeast protein from independentyeast strains containing genomic integrations of the plasmidpUDHIL208 with (lanes 1 to 5) and without insert (-). The poly-clonal PAT-BiP antiserum was used for detection. The yeastswere grown at 28°C. PL, plant BiP from tobacco stem extracts,used as size marker.(C) Comparison of the temperature-dependent growth of the kar2mutant. T, transformed with the chimeric BiP gene; C, transformedwith the vector; UT, untransformed. The incubation temperaturesare indicated.(D) Protein gel blot of yeast extracts prepared from liquid cultures.Incubation temperatures and nomenclature are as in (B) and (C).

1032 The Plant Cell

region in front of the HDEL peptide in tobacco BiP supports the view that the targeting information only resides in the last 4 amino acids. However, the putative auxin receptor located in the ER lumen of maize (Hesse et al., 1989; lnohara et al., 1989; Tillman et al., 1989) and the luminal enzyme sulfhydryl endopeptidase of Vigna mungo (Aka- sofu et al., 1989) both contain the mammalian signal (KDEL) for reticuloplasmin retention. This suggests either that different retention signals are used between different plant species or that both signals are functional in plants. Cloning of BiP or other reticuloplasmins from the mentioned species followed by a comparison of the C termini would provide an answer to this question.

Analysis of the expression patterns on the mRNA level suggests that BiP is most abundant in tissues with high proportions of rapidly dividing cells. Meristematic cells secrete large amounts of cell wall proteins following the process of cell division. They also contain large quantities of ER membranes compared with cells from other tissues (J. Denecke, unpublished observations). Relative high BiP mRNA levels are also detected in flower organs that contain secretory tissues. The tapetum cell layer in anthers (Tiwari and Gunning, 1986; Mascarenhas, 1990), the secretory zone in the stigma, and the transmitting tissue in the style (Kandasamy et al., 1990) are evident candidates for such tissues. In stems and in petals, BiP mRNA levels were modest but also clearly elevated in comparison to the nonsecretory leaf tissues. It is possible that a limited number of secretory cells within these organs, similar to the cells associated with phloem cells in stems, contribute largely to the modest increase in the mRNA level. We did not observe significant changes in expression patterns when using probes that correspond to different classes. However, a complete analysis of gene-specific expression profiles requires an exact knowledge about the BiP gene complexity in tobacco. Taken together, these observations support the view that BiP is continuously required in the folding process of de novo synthesized secretory proteins in unstressed cells (Gething et al., 1986). It is expected that larger amounts are needed in cells where the rate of protein translocation through the ER membrane is high.

As in mammals, BiP mRNA levels are elevated in cells treated with tunicamycin, a potent inhibitor of N-linked glycosylation. Underglycosylation of proteins may also cause the accumulation of malfolded proteins in the ER of plant cells, but although this has been demonstrated in mammalian cells (Kozutsumi et al., 1988), it remains to be shown that the presence of malfolded proteins in the ER lumen rather than abnormal glycosylation is the direct inducing principle of BiP expression in plant cells. In contrast to mammalian BiP, no increased mRNA levels were observed upon treatment with the calcium ionophore A231 87. This may reflect the fact that calcium ionophores do not affect the retention of reticuloplasmins in tobacco (J. Denecke, unpublished observations), therefore not re- quiring increases in BiP de novo synthesis to maintain

homeostasis. Heat shock has no influence on the level of BiP mRNA in tobacco, similar to the regulatory pattern observed in mammalian cells, but unlike that in yeast cells (Normington et al., 1989; Rose et al., 1989). Whether this feature reflects the in vivo function of the different BiPs remains to be shown.

Protein translocation and folding in the lumen of the ER is one of the limiting steps in protein secretion. BiP is a key molecule in the protein maturation and transport machinery of the endomembrane system. The analysis of BiP expression patterns in response to severa1 externa1 factors or the presence of heterologous proteins in the plant endosomal system will increase our understanding of protein transport and protein maturation in plants.

METHODS

Plant Culture and Nucleic Acid lsolation

Nicotiana tabacum 'Petit Havana' SR1 plants (Maliga et al., 1973) were grown under standard greenhouse conditions. For RNA isolation, tissues from flowers, roots, stems, leaves, seeds, and germinating seeds were frozen in liquid nitrogen and stored at -70°C. Seeds were germinated in shaking liquid cultures at 24°C in culture medium containing MS (Murashige and Skoog, 1962) salts, 10 g/L glucose, and 0.5 g/L Mes and brought to pH 5.8. Germinating seeds were collected 4,5, and 6 days after imbibition start (G4, G5, G6) and frozen. For the analysis of stress conditions, germinating seeds (G5) were shaken in the same culture medium for 16 hr either under standard conditions, in the presence of tunicamycin (20 pg/mL), in the presence of calcium ionophore A23187 (5 pM), or at 4°C. In the case of heat shock treatment, incubation under standard conditions was performed for 13 hr, followed by an incubation at 42°C for 3 hr. Protoplasts for RNA isolation were prepared (Denecke et al., 1989), cultivated under standard conditions for 24 hr, treated with tunicamycin (20 pg/ mL) for 24 hr, or exposed to heat shock (3 hr at 42°C) after 21 hr of incubation at standard conditions. Total FINA from protoplasts and all plant tissues was extracted essentially as described by Dean et al. (1985) and adapted for protoplasts as described earlier (Denecke et al., 1989). Genomic DNA was prepared from tobacco leaves according to Dellaporta et al. (1983).

cDNA Cloning

One microgram of total FINA with added primers was heated to 50°C and chilled on ice. After adding the polymerase reaction buffer, avian myeloblastosis virus reverse transcriptase, Taq polymerase, and deoxynucleotide triphosphates (according to Goblet et al., 1989), the reaction mixtures were incubated at 42°C for 1 hr. Subsequently, 40 amplification cycles were performed, each consisting of 30 sec at 92"C, 2 min at 50"C, and 2 min at 72°C. The denaturation step at 92°C during the first cycle was prolonged for 5 min, and the elongation step at 72°C was prolonged for 10 min during the last cycle. A secand round of amplifications was done using 5 pL of the first reaction mixture,


which was diluted to a final volume of 50 pL with the above- mentioned reaction mixture without reverse transcriptase. The ends of the amplified DNA fragments were filled in, kinased, and inserted into the Smal site of pGEM3Z (Promega). Plasmid pDE602 contains the largest fragment with the open reading frame oriented downstream of the T7 promoter of pGEM3Z.

Poly(A)+ RNA was purified by chromatography over oligo(dT)- cellulose according to Ausubel et al. (1987). A cDNA library from style and stigma RNA was constructed according to the Amer- sham protocols “cDNA Synthesis System Plus RPNl256Y/Z” and “cDNA Cloning System XgtlO RPN 1257.” For screening, about 10,000 plaque-forming units were plated onto 14-cm plates. Rep- lica nylon filters (Hybond-N, Amersham) were lifted from each of the plates and were treated as recommended by Amersham. Filters were prehybridized overnight in 6 x SSC (1 x SSC is 0.15 M NaCI, 0.015 M sodium citrate), 5 x Denhardt’s solution (1 x Denhardt’s solution is 0.02°/0 Ficoll, 0.02% PVP, 0.02% BSA), 1% SDS, and 100 pg/mL denatured carrier DNA at 68°C. Radioactive labeled DNA fragments corresponding to the amplified fragment in pDE602 were added to the prehybridization solution to a final concentration of 106 cpm/mL, and the incubation was done 24 hr at 68OC. Filters were then washed at the same temperature in 6 x SSC, 0.1% SDS for 30 min and in 2 x SSC, 0.1% SDS for 30 min. A total of 160,000 plaque-forming units were screened. lnserts of the selected clones were isolated by EcoRl digestion and subcloned in the EcoRl site of pGEM1 (Promega).

Nucleic Acid Analysis

Radioactive labeled DNA probes for DNA and RNA gel blot analyses were prepared from gel-purified DNA fragments using a multiprime labeling system (Amersham). A probe for the screening of the cDNA library and the initial RNA gel blot analysis was prepared from the largest amplified fragment, which was directly purified on agarose gels. A BLP2 probe for RNA gel blot analysis (Figure 6) was prepared from an Alul-EcoRI fragment from BLP2. The DNA gel blot analysis (Figure 5) was done with the Aval1 fragment from BLP5, as indicated in Figure 2.

Plasmid Constructions

To overproduce the polypeptide encoded by the amplified DNA fragment in €scherichia coli, the cloned amplified fragment of pDE602 was ligated at its BamHl site to the Bglll site of pGSFRC1 (Botterman et al., 1991), yielding pDE603. The Bglll site in pGSFRC1 immediately precedes the stop codon of the bialaphos resistance gene (bar) coding region, which allows the construction of a hybrid structural gene. The bar gene encoding PAT is under control of the X pR promotor, which directs high-leve1 synthesis of a PAT-BiP fusion protein.

A restriction fragment containing the pyruvate decarboxylase promoter (EC 4.1.1.1., Kellermann et al., 1986) was placed in front of the BLP4 open reading frame, yielding pBLPC1. The open reading frame of BLP4 containing untranslated 3‘ and 5’ ends was obtained as a Dral-BamHI fragment from pBLP4. The chimeric gene was cut out as a Smal-Xbal fragment from pBLPC1 and ligated into pUDHIL208 (provided by R. Contreras, Laboratory of Molecular Biology, Gent, Belgium) that was digested with BamHI, filled in, and further digested with Xbal, thus yielding pBLPC2.

Purification of the PAT-BiP Fusion Protein and Production of Polyclonal Antisera

For overproduction of the PAT-BiP fusion protein, precultures of the strain NFl (Botterman et al., 1991), which contains plasmid pDE603, were grown at 28”C, shifted to 42OC, and grown further for 4 hr. The harvested cells from a 500-mL culture were resuspended in 12.5 mL of lysis buffer (50 mM Tris-HCI, pH 7.2, 10 mM MgCI2, 200 mM KCI, 5% glycerol) with 1.2 mL of lysozyme solution (1 O mg/mL) and left for 1 O min on ice. EDTA was added to a final concentration of 10 mM and the suspension was left for 20 min on ice. Cells were lysed in a French press (1200 p.s.i.) and centrifuged at 16,000 rpm (SS34 rotor, 4OC). The lysate was run over an S2OOHR column (Sephacryl, 2.2 x 1 O0 cm), and DNA in the combined eluate was precipitated with 0.5% streptomycin sulfate. Proteins were precipitated with ammonium sulfate, resuspended, and passed over a phenyl-Superose column (2.2 X 16 cm). PAT-containing fractions were collected and precipitated with 80% ammonium sulfate. The pellet was dissolved in 5 mL of 10 mM Tris-HCI, pH 8.6, 10 mM EDTA and finally run over a Mono Q HR 10/1 O column.

A polyclonal antiserum was obtained from injected rabbits as described by De Clercq et al. (1990). Antisera were tested by protein gel blotting with purified PAT-BiP fusion protein. The detection limit was determined to be 0.2 ng of the fusion protein.

Plant Cell Fractionation and Analysis

Tobacco leaf protoplasts were prepared and cultivated for 24 hr according to Denecke et al. (1989). Cells (10’) were floated in culture medium, concentrated, washed, and pelleted in 250 mM NaCI, resuspended in 5 mL of fractionation buffer (12% sucrose, 100 mM Tris-HCI, pH 7.9, 2 mM MgC12, 5 mM CaCh), and disrupted by passing through a 25-gauge syringe. After removal of cell debris by centrifugation (HB4 rotor, 2000 rpm, 4OC), microsomes were separated from soluble cytoplasmic proteins by 2 hr of centrifugation at 35,000 rpm in an SW50.1 rotor at 4OC. The supernatant was kept (“cytoplasmic” fraction) and the pellet was resuspended by gentle sonication (5 pm amplitude, 1 O sec) in 5 mL of buffer (“microsomal” fraction). Twenty microliters of each fraction was applied on 9% SDS-polyacrylamide gel and analyzed by protein gel blotting using the polyclonal PAT-BiP antiserum.

Five hundred milligrams of tobacco, potato, and tomato stem tissues were ground in 500 pL of PAT extraction buffer (Denecke et al., 1989), and cell debris was removed by 10 min of centrifugation at 14,000 rpm in an Eppendorf centrifuge at 4OC. The protein concentration in the supernatant was brought to 1 mg/ mL, and 20 pL was analyzed as the above-mentioned fractions.

Transformation and Culture of Yeast Strains

The plasmid pBLPC2 was linearized with Clal within the Tyl sequence element. Transformation, selection, and culture of the transformants were done as described by Zhu et al. (1986). The thermosensitive kar2 mutant was YPE1 (a , his4-Al5, ade2-1, canl , nyr ‘, kar2; Polaina, 1980).

1034 The Plant Cell

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939-950.

2250-2254.

Drs. Enno Krebbers and Allan Caplan are thanked for scientific discussion and critically reading the manuscript. Dr. Dulce E. de Oliveira is thanked for useful suggestions concerning the PCR method. Adri Van Vliet is thanked for purifying the PAT-BiP fusion protein and Annemie Van Houtven for preparing the antisera. Dr. Roland Contreras is thanked for providing the plasmid pUDHIL208 and for helpful discussion. Karel Spruyt and Vera Vermaercke are thanked for preparing the illustrations. J.D. is indebted to the Belgian lnstituut ter Aanmoediging van het Wetenschappelijk Onderzoek in de Nijverheid en in de Landbouw for a fellowship. M.H.S.G. is supported by the Brazilian Research Council (CNPq), Fellowship No. 200746/88.0.

Received April25, 1991 ; accepted July 18, 1991

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DOI 10.1105/tpc.3.9.1025 1991;3;1025-1035Plant Cell

J Denecke, M H Goldman, J Demolder, J Seurinck and J BottermanThe tobacco luminal binding protein is encoded by a multigene family.

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