Laiqiang Huang*$, Amie E. FranklinSlI, and Neil E. Hoffman*I(. From the SCarnegie Institution of Washington, Department of Plant Biology, Stanford, California ...
THE JOURNALOF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc
Vol. 268, No. 9, Issue of March 25, pp. 6560-6566,1993 Printed in U.S A .
Primary Structure and Characterization of an Arabidopsis thaliana Calnexin-like Protein* (Received for publication, November 2, 1992)
Laiqiang Huang*$, Amie E. FranklinSlI, andNeil E. Hoffman*I( From the SCarnegie Institution of Washington, Department of Plant Biology, Stanford, California 94305 and the llDepartment of Biological Sciences, Stanford University, Stanford, California 94305
A cDNA clone(pTE-83)encoding a protein (CNXlp) Possible functions for calnexin have been suggested by a related to the microsomal Ca’+-binding protein, cal- number of recent studies. Wada et al. (1) coimmunoprecipinexin, wasisolated froman Arabidopsisthdiana tated and copurified calnexin in a complex with three other expression library. Southern and Northern hybridizatransmembrane polypeptides. Two of the polypeptides, SSRa tion indicated thatCNXl is a single-copy gene encod- and SSRD, had been previously identified (3) and were proing a message of 1900 nucleotides. The open reading posed to be involved in translocating nascent polypeptides frame encodes a polypeptide with 530 amino acids, a into the E R the third protein had not been previously charmolecular mass of60.5 kDa, andoverall 48%identity acterized. SSRa was found to also bind Ca2+ ion and be todog calnexin. Bothanimal calnexin and CNXlp phosphorylated in the cytosolic C-terminal region only (1). contain a large luminal domain followed by a single potential membrane-spanning domain near theC ter- As a result, Wada et al. (1)suggested that the complex may act as a signal transducer sensing or affecting changes in minus and a small C-terminal domain exposed to the cytoplasm. The in vitro translation product from the intraluminal calcium levels. Based on the observations that cloned cDNA yielded a polypeptide67 ofkDa thatwas depletion of lumina1 Ca2+ led to the secretion of soluble resident ER proteins (4-6), Wada et al. (1)also suggested that co-translationally imported into dog microsomes and processed to a 64-kDa product. Antibodies generated calnexin may play a role in the Ca2+-dependentretention of against the C-terminal half of the protein cross-reactthese proteins. with an identically sized protein present in the micro- Other studies have indicated that calnexin functions as a molecular chaperone. Degen and Williams (7) described an somal fraction from Arabidopsis. Both the imported and native proteins are cleaved by trypsin to a 59-kDa 88-kDa ER protein (p88) that participates in the assembly of product indicating that the gene product was indeed murine class I histocompatibility molecules. They suggested that p88 may either promote proper assembly of class I correctly processed and translocated into dog microsomes and that the membrane topology ofCNXlp re- molecules or retain themwithin the ER untilassembly of the sembles that of dog calnexin. The presence of a cal- ternary complex of heavy chain, µglobulin, and peptide nexin-like protein within the plant kingdom indicates ligand is complete (8). It was recently revealed that p88 and that this protein is widespread and involved in proc- calnexin represent the same protein (9-11). Hochstenbach et esses fundamental toall eukaryotes. al. (10) also showed that calnexin was transiently associated with a number of partially assembled membrane complexes. Hochstenbach et al. (10) and Ahluwalia et al. (9) have noted that calnexin may function analogously to the immunoglobCalnexin is one of the major integral membrane proteins ulin-binding protein, BiP, as a general molecular chaperone. localized in the endoplasmic reticulum (1).It has a single BiP is a soluble luminal protein that facilitates folding and transmembrane domain near the C terminus, a long N-ter- assembly of proteins during theirtransit through the ER and minal domain within the ER’ lumen, and a short C-terminal retains proteins within the ER, in a Ca2+-dependentmanner, domain in the cytosol. The cytosolically exposed domain can until folding is complete (6, 12). The currentknowledge about calnexin is quite limited with be serine-phosphorylated in uitro. Calnexin binds Ca2+and is similar in sequence to calreticulin (2), a major Ca2+-binding regard to its actual cellular function, its structural features, and its regulation. It remains to be demonstrated whether protein in the lumen of the ER. calnexin occurs ubiquitously in eukaryotic organisms. If so, * Support for this work was provided by National Institutes of the common features may provide insight into its molecular Health Grant GM42609-02 (to N. E. H.). The costs of publication of structure and function. this article were defrayed in part by the payment of page charges. Inthe present study, we report the primary structure, This article must therefore be hereby marked “advertisement” in deduced from cDNA sequence, of the calnexin-like protein, accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. CNXlp, in the higher plant Arabidopsis thulianu. The plant The nucleotide sequence(s) reported in this paper has been submitted totheGenBankTM/EMBL Data Bankwith accession number(s) protein exhibits high sequence identity to mammalian calnexin. Immunoblot analyses and in vitro import studies indi218242. 5 Present address: Dept. of Biological Sciences, Stanford Univer- cate that CNXlpis localized in the microsomal fraction. The sity, Stanford, CA 94305. in uitro import studies also confirm its predicted membrane 11 To whom correspondence should be addressed Carnegie Insti- topology and indicate thattheplant calnexin containsa tution of Washington, Dept. of Plant Biology, 290 Panama St., functional signal sequence. Stanford, CA 94305. Tel.: 415-325-1521;Fax: 415-325-6857. The abbreviations used are: ER, endoplasmic reticulum; bp, base EXPERIMENTALPROCEDURES pair(s); BiP, immunoglobulin heavy chain binding protein; SSRCY/B, Materials-The A. thulianu (L. cv. Columbia) cDNA library, conCY and 6 subunits of the signal sequence receptor; PAGE, polyacrylstructed in X YES (13), was a gift of Dr. John Mulligan, Department amide gel electrophoresis; TBS, Tris-buffered saline; kb, kilobase($
6560
Calnexin-like Protein A of Biochemistry, Stanford University. Sequenase (version 2.0 T7 DNA polymerase) for sequencing and goat anti-rabbit IgG-alkaline phosphatase were from U. S. Biochemical Corp. a-"S-dATP, ["SI methionine, and[cY-~*P]~CTP were from Amersham Corp. The expression vector pMAL-cR1 and amylose resin were from New England Biolabs. Trypsin and 4-nitro blue tetrazolium chloride were from Boehringer Mannheim. Poly(A)-poly(U) was from Pharmacia LKB Biotechnology Inc. Cyanogen bromide-activated Sepharose 4B, 5-bromo-4-chloro-3-indolyl phosphate, and Protein A-alkaline phosphatase were from Sigma. Rabbit reticulocyte lysate (nucleasetreated) and canine pancreatic microsomal membranes were from Promega Biotech. Preparation of Antibodies-Total chloroplast envelope membranes were prepared from spinach leaves essentially according tothe method of Keegstra and Yousif (14) with the following modifications. Crude envelope membranes were pelleted by centrifugation at 75,000 rpm for 5 min in a TL100.3 rotor. Membranes were resuspended in 0.2 M sucrose containing 20 mM Tris, pH8.0, and 2 mM EDTA (TE) and layered on top of 1.1M sucrose + TE. Samples were centrifuged as above, and the yellow membranes a t the interface were collected, diluted in TE, and concentrated by centrifugation as above. The envelope polypeptides were size-fractionated by SDS-PAGE (E), and proteins of 55-75 kDa were electroeluted. Antisera against the 5575-kDa proteins were raised in a single rabbit (16). The IgG fraction was purified from serum by ammonium sulfate precipitation and DEAE-Sepharose chromatography (17). Antibodies were also generated against a peptide expressed from a portion of the calnexin-like cDNA,pTE6-83. The 615-bp SalI-HindIII fragment (bp 918-1533) from pTE6-83 (see Fig. l B ) was subcloned into theexpression vector, MAL-cRI, creating a fusion with maltosebinding protein. After verification by DNA sequencing, the fusion protein was overexpressed in Escherichia coli strain TB-1 and purified through an amylose column according to the manufacturer's directions. The fusion protein (1 mg/ml) was cleaved by incubation with factor Xa (0.01 mg/ml) a t room temperature for 3 h in a buffer containing 20 mM Tris, pH 8, 100 mM NaCl, and 2 mM CaC12. Maltose-binding protein was separated from antigen by a second passage over the amylose column, and theantigen inthe effluent was further purified by SDS-PAGE followed byelectro-elution. The eluted protein was used to generate polyclonal antibodies, and IgG was prepared as described above. Antigen was immobilized on cyanogen bromide-activated Sepharose beads (17) and used to immunoaffinity purify the IgG fraction. Antibodies were eluted from the column using 0.1 M glycine solution, pH 2.5 (17). Screening of cDNA Library and Sequencing of cDNA Clon~sRabbit IgG produced against the 55-75-kDa envelope polypeptides were used to screen the Arabidopsis cDNA X YES library expressed in E. coli strain Y1090 essentially as described (18) with the following modifications. 1) Filters were successively washed for 6 h in Trisbuffered saline ( T B S 10 mM Tris (pH 7.5), 150 mM NaCl), blocked for 30 min in TBS 2% nonfat dry milk, and rinsed in TBS prior to incubation with the primary antibody; 2) 0.2% Nonidet P-40 was used as a blocking agent in subsequent steps instead of 2% nonfat dry milk; 3) antibodies were used at a dilution of 1:250. From approximately 500,000 plaques, 18 positive clones were isolated for further characterization. Plasmids were liberated from phage DNA after infection of XKC/JM107 with the phage isolate, and cDNA insert size was characterized by EcoRI and XhoI digests. To type the phage clones by DNA hybridization, the E. coli host strain was plated onto LB-agar, and 1pl of individual phage isolates were spotted in a grid pattern. Plateswere incubated overnight at 37 "C, plaques were lifted onto nitrocellulose filters, and filters were hybridized against DNA probes made from the individual clones. These clones were grouped into seven types of non-cross-hybridizing clones. The type reported here was isolated seven independent times. Both strands of the longest clone, pTE6-83, were sequenced (19). Immunoblotting of Plant Subcellulur Fractions-Plant microsomal fractions were prepared according to Gallagher et al. (20) from 1012-day-old greening or etiolated pea or 3-week-oldArabidopsis. Chloroplasts were isolated according to Cline (21) and fractionated into membrane and stroma essentially as described previously (22). Samples were electrophoresed and immunoblotted as described (18)with slight modifications. Briefly, the nitrocellulose was blocked with 2% bovine serum albumin in TBS for 20 min at room temperature, followed by incubation with the immunoaffinity-purified antibodies (from above) at 2 pgof protein/ml in TBS containing 2% bovine serum albumin, 0.3% Nonidet P-40, 0.1% SDS, and 0.05% sodium azide for 12 h at 4 ' C. After three 5-min washes (1 x TBS, 1 x TBS
+
from Arabidopsis
6561
+
0.3% Nonidet P-40 + 0.1% SDS, and 1 X TBS), the nitrocellulose was incubated with goat anti-rabbit IgG coupled with alkaline phosphatase (1:10,000 dilution) in 1 X TBS containing 2% bovine serum albumin, 0.2% Nonidet P-40, and 0.05% sodium azide for 1h at room temperature. It was then washed and developed as described (18). I n Vitro Translation and Import-The insert from pTE6-83 was subcloned into the EcoRI and XhoI sites of pSP73, transcribed in uitro using SP6 RNA polymerase (23), and translated in uitro in rabbit reticulocyte lysate in the presence of [36S]methioninewith or without dog pancreatic microsomal membranes following procedures suggested by Promega. Pepstatin, 10 p ~was , added during translation to reduce proteolysis of the precursor. After 60 min, 5% of the reaction (1.25 pl) was incubated in 15 pl (final volume) of 25 mM Hepes pH 8.0 containing 250 mM sucrose for 30 min at 0 "C. Where indicated, trypsin and TritonX-100 were added to 0.1 mg/ml and 0.2%, respectively. Reactions were terminated by the addition of SDS solubilization buffer followed by immediate boiling for 5 min. Samples were run on SDS-PAGE,and gels were dried and autoradiographed. Northern Blot Analysis-Total RNAwas isolated according to Verwoerd et al. (24) from green shoots of A. thnliuna (L.cv. Columbia) grown under continuous light. Total RNA was separated electrophoretically (25 pg/lane) in a 1.2% (w/v) agarose gel containing 6% (v/v) formaldehyde, blotted onto Hybond-N membrane by capillary transfer, and hybridized to full-length pTE6-83 insert (2 X 10' cpm/ ml in solution containing 0.75 M NaCl, 0.075 M sodium citrate (5 X SSC), 0.5% SDS, 5 X Denhardt's solution (25), and 50 pg/ml salmon sperm DNA) a t 63 "C for 15 h. The final posthybridization wash was 0.1 X SSC containing 0.1% SDS at 63 "C for 30 min. Genomic DNA Analysis-Genomic DNA was isolated according to Saghai-Maroof et al. (26) from whole plants ofA. thaliuna (L. cv. Columbia) grown aseptically in liquid culture inMurashige and Skoog salts supplemented with 0.2% sucrose under continuous light, at 18 "C, with shaking. DNA was digested with EcoRI or BglII and 5 pgllane was separated on a 0.75% (w/v) agarose gel, transferred onto Hybond-N membrane, and hybridized to 32P-labeled full-length pTE6-83 insert (10' cpm/ml in 6 X SSC, 0.5% SDS, 5 X Denhardt's solution (25), and 100 pg/ml salmon sperm DNA) for 15 h a t 68 "C. The final wash was in 0.1 X SSC containing 0.1% SDS a t 68 "C. RESULTS
Sequence and Structural Analyses-We have been using an immunological approach to isolate clones encoding chloroplast envelope polypeptides. While screening an Arabidopsis cDNA expression library with polyclonal antibodies made against envelope polypeptides in the apparentmolecular mass range of 55-75 kDa, we isolated seven types of non-crosshybridizing clones. One of these types encodes a putative ATPase from the chloroplast inner envelope and willbe described elsewhere. A second type was isolated seven independent times and is the subject of the present report. The longest of the clones of the second type, designatedpTE6-83, was further characterized by sequencing. Fig. lA shows the nucleotide and deduced amino acid sequences from the 1850bp cDNA clone. Although the open reading frame extends to the 5' end of the cDNA, we believe the first ATG, beginning with nucleotide 87, is the initiating Met for reasons that will be described below. The cDNA would encodea protein of 530 amino acids with a calculated molecular mass of 60,481 Da. Searching the GenBank data base revealed that theprotein predicted by pTE6-83 shares significant sequence identity with that of dog calnexin ( l ) , and, hence, we designated the calnexin-like gene and protein, CNXl and CNXlp, respectively. CNXlp is 47.6% identical to dog calnexin (for alignment, see Fig. 2). Like dog calnexin, CNXlp also resembles calreticulin; it shares 38.7% identity with rabbit calreticulin (2).
Dog calnexin begins with a 20-residue signal sequence (1). The deduced sequence of CNXlp also begins with what appears to be a classical signal sequence. The first M e t is followed in succession by positively charged and hydrophobic residues (27). As described below, CNXlp contains a functional signal sequence that is processed by dog microsomes.
A from Calnerin-like Protein
6562
Arabidopsis
A AM CCC G M GGA TGG TTA GAT GAT GAG CCT GAG GAG GTC GAC GAC CCC
5' CATTTTTGTTCCGATCTACTTCTCCGGCTTATCACCGATTTAGATCTMGCCTGAGGTT 59 TCCTTTTGCATTTTAGAGATTCCGAGA ATG AGA C M CGG C M CTA TTT TCC GTG 113 M R Q R Q L F S V > 9
K
P
E
G
W
L
D
D
E
P
E
E
V
D
D
P
>
Z
8
929 1
9
911 1
G M GCG ACC M G CCT G M GAT TGG GAT GAT GAG G M GAT GGT ATG TGG
TTA GCTTTC
TTT TTG C T TC T C F
L
L
L
L
A
GTT TCGTTC
F
V
S
E
TGT 161 0 2 5
CAG M G CTTTGCTAC Q K L C Y
F
A
T
K
P
E
D
W
D
D
E
E
D
G
M
D
2
GAG GCT CCT AM ATT GAC M C CCC M G TGT G M GCA GCA CCT GGT TGT 1 0 2 5
TAT G M TCGTTC
CTG GAC GAT C M ACG GTT D D Q T V L CGC TGG ATCGTTTCG R W I V S
Y
S
F
D
GAT GAG CCT TTT GAT GGC E P F D G >
N
S
D
Y
E
G
V
W
K
GCT AGG M G TAT GGA ATT GTG AM GAG CTT GAC GAG CCTCTA A R K Y G I V K E L D E P L N
K
G
T
V
V
L
Q
Y
E
V
R
251 l
S
F
Q
CTT GAG TGT GGT GGT GCT TAC TTG M G TACCTCCGTCCT L E C G G A Y L K Y L R P
P
K
I
D
N
P
TGG AGCTCACCTCTT W S S P
CCC AGA GAT ATCCCT P R D I P
M C CTC 353 L > 8 9
Q
E
T
P
Q
G
F
D
S
E
S
P
K
E
A
S
I
ATG
M
>
F
GGG CCT GAC M G TGT G G A GGT ACG M C AM GTG CAT TTC ATC
G
P
D
K
C
G
G
T
N
K
V
H
1
3
E
491 7
T
Y
R
Q
T
T
F
I
TTG 5 4 5 > I S
L
E
K
Q
K
A
E
E
K
H
K
TTCCCTCCCTCT F P P
N
S
P
V
K
S
G
E
Y
G M CAC CAT CTG
V
GTT CCT TAT GAC M G CTTTCC P K D K L S H
E
V
H
H
CAT GTC TAC ACC GCC Y T A
593
M G
L
D
>
1
8
641 5
ATC TTG AM CCC GAC M C GAG GTT AGA ATT TTG GTT GAT GGA GAG GAG 6 8 9 l L K P D N E V R I L V D G E E > Z O l
K
K
K
M T TTA CTCTCT
ATC CCT GCC I P A
A
N
L
L
S
G
GGT G M GAC TTT GAG CCT GCA TTG E D F E P A W Z
T
I
P
D
731 l l
185
G M GAC M G A M CCA GAG GAT
M G ACCATCCCTGACCCT
K
P
E
D
K
K
P
E
D
>
2
3
GAG ATT G M GAT GAG G M GCT GAG
E
I
E
D
E
E
>
3
1
3
M G GGA ATC TGG AM 1 1 2 1
K
G
I
~
K
>
J
F
E
L
D
R
P
D
3
6
A
D
2
1265 9
3
TGG M G CCC AM TTC GAT GTT GAG AM 1313 W K P K F D V E K > I O S
E
A
A
GGC TCT GCA GAT GGT CTC 1361 S A D G L > 4 2 5
G
K
S
Y
Q
U
TTA TCTTTCCTA L S F L
S
V
GTT GTA TTC GAC CTC TTG M C M G GTT GCA GAT 1 4 0 9 V F D L L N K V A D > I I l
K
A
E
Q
ACT GCC TAC M G T C T M G ATC ACG G M CTG ATT GAG 1 4 5 7
A
Y
K
S
K
GTG GTT GTA TTCTTC V V V F F
L
F
L
T
E
L
I
E
G
V
L
V
A
I
>
~
I
5
ATT 1 5 0 5 1 7 3
M G CTT ATC TTT GGT GGC AM 1 5 5 3
TCG CTCTTCCTC S
l
ATT GGT GTCCTCGTCGCC
C M CCA M C TTAACC Q P N L T I
K
L
I
F
G
G
D
4
8
9
M G GCG GCG GCA CCT GTG G M M G M G AM CCA G M GTA GCG GAG AGT 1 6 0 1 K A A A P V E K K K P E V A E S > 5 0 5
881 6 5
TCA M G AGT GGG GAT GAG GCG GAG M G M G G M G M ACC GCG GCA CCA 1619 S K S G D E A E K K E E T A A P > 5 2 1 CGC AM AGG C M CCG AGA CGT GAT M T TAGA A C M G A T T T T A M G T A G T T T T C1 1 0 2 530 R K R Q P R R D N >
GMGATGTGAMGAAMGTTCAGATTTATTTTTCTGTTTTTGGTTTTGGGTTGMTGT~GTTT 1165 TCTGCTGTAGCCTATMTGGTGTMCACCMGTTTGAGAMTTTTTGTCTTTGGGCGMGAM 1828 AGAMGGAGGMTATGTATGTG 3 ' 1850
C
B
~
1
3
TGG GAC GAA AGA GCC M G ATT CCT GAT CCA M T GCC GTG M G CCT GAG 833 W D E R A K I P D P N A V K P D 2 4 9 ATG GAC TGG GAT GAG GAT GCA CCT D W D E D A P M
C
l
AM GCC GAG C M
M G AM M G GCC
G
~ M G AGC TAC CAG M G
M T CCC M G AGT GGC GAG TAC GTT
M G CAC M G
P
TTT GAG CTA GAC AGA CCC GAT 1169
M C CCTGACTAC N P D Y
GAG AM C M M G GCA G M GAG G M GCT GCT
TTT
A
GGT ATC TTG TTT GAC M C ATC TTG ATA GCG AM GAC GAG M G GTCGCT G I L F D N I L I A K D E K V W 3
CAG G M GCT 1 4 9 A > l Z l
GGA TGG ACA CCT CAG GGA TTC GAT AGT G M TCCCCTTACTCTATC W
C
ATT GAT M C CCGGCTTAC I D N P A Y
L
GAG ACT T I C AGA CAG ACC ACT
G
K
CAG G M GGA 1 0 1 E D l O S
GM GGA ACT GTT GTT CTT CAG T I C GAG GTT CGTTTC I
>
A
T I C M G GGC AM 1013 GGT G M TGG M G AGA CCA ATG M G AGG M T CCTGCT G E W K R P M K R N P A Y K G K > 3 2 9
GTG AGT GAG M G 305 V S E D l 3
GCA M G AGT G M GGA CAT GAG GAT TAT GGA CTTCTC A K S E G H E D Y G L L
M G
H
E
209 l
I
M T AGT GAT TAC G M GGT GTA TGG M G CAT
M G
K
E
100
200
300
400
500
FIG.1. cDNA sequence and the deduced amino acid sequence (panel A ) , restriction map (panel B ) , and hydrophilicity plot of CNXlp (panel C).
Processing sites of signal sequences frequently occur after a in calreticulin as well as a number of other Caz+-bindingER small amino acid, and signal sequences are generally longer proteins (28). In contrast, the C terminus of CNXlp is basic. than 15 residues (27). Based on this information, a probable As was found for mammalian calnexin, the hydropathy plot processing site would be after Serl*. of CNXlp (Fig. IC)predicts a single transmembrane domain For the most part, the middle of the deduced calnexin near the C terminus. Although the C termini are generally proteins are highly conserved, while the first 100 and the last divergent, there are short regions of sequence identity just 150 residues are highly diverged. There are seven domains flanking the transmembrane domain (amino acids 456-465 that have been conserved between the two proteins ranging and domain F) and at thevery C terminus (domain G). The from 59 to 75% identity (labeled A-G in Fig. 2). Domain D significance of domain F isunclear, but domain G may include contains the most extensive region of amino acid identity. an ERretention signal. The following C-terminal motifs have This domain is also highly conserved between calnexin and been shown to retain transmembrane proteins in the E R -RKXX,or -=X (29, 30). It is calreticulin, where it has been called the P domain due to the -KKXX, -=X, frequency of proline residues (28). Wada et al. (1)noted that thought that the Lys at -3 is critical, because substitution of Arg at this position abolished ER retention (29, 30). It is in this domain, calnexin and calreticulin share three internally repeated sequences of KPEDWD and GXW. We note therefore interesting to note that both calnexin and CNXlp that both of these repeats arepart of two larger motifs having have Arg at the -3 position. It remains to be established the consensus sequences DP(E/D)(A/D)XKPEDWD(D/E)whether the basic residues in domain G contributeto retaining and GXWXXPXIDNP, respectively. In domain D of calnexin calnexin or CNXlp in theER. Southern Blot Analysis-GenomicDNA isolated from and CNXlp, the firstmotif is repeated three times followed whole plants of Arabidopsis wasdigested with EcoRI or EglII by four repeats of the second motif (Fig. 2). CNXlp (predictedp1 = 4.62) is less acidic than dog calnexin and subjected to Southern blot analysis using the full-length (predicted PI = 4.27) and lacks the acidic domains found at insert from pTE6-83 as aprobe. With EcoRI, which does not the N and C termini (Fig. 2). An acidic C terminus is found cut internal to the cDNA, only a single 7.9-kb band was
1
S
A Calnexin-like Protein
from Arabidopais
6.563
CNXlp CALNEX
MRQRQLFSVFLLL-LAFVSFQK-----LCYCDDQ----------------------TVLYES-34 .EGKW.LCML.V.GTTI.QAHEGH~~R)MIRIE..R~R~~EE~ERSKSKPDTSAPTSPK.T.KAPV 65
CNXlp CALNEX
------ FDEPFD-----GRWIVSKN---------SDYEGVWK-HAKSEG--HEDYGLLVSEKARK PSGEVY.ADS..RGTLS.-..L..AKKDDTDDEIAK.D.K.EVDEMK.TKLPG.K..VLHSR.KH
.........
.................... A (75%)
CNXlp CALNEX (64%)
FIG. 2. Comparison of CNXlp with dog calnexin (1). Aminoacids
C CNXlp CALNEX
76 129
YGIVKELDEPLNLKEGTWLQYEVRFQEGLECGGAYLKYLKYLRPQEAGWTP~FDSESPYSIMFGPD 141 N . . N.1 . . . . . .V.L .-SKTPELNLDQ.HDKT . .T.. . . . . 193 HA.SAK.NK.FLFDTKPLIV
....
......................
.................. B (74%)
KCGGTNKVHFILKHKNPKSGEWEHHLKFPPSV“PY--PY--DKLSHVYTAILKPDNEVRIL~GEEK 202 EDY.L ...F R . . . . .T.V.E.K.A.R.DADLKT.FT . .KT.L L..N...SFE....QSIV 258
are numbered beginning with the initi... .. ating methionine.Dots indicate sequence identity.Gapsintroduced to optimize KKANLLSGEDFEPALIPAKTIP-RAKIPW-~DAPMEIEDEEAEKP 267 alignments are shown with dashes. Con- CNXlp ......,. P...,.D....D.SL...AK.P....T.. 321 served domains(A-G) are indicated with CALNEX NSG ...N--.MT.PVN.SRE.E-O heavy dots above the sequence. The numD(75%1 bers in parentheses are the percentage of identical amino acids between the two EGWLDDEPEEVD-EED-KCEAAPGC-YK332 proteins in the indicated domain. Trans- CNXlp . . .0LB, . . .S . . . .V, .0 . . m. . 2 3 68 CALNEX D . . . . . . . .Y .P a . .... A D M . membrane sequences are double underscored. Repeated motifs in domain D are E (59t) underscored. Acidic domains in calnexin are underscored with a dotted line. Align- CNXlp P L I D N P A Y K ~ D Y F E - L D R P D Y E P I A A I G I E I W T M Q D G I L F D N I L I A K D E K V A E T Y 396 ment was performed using the algorithm CALNEX O.NM. ........ F..D.EPFKMT.FS...L.L.S.TSD.F...FIVCG.RR.VDDW 451 of NeedlemanandWunsch(Genetics Computer Group “Gap” program). R Q T T W K P K F D V E K E K Q K A E E E A A G S A D G L K S Y ~ ~ F D L L N K V A D L S F L S A Y K S K I T E L I E K A E461 Q CNXlp
...........................................
.................................................................
........................
CALNEX
ANDG
.................
.G L .--------.,ADG..EPG----------------------------WGQM..A,.E
480
............ F (67%)
CNXlp CALNEX
QPNLTIGVLVAIVWFFSLFLKLIFGGKKAAAPVEKKKKPEVAESSKSGDEAEKKEETA-------
.
R.W.WWWLTVALPV. LVI -FCCS
519
. . .QSS . . .Y . . TDAPOPQVKEE.,E.... E.... PQKGQEEEE 544
.......... G (700.)
CNXlp CALNEX
.......................................
APRKRQPRRDN 530 S ; A B ~ ’ k B B ~ ~ . S R F B R G G T A S Q E E D D R K P K A E E D E I L. N. N.K R S . . . . - 593
detected (Fig. 3A). BgnI-digested DNA cuts thecDNA at two internal sites: 45 and 1200 nucleotides from the 5’ end of the cDNA(Fig. 1R). Only two hybridizing fragments were detected at 1.7 and 1.9 kb. Given the short overlap,it is unlikelv that the probe would hybridize to sequences 5’ to the first RglII site. The 1.7- and 1.9-kb fragments most likely correkB spond to DNA sequences 5’ and 3’ to the second BglII site. 1.9 The fact that both fragments arelarger than 1200 bp indicates that at least one intron occurs between the two HgnI sites. The Southern blot analysis also indicates that C,VXI is a single-copy gene. RNA Northern Blot Analysis-RNA extracted from lightgrown Arabidopsk was electrophoresed, blotted, and probed -1.C in order to determine the size of the CNXI transcript. Using ”1.7 the entire cDNA as a probe, a single message of 1.9 kb was detected at high stringency (Fig. 3 H ) . The size of the transcript is roughly the size of the largest cDNA we obtained. Immunodptection of Native Calnain-like Protein-“ammaliancalnexin is an ER membraneprotein (1). From its deduced sequence, CNXlp appears tohave a signal sequence FIG.3. Southern (panel A ) and Northern (panel B ) blot and not a chloroplast transit peptide. Since we isolated the analysea of nucleic acids from Arabidopsis. In panel A, Arabi- clone using antibodies prepared against chloroplast proteins, dopsis DNA (5 p g ) was digested with EcoRI or BfflI, separated on an we sought to establish whether we could detect native calagarose gel, and transferred onto anylon membrane as described nexin-like protein in plant microsomes or chloroplast fracunder “Experimental Procedures” using the entire insert from pTE6-tions using antibodies directed against CNXlp. A n antiserum 83 as a probe. The size of the bands was estimated using X DNA digested with BstElI as a standard. In pane1 R , total RNA (25 pg) against the purified peptide expressed from a fragment of the cDNA corresponding to amino acids Aspz’*-Lvs4~(Fig. L4 ) was separated in a formaldehyde-agarose gel, transferred to a nylon membrane, and hybridized to the pTE6-&? insert as described under was generated, immunoaffinity-purifiedwith the antigen, and “Experimental Procedures.” The transcriptsize was estimated using used for immunoblot. analvses of microsomal and chloroplast RNA standards from Rethesda Research Laboratories. fractions of both Arahidopsk and pea leaves. Fig. 4 shows the results of a representative experiment. Preimmuneserum did
6564
FIG.4. Antiseraagainst C N X l p specifically recognizes a peptide in Arabidopsie and peamicrosomes. Lune I contains 1 gg of antigen for Coomassie Blue staining and 120 ng of antigen for blotting. Lane 2, Arabidopsis microsomes (35 pg of protein); lane 3, pea microsomes (35 pg of protein); lane 4, chloroplast membranes (35pg of chlorophyll); lane 5, chloroplast stroma (35 pg of protein). Preimmune sera (unpurified, 10 pg/ml) and antisera (affinitypurified I& fraction, 2 pg/ml) were used to probe the samples after transfer to nitrocellulose. Bio-Rad molecular mass markers are indicated on theleft in kDa.
A Calnexin-like Proteinfrom Arabidopsis
97.466.2-
-
-
45-
-
31
21.514.4-
-
Microsomes - + + + not recognize the antigen, though it did recognize some low Trypsin - + + 4 molecularweight proteins in bothplantsandan80-kDa Detergent - 4 polypeptide in the Arabidopsis microsomalfraction.These 1- 2 3 4 5 bands are stronger in the preimmune blot, presumably because the preimmune serum was not affinity-purified. The (lane I ) and antiserum recognized the26-kDaantigen Arabidopstrongly cross-reacted witha 64-kDa protein in the Precursor b sis microsomal fraction ( l a w 2 ) and a 69-kDa protein in the Mature pea microsomal fraction (lane 3 ) . Neither of these bands was detected by the preimmune serum. Antiserum did not crossreact with any proteins in the membrane or soluble chloroplast fractions. Thisprovides strong evidence that CNXlpis a microsomal protein. Furthermore, pea microsomes contain a n antigenically related protein that has a different electrophoretic mobility than CNXlp. In Vitro Translation and Import Studies of C N X l Gene FIG.5. Co-translational import of C N X l p into dog microProduct-To establish if CNXlp containsa functional signal somes. The insert of pTF;fi-R.'Iwas transcrihed in oifro using SP6 sequence, we examined whetherthe in vitro translated protein polymerase. Approximately 1 pg of RNA was translated in rabbit would be imported into dog microsomes. For comparison, we reticulocyte lysateinthepresence of 20 pCi of ["'SIMet with or also examined whether the protein would be imported into without 3.6 units of dog microsomes. Where indicated, samples were peachloroplasts.ThecDNAencodingCNXlp was tran- treated with 0.1 mg/ml trypsin and 0.2%Triton X-100 and analyzed by SDS-PAGE and autoradiography asdescribed under "Experimenof microsomes t a l Procedures." Each lane contains 5% of the reaction mixture. The scribed in vitro and translated in the presence or chloroplasts to test for co-translational import. TheRNA sizes of the precursor and mature forms of the protein are indicated was also translated in the absence of membranes to test for by arrows on the left. posttranslational import into the twoorganelles. Uptake was established by treating the reaction mixture with trypsin and chloroplast protein despite how it was originally isolated. analyzing samples on SDS-PAGE. T o compare the topology of the native protein with that of Translation of the mRNA from p T E 6 - 8 3 in the absenceof radiolabeled C N X l p imported into microsomes, dog microthe microsomes yielded a 67-kDa radiolabeled protein as the somes containing imported CNXlp were mixed with Arabipredominantproduct (Fig. 5, lane I). Rabbit reticulocyte dopsis microsomes, and the samples were treated with or lysate appears to have some processing activity since someof without trypsin and analyzed by both immunoblot and autothe translation product is cleaved to a 64-kDa peptide, the radiography (Fig. 6 ) . The arrows in Fig. 6 point to the bands same size as the mature protein. The processing could be that are specifically detected by the immune serum. These partly inhibited by inclusion of 10 p~ pepstatin during trans- bands co-electrophoresed with the radioactive bands before lation (data not shown). When the translation was carried and after trypsin treatment. This strongly suggests that the out in the presence of microsomes, most of the 67-kDa protein dog microsomes are processing CNXlp at the same site and with the same was processed to a 64-kDa protein (Fig. 5, lane 3 ) . Trypsin integratingtheproteinintothemembrane treatment completely digested both the 67-kDa precursor and topology as the native Arabidopsis protein. These data also 64-kDa peptide when microsomes were not present (Fig. 5, indicate thatpTE6-83 encodes a protein that is full-length. Expression of Plant Calnexin Protein in Different Tiwwslane 2 ) but degraded the 64-kDa protein toa 59-kDa peptide in the presence of microsomes (Fig.5, lane 4 ) . When detergent Microsomal fractions were isolated from green leaves, green was included to solubilize the microsomes, the 64-kDa band stems, and etiolated leaves of pea plants and subjected to immunoblotanalysisusingthe purified antibodiesagainst was completely digested (Fig. 5, lane 5). These results indicated that CNXlp can be imported into andprocessed by dog CNXlp. The pea calnexin-likeprotein was present in all three samples at approximately the samelevel (Fig. 7 ) . microsomes and has type I topology (27).Wefoundthat C N X l p was neither imported intomicrosomes or chloroplasts DISCUSSION posttranslationally nor was it imported into chloroplasts cotranslationally (data not shown). These results provide furWhile screening an A . t h u l i u mexpression library for cDNA ther evidence that CNXlpis a microsomal protein and not a clones that encode chloroplast envelope polypeptides. we re-
- -
~
-
-
"
A Calnexin-like Protein Immune-Blot Autored
from Arabidopsis
6.565
able signal sequence, and, third, after import into microsomes, C N X l p co-electrophoreses with the native Arahidopsis pro1 2 3 4 tein. " " " Mammaliancalnexin is clearly a Ca"-binding protein, though it lacks the Ca2'-binding EF-hand motif of calmodulin (31). Likewise, no EF-hand motif is present in CNXlp. Calnexin and CNXlp show their highest amino acid identity in domain D. This region is also conservedbetween calnexin and calreticulin, where it has been shown to contain a low capacity high affinity Ca2'-binding domain (82). Therefore. C N X l p is alsolikely to be a Ca2'-hinding protein. Theprecise site responsible for Ca2+ binding hasyet to he determined in any of the aforementioned proteins. Calnexin, calreticulin, and CNXlp all contain at least two types of repeats in this domain, DP(E/D)(A/D)XKPEDWD(D/E) and G X W X X P XIDNP, that may play a role in this function ( 2 8 ) . Like calnexin, CNXlp contains a single transmembrane FIG. 6. Co-electrophoresis of the CNXlp in vitro import product with the native plant microsomal protein. Co-transla- domain near the C terminus. Thisis predicted from sequence tionally imported protein was produced as described in Fig. 5. and analysis and supportedby tryptic digestions of native protein 2.5% of the reaction mixture was mixed with 36 pg of Arabidopsk in plant microsomes and protein imported into dogmicromicrosomes. Where indicated, samples were treated with 0.1 mg/ml somes in uitro. Trypsin cleavage results in a 5-kDa reduction trypsin as described under "Experimental Procedures." Samples were in the apparent molecular mass of the mature protein. Rased separated by SDS-PAGE, transferred to nitrocellulose, probed with immune serum asdescribed in Fig. 4, and then blotswere exposed to on trypsin cleavage at Lys'", the first site after thepredicted transmembrane domain, the calculated product would have a Kodak XAR film. 4.6-kDa shift in molecular mass in good agreement with the observed result. Thus,it is likely that thecytosolicallv exposed Stain Pre-Immune Blot lmmuno Blot C-terminal region is 42 amino acids in C N S l p , whereas this IMW1 2 3 4 11 2 3 4 I 1 2 3 4 1 domain is 89 aminoacids in dog calnexin. ManyCaz+-bindingproteins, including mammaliancalnexin, calreticulin. calsequestrin, RIP, endoplasmin, and protein disulfide isomerase, have clusters of acidic residues at theirCtermini(28). In calreticulin,theC-terminal acidic 31domains have been shown to bind Ca2+with low affinity but high capacity (32). Although calnexin has an acidic C terminus, this domain is localized in the cytosol where the Ca" levels are low and, hence, is unlikely to function as a low FIG. 7. Constitutive expraaeion of pea calnexin-like protein affinity binding site. Theacidic domain at the N terminus of in etiolated and light-growntissue. Lane I contains 1 pg of calnexin is in the lumen of the ER and conceivably could be antigen for Coomassie Bluestainingand 120 ng of antigenfor involved in low affinity Ca" binding. CNXlp lacks both of blotting. Microsomes ( 3 5 pg of protein) fromgreen leaves were loaded the acidic domains found in calnexin and, hence, is unlikely in lane 2. Lane 3, from light-grown shoots; lane 4, from etiolated buds. to be involved in low affinity Ca?' binding. Because plants Samples were blotted as described in Fig. 4. The arrow on the right store much of their Ca" in the vacuole and cell wall rather points to thepea calnexin-like protein. Bio-Radmolecular size markthan the ER as animals do (83), plantsmay have a different ers are indicated on theleft. demand for low affinity Ca2'-binding sites in the ER. A numberof investigators haveobserved a large discrepancy peatedly isolated a cDNA clone with an open reading frame between the predicted molecular mass of Ca"-binding proencodingacalnexin-likeprotein. However, inmammalian cells, calnexin is localized in the ER. Using antibodies gen- teins and the apparentmolecular mass based on SDS-PAGE erated against the C terminusof CNXlp, we observed that a (1, 28). In the case of dog calnexin, the mature protein runs single peptide from plant microsomes specifically cross-re- as a 90-kDa (hence called pp90) band, 24 kDa greater than acted with the immune serum, but we were unable to detect its sequence-predicted molecular mass of 65 kDa (11. I t has any antigenically related polypeptides in the membrane or been suggested that the discrepancy arises due to the abunsoluble fraction from chloroplasts. We further demonstrated dance of acidic residues in the protein that presumably interthat CNXlp contains a functional signal sequence but not a fere with SDS binding, therebyreducing the protein mobility in SDS-PAGE (1). Consistent with this idea, C N S l p , which functional chloroplast transit peptide. Together these data indicate that CNXlp is localized in the microsomes and not lacks two major acidic domains found in dog calnexin, runs 6.5 kDa grenter than its as a 64-kDa mature protein, only the chloroplast. It is possible that the chloroplasts used for the preparation of an igen were contaminated with ER, and sequence-predicted molecular mass. Calnexin has so far been found in dog. mouse, and human because C N X l p is very abundant andpossibly very antigenic, (1, 9-11). We now reporttheexistence of acalnexin-like the corresponding cDNAwas isolated a t a high frequency. Although we did not explicitly determine whetherpTE6-83 protein in two plant species. The high sequence identity and represents the entire cDNA, thefollowing observations sup- conserved structural features between C N S l p and calnexin as well as localization of both the plant and animalpolypepport theconclusion that the cDNA encodes the entire protein and 86 nucleotides of the 5'-untranslated region. First, from tides to microsomes make it likely that CNSlp is a plant homologue of calnexin. We expect that calnexin is prohablv Northern analysis, we observed a single hybridizing species among eukaryotic organisms and conserved that was approximately the same size as the cDNA. Second, ubiquitous the transcript encodes a protein witha functional and cleav- through evolution. We found it similarly expressed in differTrypsin
I
+
- I
+
-
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6566
A Calnexin-like from Protein
ent tissues (leaves and shoots) andat different developmental stages (etiolated buds and light-grown leaves). These findings hint thatcalnexin is constitutively expressed, and itsfunction is fundamental to many different types of cells. As calnexin has been studied entirely in mammalian tissues, it was primarily observed in a specialized role, namely as the major histocompatibility complex class I antigen-binding protein (7-11). Recent studies have shown that calnexin is transiently associated with a number of other membrane proteins, prompting the speculation that calnexin functions as a general molecular chaperone. Consistent with this idea, we observe that calnexin is an abundant component of plant microsomes, suggesting that it is involved in processes common to both plants and animals. Mammalian calnexin proteins are conserved to a degree that it is not possible to identify conserved domains that contribute to calnexin function. The deduced plant sequence is sufficiently diverged from its mammalian counterparts that conserved sequences are now evident and can be further explored by structure-function studies.
Arabidopsis
D. (1991)J. CeU BWL 114,189-205 7. Degen, E., and Williams, D. B. (1991)J. Cell BWL 112.1099-1115
8. Degen,E., Cohendoyle, M. F., and Williams, D.B. (1992)J. Exp. Med. lfi5Xlfifil -17K. __,”” 9. Ahluwdia, N., Ber eron, J. J.M., Wada, I., Degen, E., and Williams, D. B. ””
(1992)J.BWl. C L m . 267,10914-10918 10. Hochstenbach, F:, David, V., Watkins, S., and Brenner, M. B. (1992)P m . Natl. Acad. Sct. U.S. A. 89.4734-4738 11. Galvin K Krishna S Ponchel F Frohlich, M., Cummings, D. E., Carfson,”R., Wands, R., Isaeliacier, K. J., Pillai, S., and Ozturk, M. (1992)P m . NatL Acad. Sci U.S. A. 89,8452-8456 12. Pelham, H. R. B. (1989)Annu Reo. Cell BwL 6,l-23 13. Ell e S. J., Mulligan, J. T., h e r , S. W., Spottswood, M., and Davis, R%. (1991)P m .NatL Acad. Sei. U.S. A. 88,1731-1735 14. Keegstra, K., and Yousif, A. E. (1988) in Methodp for Plant Molecub Bwlogy (Weissbach, A., and Weissbach, H., e&) pp. 173-182, Academic Press, Inc., San Diego 15. Laemmli, U.(1970)Nature 227,680-685 16. Hovanessian. A. G.. Galabru. J.. Riviere.. Y... and Montaenier. , L. .(1985) . Z m m u ~ lToda . 9 161-162 ’ 17. Harlow. E.. and L e . D. (1988)Antibodies: A Laboratorv M a d . Cold Spring Harbor LabdratoG, Coid S ring Harbor NY 18. Hoffman, N. E., Pichersk , E , M a d V. , S., Castksana, C.,, KO,K., Darr, S. C., and Cashmore, R. (1987)P m . NatL Acad. Sct. U.S. A. 84, 8844-8848 19. San er, F., Nicklen, S., and Coulson, A. R. (1977)P m . Natl. Acad. Sei. d S . A. 74,5463-5467 20. Gallagher, S., Short, T. S., , P. M., Pratt, L. H., and Brigga,W.R. (1988)P m .NatL Acad. Scr S. A. 86,8003-8007 21. Cline, K. (1986)J. BWL Chem. 261,14804-14810 22. Huang, L., Adam, Z., and Hoffman, N. E. (1992)Plant PhysWL 99, 247Acknowledgments-We express gratitude to Drs. Philippe R e y 255 mond and Ken Poff for providing microsomal fractions from pea and 23. Krieg, P. A., and Melton, D. A. (1984)Nucleic Acids Res. 12, 7057-7070 24. Verwoerd, T. C., Dekker, B. M. M.,and Hoekema, A. (1989)Nucleic Acids Arabidopsis and to Michael Schaeffer and Tom Berkelman for critiRes. 17,2362 cally reading the manuscript. 25. Sambrwk, J., Fritsch, E. F. and Maniatis, T. (1989)Molecular Cloni Labomtory Manual, Cold $ring Harbor Laboratory, Cold Spring Ha%: NY REFERENCES 26. Sa hai Maroof, M. A., Solime, K. M., Jorgensen, R. A., and Allard, R. W. 1. Wada, I., Rindress, D., Cameron, P. H., Ou, W. J., Doherty, J. J.,Louvard, f1984)P m .Natl. Acad. Scr U.S.A. 81,8014-8018 D.,Bell, A. W., Dignard, D., Thomas, D.Y., and Bergeron, J. J. M. 27. Pugale ,A P. (1989)Protein Targeti Academic Press, Inc., San D i e p (1991)J. BWL Chem. 266,19599-19610 28. Michdk, M., Milner, R. E., Burns, and Opas, M. (1992)Bimhem. J. 2. Flie 1, L , Burns, K., MacLennan, D.H., Reithmeier, R. A. F., and 286,68-692 h&dak. M. (1989)J. BWL Chem. 264.21522-21528 29. Jackson, M. R., Nilsson, T., and Peterson, P. A. (1990)EMBO J. 9,31533. Gorlich, D.,’Prehn,S.; Hartmann, E., Hen, J., O t t o , A., Kraft, R., Wied3162 mann, M., Knespel, S., Dobberstein, B., and Rapoport, T. A. (1990)J. 30. Shin J. Dunbrack, R. L. Lee, S., and Strominger, J. L. (1991)P m . NatL Cell BWL 111,2283-2294 ~ c b d . ’ ~ cu. i . s.A. 88, ’1918-1922 4. Booth, C., and Koch, G. L. E. (1989)CeU 69,729-737 31. Babu, Y. S., Sack, J. S., Greenhough, T. J., Bugg, C. E., Means, A. R., and 5. Rudolph, H.K., Antebi, A., Fink, G.,Buckley,C.M., Dorman, T. E., Cook, W. J. (1985)Nature 316,37-40 Levitre, J., Davidow, L. S., Mao, J., and Moir, D. (1989)Cell 68, 133- 32. Baksh, S.,and Mjchalak, M. (1991)J. Bwl. Chem. 266,21458-21465 33. Evans. D.E.. Briars. S. A,. and Williams, L. E. (1991)J. E m . Bot. 42. 145 285-303 ’ 6. Suzuki, C. K., Bonifacino, J. S., Lin, A. Y., Davis, M. M., and Klausner, R.
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