Download Cells without the calnexin/calreticulin central region are viable

4449
Journal of Cell Science 112, 4449-4460 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
JCS0763
Although calnexin is essential in S. pombe, its highly conserved central
domain is dispensable for viability
Aram Elagöz1, Mario Callejo1, John Armstrong2 and Luis A. Rokeach1,*
1Département de biochimie, Université de Montréal, CP 6128, succ. Centre-ville, Montréal,
2School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK
Québec H3C 3J7, Canada
*Author for correspondence (e-mail: [email protected])
Accepted 22 September; published on WWW 17 November 1999
SUMMARY
In mammalian cells, the calnexin/calreticulin chaperones
play a key role in glycoprotein folding and its control
within the endoplasmic reticulum (ER), by interacting with
folding intermediates via their monoglucosylated glycans.
This lectin activity has been mapped in mammalian
calnexin/calreticulin chaperones to the central region, which
is a highly conserved feature of calnexin/calreticulin
molecules across species. The central domain has also been
implicated in Ca2+ binding, and it has been proposed to be
involved in the regulation of calcium homeostasis in the ER.
Herein, we show that although the Schizosaccharomyces
pombe calnexin is essential for viability, cells lacking its 317amino-acid highly conserved central region are viable under
normal growth conditions. However, the central region
appears to be necessary for optimal growth under high ERstress, suggesting that this region is important under
extreme folding situations (such as DTT and temperature).
The minimal length of calnexin required for viability spans
the C-terminal 123 residues. Furthermore, cells with the
central domain of the protein deleted were affected in their
morphology at 37°C, probably due to a defect in cell wall
synthesis, although these mutant cells exhibited the same
calcium tolerance as wild-type cells at 30°C.
INTRODUCTION
containing the Glc1Man9GlcNAc2 glycan). Monoglucosylated
glycoproteins arise from the consecutive action of the
glucosidase I and II, or by reglucosylation of
Glc0Man9GlcNAc2-containing
proteins
by
UDPGlc:glycoprotein glucosyltransferase (GT; reviewed in Parodi,
1998). A distinctive feature of GT is that, in vitro, it can only
reglucosylate unfolded proteins (Sousa and Parodi, 1995).
These observations have led to one of the current models on
quality control of glycoprotein folding in the ER, in which
calnexin/calreticulin and GT constitute key elements
(Trombetta and Helenius, 1998, and references therein).
According to this model, the mammalian calnexin/calreticulin
chaperones interact with monoglucosylated, newly synthesized
proteins exclusively through their oligosaccharide moieties in
a manner similar to lectins, without peptide contacts.
Numerous publications have supported this model (reviewed
in Trombetta and Helenius, 1998). However, peptide contacts
also appear to be important for the interaction of calnexin with
certain substrates. For instance, Ware et al. (1995) have shown
that once the glycoprotein-calnexin complex is formed in vitro,
the glycans can be removed without disrupting the interaction.
Thus, these authors proposed a two-step model for calnexinligand interaction, where the first contact occurs through the
Glc1Man9GlcNAc2 oligosaccharide, while the second step is
peptide-mediated, probably through exposed hydrophobic
patches, in a mechanism akin to other chaperones such as BiP
(Ware et al., 1995; Williams, 1995). Moreover, a number of
Membrane-bound calnexin and its soluble homologue
calreticulin define a new family of molecular chaperones that
have been implicated in the folding of glycoproteins in the ER
of mammalian cells, such as the cystic fibrosis transductance
regulator (CFTR) and the T-cell receptor (reviewed in Bergeron
et al., 1994; Williams, 1995; Trombetta and Helenius, 1998;
Parodi, 1998). The lumenal domain of calnexin shares
extensive similarity with calreticulin. Both proteins contain a
highly conserved central region comprising two series of
tandemly repeated motifs. Motif 1 (I-DPD/EA-KPEDWDD/E)
is involved in Ca2+-binding (Wada et al., 1991; Tjoelker et al.,
1994). Ca2+ may be important for calnexin function since
chelators disrupt its interaction with ligands in vitro (Bergeron
et al., 1994; Le et al., 1994; Loo and Clarke, 1994; Vassilakos
et al., 1998). In mammalian calnexin, Motif 2 (G-W- -P-I-NPY), along with Motif 1, were shown to be required for binding
glycoproteins through their N-linked glycans (Vassilakos et al.,
1996, 1998).
The use of the α-glucosidase inhibitors castanospermine and
1-deoxynojirimycin in vivo (reviewed in Bergeron et al., 1994;
Williams, 1995; Trombetta and Helenius, 1998; Parodi, 1998)
and purified glycans in vitro (Ware et al., 1995; Vassilakos et
al., 1996; Vassilakos et al., 1998), demonstrated that the
calnexin/calreticulin family displays a lectin-like activity with
unique selectivity for monoglucosylated glycoproteins (i.e.
Key words: BiP, Chaperone, Protein folding, Yeast genetics,
Schizosaccharomyces pombe
4450 A. Elagöz and others
publications reported that calnexin/calreticulin can bind nonglycosylated proteins (reviewed in Williams, 1995; Jannatipour
et al., 1998). Also, castanospermine and tunicamycin (a potent
inhibitor of glycosylation) reduced but did not eliminate the
binding of certain glycoproteins to calnexin/calreticulin
(Pipe et al., 1998; Keller et al., 1998). Hence, the
calnexin/calreticulin chaperones appear to have multiple
modes of interaction with their ligands that may vary for
different proteins. Calnexin and calreticulin are not the only
chaperones involved in glycoprotein folding. The
calnexin/calreticulin family was shown to cooperate with other
chaperones such as BiP, PDI and Erp57, in the conformational
maturation of folding intermediates (e.g. Kim and Arvan,
1995; Oliver et al., 1997; Elliott et al., 1997; Zapun et al., 1998;
Hammond and Helenius, 1994; Tatu and Helenius, 1997).
Schizosaccharomyces pombe encodes the basic components
involved in the quality control of glycoprotein folding found
in the mammalian ER (Jannatipour and Rokeach, 1995; Parlati
et al., 1995; Fernández et al., 1996; Fanchiotti et al., 1998).
Therefore, this fission yeast represents an ideal model
organism for the genetic analysis of the mechanisms of
glycoprotein folding and its quality control. We and others
have isolated cnx1+, the S. pombe calnexin homologue, and
have shown that it encodes a protein essential for viability
(Jannatipour and Rokeach, 1995; Parlati et al., 1995). Like its
mammalian counterparts, Cnx1p is a type I ER-membrane
protein containing the characteristic highly conserved central
region including motifs 1 and 2, and a cytosolic tail
(Jannatipour and Rokeach, 1995; Parlati et al., 1995). As could
be expected, Cnx1p was shown to bind Ca2+ (Parlati et al.,
1995; our unpublished results). Although a lectin activity for
S. pombe calnexin has not been explored as yet, the presence
of four copies each of motifs 1 and 2 may suggest that the
central region might also be involved in glycan binding.
Like the mammalian GT enzyme, Gpt1p in S. pombe was
shown to reglucosylate unfolded proteins in vitro, but its
presence is dispensable for cell life under normal growth
conditions (Fernández et al., 1994, 1996). From these
observations it was possible to infer the existence in the fission
yeast of a GT-independent mechanism for quality control of
protein folding (Jannatipour et al., 1998). We have recently
shown that Cnx1p associates with newly synthesized molecules
of the glycoprotein acid phosphatase, independently of glucose
trimming and reglucosylation by GT (Jannatipour et al., 1998).
Thus, in spite of the essentiality of Cnx1p for fission yeast
viability, the glucose trimming and reglucosylation cycle do not
appear to be indispensable for protein folding in S. pombe. This
notion is further supported by the fact that fission yeast cells
genetically depleted of both Gpt1 and glucosidase II (Gls2p) are
viable (Fanchiotti et al., 1998).
Based on the sequence conservation and the role of the
central region in mammalian calnexin, it could be predicted
that this domain might encode the essential function(s) for S.
pombe viability. However, as described above, in the fission
yeast the other key elements in the calnexin/calreticulin cycle
do not seem to be necessary for viability under standard growth
conditions. In this study, we wished to delimit the Cnx1p
region required for cell viability and assess the relevance of the
highly conserved calnexin/calreticulin central domain. Our
results showed that the 317-amino-acid (aa) central region is
dispensable under normal growth conditions but is required in
situations of high-folding stress in the ER. Thus, overall our
observations further suggest that the mechanism of
glycoprotein folding in S. pombe might be different from that
in mammalian cells.
MATERIALS AND METHODS
Strains and medium
The S. pombe strain SP6089 was used for all plasmid shuffling
experiments (see below for construction and genotype). S. pombe strain
SP556 (h+ ade6-M216 ura4-D18 leu1-32) was used as a wild-type
control in various experiments. S. pombe transformations were
performed by the lithium acetate procedure, and genomic DNA
extraction was as previously described (Moreno et al., 1991). Strains
were grown at 30°C (except where indicated) in EMM minimal medium
supplemented with nutrient requirements (Moreno et al., 1991). The E.
coli strain Top10, from TOPO XL PCR Cloning Kit (Invitrogen Co.
CA), was used for cloning PCR products and the E. coli strain AP401
(lon::mini tetR ara− ∆lac-pro nalA argEam rifR thiI [F′ pro AB lacIq Z
M15]) was used for plasmid amplification and isolation.
Construction of the cnx1::his3 haploid strain SP6089
To construct a haploid S. pombe deleted of entire Cnx1p coding
sequences, a plasmid containing a 4.5-kb PstI-PstI genomic fragment
encompassing the cnx1 gene was linearized with NcoI and progressive
bi-directional digestion was made with ExoIII. The his3 gene was
ligated to create the plasmid pSPCA3282, in which the cnx1 coding
sequences (955-2805, according to EMBL U13389) were deleted, and
this was used in the subsequent steps to obtain cnx∆ strains.
Disruption of cnx1 in the haploid strain was obtained by simultaneous
transformation of the haploid S. pombe strain 248 (h− his3-D1 ade6M216 ura4-D18 leu1-32; Burke and Gould, 1994); with the PstI-PstI
fragment from plasmid pSPCA3282 (containing the cnx1 deletion
marked with his3) and the plasmid pSPCA3261 containing Cnx1p
coding sequences in the vector pREP42 (see below). Transformants
were selected for histidine prototrophy, and selected clones were
analyzed by Southern blotting and PCR to ascertain correct disruptive
integration. Strain SP6089 had the correct ∆cnx1::his3 complemented
by episomal copies of cnx1 (plasmid pSPCA3261). The genotype of
strain SP6089 is: h− his3-D1 ade6-M216 ura4-D18 leu1-32
∆cnx1::his3[pSPCA3261].
DNA manipulation and analysis
Procedures used for DNA manipulation and analysis (purification,
digestion, electrophoresis, transformation, etc.) were as previously
described (Sambrook et al., 1989). The DNA sequences of both
strands of mutant cnx1-containing clones were determined by the
dideoxy chain-terminating method using a T7 DNA Polymerase Kit
(Amersham Pharmacia Biotech, Inc. NJ).
Polymerase chain reaction
Site-directed mutagenesis experiments were done by polymerase
chain reaction (PCR) using the overlap extension method, as
described (Higuchi and Krummel, 1988), with Pfu DNA polymerase,
using the manufacturer’s conditions (Stratagene, La Jolla, CA). In
oligonucleotide sequences (see below), the NdeI restriction site is
underlined, the BamHI site is shown in bold characters, the
corresponding nucleotide sequence of the ER retention signal ADEL
is shown in italics, and stop codons are doubly underlined. Nucleotide
numbers correspond to the position on cnx1 sequence (Jannatipour
and Rokeach, 1995) as in the database (EMBL accession number
U13389). Oligonucleotides used for the construction of each mutant
(Fig. 1B) were: primers A, 5′-CCA CCC AAC ACG TGC ATA TGA
AGT ACG GAA AG-3′ (1049-1080 nt) and B, 5′-CGG GAT
CCT TAC CCA ATT TCA GGA GTC TCG ATG-3′ (2532-2511 nt)
Cells without the calnexin/calreticulin central region are viable 4451
for lumenal (#3) mutant; primers A and C, 5′-CGG GAT CCT TAA
AGC TCG TCA GCC CCA ATT TCA GGA GTC TCG ATG-3′ (25322511 nt) for lumenal+ADEL (#16); primers A and D, 5′-CGG GAT
CCT TAG CGG GTT CAT CAA ATT CAT AAG-3′ (1395-1376 nt)
for N-terminal (#9); primers A and E, 5′-CGG GAT CCT TAA AGC
TCG TCA GCG GGT TCA TCA AAT TCA TAA G-3′ (1395-1376
nt) for N-terminal+ADEL (#10) mutant; primers A and F, 5′-TTG
CTT TTC CAT AGG ATC AGC AAG TGA TCC CCG-3′ (1137-1117
nt) were used to amplify the first 5′ region of the mini-cnx fragment,
then primers G, 5′-ATG GAA AAG CAA TCA ATG CAT G-3′ (22772398 nt) and H, 5′-CGG GAT CCG GCT TTT AAC AGA GTC GCT
AC-3′ (2932-2913 nt) were used to amplify the 3′ part. Finally,
external A and H primers were used to amplify the whole insert of
mini-cnx (#11). For the central region mutant (#12), primers A and F
were first used to amplify the 5′ region. Primers I, 5′-ATA AAT GAA
CCA GAA AAG GAT TTG-3′ (1396-1419 nt) and J, 5′-CGG GAT
CCT TAG GAC TCT TGC TTA GAA AGT AAT TC-3′ (2376-2453
nt) were used to amplify the 3′ region, and finally external primers A
and H were used to amplify the whole insert. To add the ADEL
sequence to the central region mutant (#12+ADEL), primer K, 5′CGG GAT CCT TAA AGC TCG TCA GCG GAC TCT TGC TTA
GAA AGT AAT TC-3′ (2376-2453 nt) was used. Primers A, F and I,
and H were used for the construction of cnx-N-terminal (#14) mutant,
and primers A, F and I, B for luminal-N-terminal (#15) mutant. The
various cnx1 mutants were constructed by cloning the PCR products
(see above) in pCR-XL-TOPOTM (Invitrogen Co. CA) following the
manufacturer’s conditions. The construction of deleted-cnx (#4)
fragment was carried out using in vitro mutagenesis system Altered
Sites II™ (Promega Co., WI), as described by the manufacturer.
Briefly, DEL-CNX1 5′-GAA TTT GAT GAA CCA TGG ATG AAC
CAG AAA AGG-3′ (1381-1414 nt), primer was used to create a NcoI
site at position 2942 nt (the restriction site is in bold italic characters,
and the mutagenized bases are underlined) on wild-type cnx1, then
the internal 981-bp NcoI-NcoI fragment was removed.
Plasmids
pREP41 is an S. pombe expression multicopy vector containing the
LEU2 marker and the ars1 origin of replication. Expression in this
plasmid is under the control of the thiamine repressible nmt41
promoter (induction ratio 25×), flanking a polylinker site (Maundrell,
1993). In the case of pREP42, the ura4 marker is used for selection
instead of LEU2. The full-length and the various cnx1 mutant genes
were inserted in pREP41 digested with NdeI and BamHI. The
plasmids constructed with pREP41 were designated as follows:
pSPCA3220 (#2; full-length cnx1), pSPCA3221 (#3), pSPCA3222
(#4), pSPCA7092 (#9); pSPCA7093 (#10), pSPCA7094 (#11),
pSPCA7102 (#12), pSPCA7096 (#13), pSPCA7097 (#14),
pSPCA7100 (#15) and pSPCA7095 (#16). The construct containing
full-length wild-type cnx1 (#2) in pREP42 was designated
pSCPCA3261. Constructs containing inserts #17 and #18 were cloned
in pREP42 and the resulting plasmids were designated pSPCA7178
and pSPCA7180, respectively. These last plasmids were constructed
by replacing the ClaI-BamHI fragment pSPCA7136 (pREP42-#16)
and replaced with the ClaI-BamHI of constructs #12 and #13. All S.
pombe cultures in this study were grown in EMM medium under
induced conditions (absence of thiamine).
Plasmid shuffling experiments
S. pombe SP6089 strain containing wild-type cnx1+ inserted on
pREP42 (ura4 marker) was transformed with 2 µg of pREP41 (LEU2
marker) containing either full-length wild-type cnx1+ or mutant cnx1.
Transformants were grown for 6 days at 30°C in 5 ml liquid EMM
supplemented with adenine (Ade) and uracil (Ura) to chase the ura4containing pREP42 plasmid, then cells were plated onto solid
EMM+Ade+Ura. After 3-4 days at 30°C, colonies were replica-plated
onto EMM+Ade+Ura and EMM+Ade-Ura. Cells which shuffled
wild-type cnx1 are Leu+/Ura− and contain mutant cnx1. In the case of
pSPCA7178 (#17) and pSPCA7180 (#18), the plasmid shuffling was
carried out against pREP41-cnx1+ (pSPCA3220) in EMM+Ade+Leu.
Spheroplast and membrane preparations
S. pombe wild-type, lumenal and lumenal+ADEL strains were
grown at 30°C in 250 ml EMM+Ade+Ura containing 0.5% glucose,
to an OD595 of 0.5. Deleted-cnx and mini-cnx strains were grown
under same conditions, except that 1 l of medium was used. For each
strain, 109-1010 cells were harvested by centrifugation, washed in 1
ml of citrate buffer I (20 mM citrate phosphate, pH 5.6, 40 mM
EDTA), and resuspended in 5 ml of citrate buffer II (50 mM citrate
phosphate, pH 5.6, 1.2 M sorbitol). Cells were spheroplasted with
25 mg of NovoZym (Sigma Chemicals Co.) by a 45-minute
incubation at 37°C. Spheroplasts were spun at 500 g for 5 minutes
at 4°C, washed twice in citrate buffer II containing 1.2 M sorbitol.
The pellet was resuspended in 2 ml of lysis buffer (0.1 M sorbitol,
20 mM Hepes, pH 7.5, 20 mM potassium acetate, pH 7.4) containing
protease inhibitors (1 mM phenylmethylsulfonyl fluoride: PMSF),
10 mM iodoacetamide: IAA), 300 µg/ml pepstatin A, 300 µg/ml
leupeptine, 300 mg/ml phenanthroline). Lysates were prepared in a
Potter-Elvejhem homogenizer and cleared at 1000 g by spinning for
5 minutes at 4°C. The supernatant was spun at 15,000 g for 15
minutes at 4°C in an SW 50.1 ultracentrifuge (Beckman
Instruments, Fullerton, Ca). The supernatant from this spin was
centrifuged at 100,000 g for 1 hour at 4°C in the SW 50.1
ultracentrifuge, and the resulting pellet was resuspended in 2.5 ml
of ice-cold lysis buffer.
Membrane extraction
S. pombe microsomal membranes (as described above) were treated
for 15 minutes at 4°C by mixing with 1 vol. of either 1 M NaCl, 0.2%
SDS, 0.2 M sodium carbonate, pH 11.5, or 3% Triton X-100.
Membrane lysates were spun at 80,000 g for 1 hour at 4°C in an SW
50.1 ultracentrifuge, then the pellet from this spin was resuspended in
0.1 ml of 3× Laemmli’s sample buffer (Laemmli, 1970) (P fraction).
Proteins in the supernatant fraction were treated for 30 minutes at 4°C
in the presence of 6% ice-cold tricholoroacetic acid (TCA) and spun
at 2,000 g for 45 minutes at 4°C. The pellet was washed twice in icecold 80% acetone and dissolved in 0.1 ml in sample buffer (S
fraction). Before SDS-PAGE, samples were boiled for 5 minutes.
Cell-wall resistance to lytic enzymes
20 ml cultures of wild-type or mutant cnx1 cells were grown in liquid
EMM+Ade+Ura medium at 30°C to an OD595 of 0.2-0.5. Cells were
harvested, washed in 10 ml of citrate buffer I (20 mM citrate
phosphate, pH 5.6, 40 mM EDTA, pH 8.0), spun and resuspended in
5 ml of citrate buffer II (50 mM citrate phosphate, pH 5.6, 0.1 M
sorbitol). 2.5 mg of NovoZym (Sigma Chemicals Co.) was added and
cells were incubated at 37°C. Cell wall degradation was monitored by
measuring cell density at OD595 and taking samples every 20 minutes.
Cell viability was also followed at the same time, by plating an
appropriate amount of cells on solid EMM+Ade+Ura.
Confocal microscopy
Confocal immunofluorescence using anti-Cnx1p or anti-BiP
antibodies (diluted 1:100) was carried out essentially as previously
described (Pidoux and Armstrong, 1993; Jannatipour et al., 1998).
Briefly, cells were grown for 8 hours in EMM at 37°C before fixation
and labeling with anti-Cnx1p and anti-BiP antibodies.
Calcofluor staining of cell wall
Exponentially growing cells were washed once with 1× PBS (130 mM
NaCl, 2.5 mM KCl, 5 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) and
resuspended in 100 µl of the same buffer containing 20 mg/ml of
Calcofluor white (Sigma, F-3543). The cells were subsequently washed
in 1× PBS and placed on a microscope slide and covered with a
coverslip. Cells were observed under UV light or Nomarski interference.
4452 A. Elagöz and others
Fig. 1. S. pombe calnexin (cnx1) mutants
ER lumen
Cytosol
C.terminal
used in this work and viability of
A
N.terminal (86aa) Conserved central-domain (317aa)
(52aa) TM (48aa)
phenotypes. (A) Schematic representation of
1 1 1 12 2 2 2
100 aa
conserved amino acids of the central domain
NH2
COOH
of Cnx1p. The amino acid sequences of
121 QYEVNpEEGL nCGGAYLKLL aepthgemsn sidYrIMFGP DKCGvndRVH
calnexin homologues were aligned as
172 FIFKHKNPlT GEYSEKHLds rpasllkpgi tnlytlivkp dqtfevring
described in Materials and Methods. The
221 dvvrqgslfy dfiPPVLPPV EIyDPEDiKP aDWvDePeIP DPNAVKPDDW
highly conserved central region extends
271 DEDAPrmIPD pDAvKPEdWL EDEPlYIPDP EAqKPEDWDD EEDGDWiPse
from residues 121 to 437. A consensus was
made with the sequences aligned (see
319 IiNPKCiEgA GCGEWKpPWI RNPNYRGpWs PPMIpNPefi GeWYPRKIPN
Materials and Methods), with similarities
371 PDYFDddhps hfgPlygvGf ELWTMQpnIr FsNiyVghsi edaerlgNET
and identities denoted as follows: bold
421 FLpKLKaere llskqEs
letters for 100% identity; capital-underlined
Viability*
B
letters for 75%, capital letters for 50%, small
pREP41 (no insert)
wild type (#2) SP
caps for 25%, and 0% identical residues
+
(A)
were given by lower case letters. Open
lumenal (#3)
+
(B)
2+
boxes, putative Ca -binding regions (motif
ADEL
lumenal +ADEL (#16)
+
(C)
1); double-headed arrows, conserved
deleted_cnx (#4)
+
oligosaccharide-binding domains (motif 2;
N.terminal (#9)
(D)
see Tjoelker et al., 1994; Vassilakos et al.,
ADEL
N.terminal+ ADEL (#10)
(E)
1998; reviewed by Trombetta and Helenius,
(G)
mini_ cnx (#11)
(F)
(H) +
1998); TM, the transmembrane domain; the
(I)
central region (#12)
(J)
‘tree’, the N-glycosylation site; black boxes,
ADEL
#12+ADEL (#13)
potential phosphorylation sites by protein
(K)
cnx-N.terminal (#14)
kinase C (PKC); black bars, potential
phosphorylation site by casein kinase II
lumenal -N.terminal (#15)
(CK2); SP, the signal peptide; N.terminal,
lumenal -C.terminal (#17)
C.terminal, indicate deletion of the 86-aa N
ADEL
lumenal -C.terminal (#18)
terminus or 123-aa C terminus, respectively,
of mature Cnx1p. (B) Plasmid shuffling
experiments to study the cnx1 mutants were carried out as described in Materials and Methods. Viability*, cells which exchanged mutant cnx1
against wild-type cnx1+and contain only mutant cnx1. Primers used for the construction of cnx1 mutants by the overlapping PCR strategy are
shown by arrows
Immunoprecipitations and immunoblotting
Immunoblots and immunoprecipitations were carried out essentially
as previously described (Jannatipour et al., 1998).
Protein sequence alignments
Protein sequence alignments were realized by using the Block
Maker multi-alignment program from BCM Search Launcher
(http://dot.imgen.bcm.tmc.edu:9331), Human Genome Center
(Baylor College of Medicine, Houston, TX). The sequences aligned
to obtain the consensus were: A. thaliana (U08135), C. elegans
(Z22181), Canis familiaris (P24643), D. melanogaster (U30466),
Glycine max (U20502), H. sapiens (P27824), Mus musculus
(P35564), Rana rugosa (D78590), Heliantus tuberosus (Z35108), S.
cerevisiae (U12980) and S. pombe (U13389).
RESULTS
Cells deleted of the highly conserved central region
of calnexin/calreticulin are viable
Disruption of the cnx1 is lethal in S. pombe (Jannatipour and
Rokeach, 1995; Parlati et al., 1995). In order to identify the
Cnx1p sequences required for cell viability, we undertook a
deletion approach. The central region is a highly conserved
feature in the calnexin/calreticulin family of chaperones (see Fig.
1A). Because in mammalian cells, this segment was shown to
encompass the ligand (glycan)-binding and the Ca2+ domains
(Vassilakos et al., 1996, 1998; Tjoelker et al., 1994), it could be
expected that since Cnx1p contains four copies of the repeated
motifs 1 and 2, the deletion of this region would be lethal. To test
this assumption, we constructed mutant #4 (deleted-cnx; Fig. 1B),
and assessed the importance of this region by plasmid shuffling
in the strain SP6089, in which genomic cnx1 was deleted and
replaced with his3 (see Materials and Methods). Viability in this
strain SP6089 is ensured by complementation with the plasmid
pSPCA3261, bearing cnx1+ under the control of the nmt1
promoter (see Materials and Methods). The construct encoding
full-length, wild-type Cnx1p (#2) was used in this experiment as
a control (see Fig. 1B), and conferred viability as expected.
Surprisingly, the cells harboring construct #4 were viable in the
absence of a wild-type copy of cnx1. Western blot, Southern blot
and PCR analyses confirmed that the viable phenotype of strain
#4 was not due to gene conversion between wild-type and mutant
genes, nor to integration of the wild-type sequences into the
genome (not shown). From these results it is then possible to
conclude that even though deletion of cnx1 is lethal, the
calnexin/calreticulin highly conserved region is not essential for
viability in S. pombe when grown under standard laboratory
conditions (minimal medium, 30°C). In order to delimit the
Cnx1p essential sequences and to evaluate whether the central
region by itself can confer viability, a set of cnx1 deletion mutants
was constructed and transformed into strain SP6089 (see Fig.
1B). For certain constructs encoding soluble proteins (#9, #10,
#12, #13, #15, #17 and #18), versions with or without the ADEL
ER-retention signal (Pidoux and Armstrong, 1992) were made in
order to ensure that lack of viability was not due to escape of the
mutant Cnx1p proteins from the ER. The functionality of the
ADEL retention signal was verified by western blotting on
supernatants from cultures (see Fig. 2G). The synthesis of all the
Cells without the calnexin/calreticulin central region are viable 4453
Fig. 2. Localization of Cnx1p mutants and
cell-wall sensitivity. Membranes from wildtype (A), deleted-cnx (C) and mini-cnx (E)
cells were prepared as described in
Materials and Methods and extracted with
Tris-buffered saline, pH 7.5, alone (Mock),
or supplemented with either 0.1% SDS,
1.5% Triton X-100, 0.1 M sodium
carbonate, pH 11.5, or 0.5 M NaCl (high
salt). This was followed by centrifugation
(1 hour, 100,000 g) resulting in pellet (P)
and supernatant (S) fractions. The fractions
were separated by 10% SDS-PAGE for
wild-type fractions and by 14% SDS-PAGE,
containing 4 M urea, for deleted-cnx and
mini-cnx extracts. Fractions were than
analyzed by immunoblotting using
polyclonal anti-Cnx1p antibodies. Note that
the mini-Cnx1p and deleted-Cnx1p proteins
repeatedly appear as doublet bands on highresolution gels, the highest mobility species
probably being degradation products.
Arrows indicate the position of mutant
Cnx1p bands. Asterisk, diffusion of Cnx1p
from #4 and #11 extracts was repeatedly
observed. Confocal immunofluorescence on
wild-type (B), deleted-cnx (D) and mini-cnx
(F) cells was carried out with anti-Cnx1p
antibodies as described in Materials and Methods. Labeling of the nuclear envelope and reticular structures through the cell and under the
plasma membrane is apparent in each case. (G) Secreted and intracellular lumenal and lumenal+ADEL Cnx1p (lanes 1-7) were analyzed by
western blotting with anti-Cnx1p antibodies. Molecular mass markers in kDa are indicated (MW). (H) Sensitivity to lytic enzymes (NovoZym
234) on cells grown at 37°C was monitored as the decrease in OD595, and converted to percentage of intact cells. The time required to reach
50% lysis is indicated as t1 (55 minutes) for mini-cnx, t2 (90 minutes) for deleted-cnx, and t3 (120 minutes) for wild type (values are the
average of 3 experiments). Sensitivity to hygromycin B was done by drop test (as described in Fig. 6) using 50 µg/ml of the antibiotic at 30°C,
on exponentially growing cells.
Cnx1p mutant proteins was confirmed by western blotting with
anti-Cnx1p polyclonal antibodies (not shown), and the ability of
the different constructs to support viability was assessed, as
above, by plasmid shuffling against pSPCA3261 encoding fulllength Cnx1p. As shown in Fig. 1B, in agreement with Parlati et
al. (1995), the lumenal constructs with or without the ADEL
retention sequence (#3 and #16) conferred viability. However,
constructs including the central region without the sequences
encoding the first 86 aa of the mature Cnx1p (#12-15) did not
exchange with pSPCA3261 expressing full-length Cnx1p. This
would suggest that although the central region is conserved in all
calnexin/calreticulin molecules, it does not encode a function(s)
sufficient for viability. On the other hand, the mini-cnx construct
(#11) encoding the signal peptide, the last 52 aa of the lumenal
domain, the transmembrane domain (TM) and the 48-aa cytosolic
domain, conferred a viable phenotype. Constructs #9 and #10
encoding the first N-terminal 86 aa of the mature Cnx1p did not
shuffle against wild type, indicating that this region alone is not
sufficient for viability. Nevertheless, the first N-terminal 86 aa
might have a role, such as in the folding of lumenal Cnx1p when
the central region is present, as constructs #12-15 did not support
viability. In this vein, Peterson and Helenius (1999) reported that
a deletion of the 83 N-terminal aa of mammalian calreticulin
inhibits its ability to bind to substrates, most likely by disrupting
its structural integrity. Finally, constructs #17 and #18, which
represent lumenal versions of Cnx1p deleted of the last 52 aa, did
not support viability.
Taken together, these results suggest that Cnx1p encodes at
least two distinct domains: (1) a non-essential central domain,
which in mammalian cells was demonstrated to bind folding
glycoproteins (via their glycans) and Ca2+ (see Bergeron et al.,
1994; Williams, 1995; Trombetta and Helenius, 1998; Parodi,
1998; Vassilakos et al., 1998); and (2) an essential domain for
cell viability, mapping within the last 123 residues of the
fission yeast molecule.
By cell fractionation studies we confirmed that wild-type
Cnx1p and the mutants #4 and #11 are membrane-bound (see
Fig. 2A,C,E), and that constructs #3 and #16 are soluble
proteins (not shown). In addition, confocal microscopy with
anti-Cnx1p antibodies showed that the mutant proteins
localized to the ER (Fig. 2B,D,F). Moreover, confocal
microscopy with anti-BiP antibodies presented the typical S.
pombe ER staining pattern (not shown), as previously reported
for cnx1+ cells (Pidoux and Armstrong, 1992; Jannatipour et
al., 1998). Mutants supporting viability were studied further.
The growth rates of deleted-cnx and mini-cnx cells
are reduced at 37°C but are similar to wild type at
30°C
As a first step to assess how the different cnx1 mutants affect
the physiology of S. pombe, we determined the growth rates of
cells bearing the constructs lumenal (#3), lumenal+ADEL
(#16), deleted-cnx (#4) and mini-cnx (#11). For comparison,
we also determined the growth rates of the strain SP556
4454 A. Elagöz and others
A
6
30ºC
5
C
g time in min
3
m
OD595n
4
2
SP556
lumenal (#3)
lumenal+ADEL (#16)
deleted-cnx (#4)
mini-cnx (#11)
wt (#2)
1
800
600
400
200
0
0
B
500
1000
1500
2000
0
2500
Time (min)
5
30
32
34
37
ºC
37ºC
4
wt (#2)
deleted-cnx (#4)
mini-cnx (#11)
D
2
1
mini-cnx (#11)
deleted-cnx (#4)
wt (#2)
g time in min
m
OD595n
3
800
600
Temp. shift
+ Glycerol
400
200
0
0
0
1000
2000
3000
4000
5000
6000
Time (min)
7000
37
30
ºC
Fig. 3. Cells deleted of the central domain show temperature-sensitive reversible growth rates. Cells were cultured to late log phase and diluted
into 25 ml of fresh EMM+Ade+Ura medium to OD595 of 0.02 and growth rates were determined. (A) Growth curves of SP556, wild-type (#2),
lumenal (#3), lumenal+ADEL (#16), deleted-cnx (#4), and mini-cnx (#11) cells grown at 30°C. (B) Growth curves for cells grown at 37°C for
70 or 115 hours. (C) Generation (g) times for wild-type (#2), deleted-cnx (#4) and mini-cnx (#11) cells were calculated on the exponentialphase portions of the growth curves and determined at temperatures ranging from 30°C to 37°C. (D) Generation times for wild-type (#2),
deleted-cnx (#4), and mini-cnx (#11) cells were determined after a temperature shift from 37° to 30°C. Late log-phase cells, cultured at 37°C,
were diluted into fresh EMM+Ade+Ura medium to an OD595 of 0.02, and the growth temperature was shifted down to 30°C. Generation times
were also calculated in the presence of 4% (approx. 0.4 M) glycerol at 30°C and 37°C (dotted lines). Cell growth was monitored by measuring
OD595. The values represent the average of three experiments.
carrying an intact genomic copy of cnx1+ and the strain
carrying episomal copies of full-length cnx1+ (#2). As shown
in Fig. 3A, all the strains grew at about the same rate at 30°C
without a major lag phase. However at 37°C, while strains #3
and #16 grew at essentially the same rate as SP556 and (not
shown) strain #2, deleted-cnx (#4) and mini-cnx (#11) cells
grew at considerable slower rate, reaching stationary phase
after 3000 minutes and 6000 minutes, respectively (see Fig.
3B). Moreover, at 37°C, the lag phase for #2 was 100 minutes,
but 500 minutes for deleted-cnx and 1500 minutes for mini-cnx
cells. A prolongation of the lag phase may correspond to a
reversible adaptation of the organism to the stress conditions
or may represent the time required for the selection of mutant
populations of cells able to grow at 37°C. To discriminate
between these two possibilities, we reasoned that if the first
hypothesis was correct, the cells growing at 37°C when shifted
back to 30°C would grow at the same rate as wild type (#2).
On the other hand, if the second possibility was true, we
expected that these mutant cells would grow at an altered rate
when shifted down to 30°C. As shown in Fig. 3D, the growth
rates of strains #4 (deleted-cnx) and #11 (mini-cnx) after the
shift-down (i.e. from 37°C to 30°C) were practically
indistinguishable from those carrying wild-type cnx1+ (#2).
Moreover, when these mutant strains that were shifted down to
30°C were recultured at 37°C, they exhibited the same lag
phase and growth rates as in the first experiment (not shown).
In addition, when these mutant cells grown at 37°C were plated
on solid medium they appeared homogenous without papillae,
thus excluding the possibility of selection of mutated
populations.
To further explore the hypothesis of adaptation of strains #4
and #11 to the temperature stress, we measured the growth
rates at intermediate temperatures. As shown in Fig. 3C, while
the generation time for the wild-type strain remained
practically invariant between 30°C and 37°C, that of strains #4
and #11 increased significantly with temperature. Furthermore,
Cells without the calnexin/calreticulin central region are viable 4455
Fig. 4. S. pombe BiP/Cnx1p levels and
co-immunoprecipitation in different
strains. (A) Exponentially growing S.
pombe cells expressing wild-type or
mutant Cnx1p were incubated at 30°C
and 37°C, with or without 4% (approx.
0.4 M) glycerol. Cell extracts were
immunoprecipitated with anti-Cnx1p
antibodies (lanes 1-6 and 9-14), with
pre-immune rabbit serum (lane 7), or
with rabbit pre-immune without
proteinA-Sepharose (lane 8). The
immunoprecipitated material was
resolved by 12% SDS-PAGE and
immunoblotted with anti-BiP antibodies.
Molecular mass markers in kDa are
indicated (MW). Note that S. pombe BiP
appears as a doublet, with an upper band
being the glycosylated form (approx.
10% of molecules), and the lower band
the non-glycosylated form (Pidoux and
Armstrong, 1993). The lower part of A
at 30°C and 37°C, is the anti-Cnx1p
western blotting of the immunoprecipitated material.
The asterisk indicates that the deleted-Cnx1p and
mini-Cnx1p bands correspond to a different part of
the same gel. (B) Wild-type cnx1+ or mutants #4 and
#11 were grown at various temperatures and BiP and
Cnx1p were detected by western blotting with antiBiP or anti-Cnx1p antibodies on 5 µg of protein
extracts. (C) Cells from wild-type cnx1+ or mutants
#4 and #11 adapted at 37°C were shifted down to
30°C. Extracts were made from cells grown at both
temperatures and the BiP and Cnx1p were detected
by western blotting with ant-BiP or anti-Cnx1p
antibodies. (D) Mutant strains #4 and #11 were
cultured at 30°C or 37°C in the presence (+) or
absence (−) of glycerol, as indicated. Western blot
analyses to detect Cnx1p were done with 15 µg of
protein extract instead of 5 µg using anti-Cnx1p
antibodies. (E) Accumulation of BiP was quantified
(Scion Image software, NIH) in different cnx1 mutants (#3, #16, #4 and #11) with respect to wild type (#2) at 30°C and at 37°C. 5 µg of protein
extracts were used to quantify BiP levels by western blotting. The ratio with respect to wild-type cnx1+ (#2) is shown as a bar graph, and
represents the average of two experiments at each temperature.
the slow growth phenotype of cells #4 and #11 was reverted
by retransformation of episomal copies of wild-type cnx1+
(#2) into these strains (not shown). Taken together, these
results support the notion of a gradual adaptation of the
physiology of the deleted-cnx and mini-cnx cells to the
temperature stress, the mini-cnx strain being more affected
than the deleted-cnx strain.
52 amino acids of the lumenal region of mini-Cnx1p
are sufficient to form a complex that includes BiP
We have previously shown that Cnx1p and BiP are found in a
complex, under normal and ER-stress conditions, that may be
involved in the folding of glycoproteins and non-glycosylated
proteins (Jannatipour et al., 1998). Moreover, KAR2/BiP and
CNE1 (the calnexin homologue) were shown to genetically
interact in S. cerevisiae (Brodsky et al., 1999). To examine
whether the calnexin mutants interact with BiP, we carried out
immunoprecipitations with polyclonal anti-Cnx1p antibodies.
After separation of the immunoprecipitated extracts by SDS-
PAGE, the presence of BiP was analyzed by western blotting
using polyclonal anti-BiP antibodies. As shown in Fig. 4A
(lanes 1-6), BiP coprecipitated with full-length and mutant
Cnx1p, including deleted-cnx and mini-cnx. This interaction
is specific since no BiP was precipitated from SP556 total
protein extracts with or without pre-immune serum (Fig. 4A,
lanes 7 and 8). Moreover, since identical results were obtained
after 2 hours incubation with cycloheximide, it seems unlikely
that mini-Cnx1p could bind as a misfolded substrate to BiP.
The decrease in the amount of BiP coprecipitated with miniCnx1p (#11) could be the consequence of the lower amounts
of BiP (see Fig. 4E, lane 5) and/or lesser efficiency of miniCnx1p precipitation due the loss of epitopes in this mutant. As
shown in Fig. 4A (lower panel of lanes 1-8) the anti-Cnx1p
western blotting of the immunoprecipitates detected lower
amounts of mini-Cnx1p. Overall, these observations suggest
that these 52 aa of the lumenal region of the mini-Cnx1p are
sufficient for the formation of a complex containing both
Cnx1p and BiP.
4456 A. Elagöz and others
Fig. 5. Cells depleted of the calnexin’s highly
conserved central region exhibit S. pombe
morphology that is reversible by 1.2 M sorbitol.
Late-log phase S. pombe cells, grown at 30°C,
were diluted into fresh EMM+Ade+Ura liquid
medium and grown for 20 hours at 30°C. For
cultures at 37°C, cells were grown for 20 hours
for wild type, 30 hours for deleted-cnx, and 70
hours for mini-cnx. Phase-contrast micrographs
of cells were then done on cells grown at 30°C
(A), 37°C (B) or at 37°C in the presence of 1.2
M sorbitol (C). Deleted-cnx and mini-cnx strains
were transformed with wild-type cnx1+, and
grown in EMM+Ade at 37°C for 20 hours (D).
Arrows indicate the vesicles appearing at 37°C.
Phase-contrast microscopy was done with a
Zeiss optical microscope, at a 2500×
magnification on the film. Bar, 10 µm.
The calnexin levels in deleted-cnx and mini-cnx
strains drop at 37°C but BiP levels remain similar to
those in cnx1+ cells
To obtain clues at the molecular level regarding the mechanism
allowing adaptation of deleted-cnx and mini-cnx cells to
temperature stress (see Fig. 3), we performed western blot
analyses with anti-BiP antibodies with extracts of strains #2,
#4 and #11 grown at the exponential phase at 30°C, 32°C, 34°C
and 37°C. Fig. 4B shows the results of these experiments. In
strain #2 (full-length Cnx1p), the calnexin levels remain
unchanged at different temperatures since its expression is
driven by an heterologous promoter (nmt1). However, in strains
#4 (deleted-cnx) and #11 (mini-cnx) the calnexin levels
dropped considerably at 37°C (see arrows in Fig. 4B). Western
blotting with anti-Cnx1p antibodies using 15 µg of cell extract
instead of 5 µg confirmed the presence, albeit diminished, of
the deleted-Cnx1p and mini-Cnx1p in cells grown at 37°C (see
Fig. 4D, lanes 3 and 4). The reduction in the levels of mutant
Cnx1p at elevated temperatures could be due to instability of
these proteins in vivo or to proteolytic degradation in vitro.
However, the second possibility could be excluded since the
levels of wild-type Cnx1p (#2) remained unchanged under
these conditions (see Fig. 4B). As expected for a heat-shock
protein, BiP synthesis was increased at 37°C in the cnx1+ strain
(see Fig. 4B). Interestingly, at 30°C the BiP levels were higher
in mutants #4 and #11 than in wild type, but remained
unchanged at different temperatures. The BiP and Cnx1p levels
were practically restored when the cells adapted at 37°C were
shifted down and cultured at 30°C (see arrows in Fig. 4C).
To explore the possibility that an induction of BiP expression
could compensate for the reduction of Cnx1p levels in strains #4
and #11 during adaptation at 37°C, we quantified by western
blotting the BiP levels in the various cnx1 mutants at 30°C and
37°C. As shown in Fig. 4E, at 30°C and compared to wild type
(#2), BiP accumulation is about 1.8-fold higher in the lumenal
(#3) and lumenal+ADEL (#16), 1.6-fold in the deleted-cnx (#4)
mutant and about 1.4-fold in the mini-cnx cells (#11).
Interestingly, the BiP levels did not further increase to
compensate for the drop in Cnx1p levels in strains #4 and #11
cultured at 37°C (Fig. 4D, lanes 1-4; and see below). This may
suggest that perhaps other chaperones compensate for the
reduction in the overall chaperone efficiency in the cnx1 mutants.
Deleted-cnx and mini-cnx cells exhibit temperaturedependent altered morphology
We assessed whether the various cnx1 mutations affected the
morphology of S. pombe cells microscopically. Lumenal and
lumenal+ADEL cells presented no visible difference in their
morphology (not shown). In contrast, the deleted-cnx and minicnx cells grown at 37°C had an aberrant round shape
morphology and the majority contained large vesicles (Fig. 5B,
see arrow). The rounded shape of these cells suggested that the
synthesis of a component(s) of their cell wall could be affected.
A classical way to test this hypothesis is to grow cells in a
hyperosmotic medium (Fanchiotti et al., 1998). As shown in Fig.
5C, the aberrant morphology of deleted-cnx and mini-cnx cells
grown at 37°C was suppressed in the presence of 1.2 M sorbitol
(Fig. 5C) or glycerol at 1.2 M (not shown), or by transforming
the plasmid encoding wild-type Cnx1p (Fig. 5D). In order to
study the cell wall integrity of the cnx1 mutants, exponentially
growing cells were treated by NovoZym 234, an enzyme
complex able to degrade S. pombe cell wall polymers (Ishiguro
et al., 1997). As shown in Fig. 3H, deleted-cnx and mini-cnx
cells exhibited increased sensitivity to the lytic enzymes at 37°C
when compared to cnx1+ cells. The assembly of the yeast cell
wall requires the biosynthesis and transport of glycoproteins and
Cells without the calnexin/calreticulin central region are viable 4457
Fig. 6. Calcofluor staining of cnx1
mutants. Late-log phase S. pombe
cells, grown at 30°C, were diluted
into fresh EMM+Ade+Ura medium
and grown for 20 hours at 30°C. For
cultures at 37°C, cells were grown for
20 hours for wild type and mini-cnx
strain transformed with wt cnx1+, 30
hours for deleted-cnx, and 70 hours
for mini-cnx. Exponentially growing
cells cultured at 30°C or at 37°C were
stained with Calcofluor White (see
Materials and Methods) (A,C).
Nomarski interference images show
the same field of cells stained with
Calcofluor (B,D). Arrows indicate
vesicles labeled with the dye.
Microscopy was done with a Zeiss
microscope, at a 1500× magnification
on the film. Bars, 10 µm.
β-glucans (Orlean, 1997). The antibiotic hygromycin B was
reported to affect the viability of yeast cells with mutations
affecting the early stages of glycoprotein biosynthesis, in
particular at the ER (Dean, 1995; Silberstein et al., 1998). Hence,
we tested the effect of this antibiotic on mutants #4 and #11. As
it can be seen in Fig. 2H (right panel) at 30°C, the mutant strains
showed increased sensitivity, while none of the strains grew at
37°C in the presence of the drug. To further study the cell wall
in the cnx1 mutants, we used Calcofluor White. This fluorescent
dye stains an unknown cell wall component of S. pombe cells
that concentrates in the septum of dividing cells (Robinow and
Hyams, 1989). The results of the microscopic observations are
presented in Fig. 6. Calcofluor stained the septa of mutants #4
and #11 at 30°C and 37°C (Fig. 6A,C) in a similar manner to
wild type (#2) or the mini-cnx cells also containing a plasmid
expressing cnx1+. However, the large vesicles observed at 37°C
in mutants #4 and #11 (see Figs 5, 6) were also labeled by the
dye, thus suggesting that these vesicles might accumulate a
Calcofluor-stainable cell-wall component.
Taken together, these results suggest that the conserved
central domain of Cnx1p may be required, directly or indirectly,
for correct cell wall synthesis and morphology at 37°C.
The slow growth phenotype of the deleted-cnx and
mini-cnx strains can be rescued by glycerol and
sorbitol
The growth rate of deleted-cnx and mini-cnx cells is reduced
at temperatures higher than 30°C (Fig. 3B,C). We observed,
however, that these mutant cells grew faster in the presence of
glycerol and sorbitol at 1.2 M. Therefore, we wished to
examine in more detail the effect of these polyols on the growth
of cnx1 mutants. Accordingly, cells cultured exponentially
were subjected to the drop test for their ability to grow at 30°C
and 37°C on minimal medium (EMM) or EMM supplemented
with glycerol or sorbitol.
The slow growth of deleted-cnx and mini-cnx cells at 37°C
was rescued in the presence of 4% (approx. 0.4 M) glycerol (see
Figs 7, 3D). It should be noted that in this case, glycerol does
not act as a source of carbon since EMM contains 2% glucose.
The same effect was observed in the presence of 0.4 M sorbitol
(not shown). Because the osmolality of the growth medium was
reported to affect the levels of molecular chaperones and the
stability of certain proteins (Kültz et al., 1997), we next analyzed
the accumulation of Cnx1p and BiP in the presence of glycerol
or sorbitol. As shown in Fig. 4D (lanes 3-6) the levels of deletedCnx1p and mini-Cnx1p at 37°C were restored to those at 30°C
when cells were cultured in the presence of 0.4 M glycerol or
sorbitol (not shown). Interestingly, the addition of these polyols
resulted in the enhancement of coprecipitated BiP with Cnx1p
(see Fig. 4A, lanes 9-14).
The deleted-cnx and mini-cnx strains cultured at
37°C are more sensitive to DTT, NaCl and Ca2+
The central-domain of the calnexin/calreticulin chaperones was
shown to bind Ca2+, and it was proposed that membrane-bound
calnexin could participate in the retention of ER-soluble proteins
by anchoring a protein-calcium gel via its transmembrane
domain (Wada et al., 1991; Tjoelker et al., 1994). As such, it
could be expected that strains deleted of the central region of
Cnx1p would be less tolerant to increased levels of Ca2+ in the
medium. To assess this possibility, we performed drop tests with
10 mM Ca2+, at 30°C and 37°C. The growth of the deleted-cnx
and mini-cnx strains remained unaffected in the presence of Ca2+
at 30°C (Fig. 7); however, it was severely inhibited at 37°C. This
inhibition was totally rescued by the addition of 0.4 M glycerol
(Fig. 7) or 0.4 M sorbitol (not shown).
4458 A. Elagöz and others
Fig. 7. Effect of ER-stress on the
growth of cnx1 mutants. Serial
dilutions of exponentially growing
S. pombe cells in liquid
MM+Ade+Ura (EMM containing
2% glucose) medium at 30°C, were
spotted on the same medium with
or without 4% (approx. 0.4 M)
glycerol and supplemented with
either 10 mM CaCl2,1 mM DTT or
50 mM NaCl. The plates were
incubated for 96 hours at 30°C or
37°C; strains used in this
experiment are indicated.
DTT is a reducing agent that induces protein misfolding in
the ER (Braakman et al., 1992). It was of interest to evaluate
the response of the various Cnx1p mutants to a DTT
challenge. As shown in Fig. 7, in the presence of 1 mM DTT
the growth of deleted-cnx and mini-cnx cells was affected at
30°C and completely inhibited at 37°C in the case of minicnx. Thus the deletion of the highly conserved central domain
of Cnx1p compromised the cell’s tolerance to the combined
effect of the two ER stresses (DTT and temperature; see
arrows in Fig. 7).
In the presence of NaCl at a concentration as low as 50 mM
in EMM, the growth of deleted-cnx and mini-cnx cells was
drastically affected at 37°C (see Fig. 7). The inhibitory effect
of NaCl suggests that these mutants cells are impaired in their
regulation of NaCl homeostasis. The sensitivity to both DTT
and NaCl was partially suppressed by the addition of 0.4 M
glycerol (see Fig. 7); however, 0.4 M sorbitol did not rescue
the growth inhibition of mini-cnx cells at 37°C in the presence
of these additives (not shown).
DISCUSSION
S. pombe cells deleted of the calnexin/calreticulin
highly conserved central domain are viable
The central region is a salient feature of the calnexin/calreticulin
molecules studied thus far. Its roles in mammalian cells in the
tethering of folding intermediates of glycoproteins through their
oligosaccharide moieties, and in the binding of Ca2+, have been
demonstrated (Tjoelker et al., 1994; Vassilakos et al., 1998).
Based on these observations we assumed that this 317-aa region
containing motifs 1 and 2 would encode the essential
function(s) of S. pombe calnexin. Our results showed that under
normal culture conditions (minimal medium at 30°C), the
fission yeast cnx1 mutants (#4 and #11) lacking the central
domain grow at the same rate as wild-type cells. Furthermore,
the central region alone could not rescue the non-viability of
cnx1∆ cells. These genetic observations may suggest that the
interaction of monoglucosylated folding intermediates with the
putative lectin determinant of S. pombe calnexin might not be
absolutely required for the folding of glycoproteins, under
normal growth conditions. Our results are consistent with four
lines of evidence supporting this notion: (1) cells deleted of
gtp1, the gene encoding glucosyl transferase, are viable at 30°C
and 37°C (Fernandez et al., 1998); (2) Cnx1p interacts with acid
phosphatase independently of glucose trimming and
reglucosylation, i.e. the two pathways that generate
monoglucosylated glycoproteins (Jannatipour et al., 1998); (3)
cells genetically depleted of GT and glucosidase II (the enzyme
that trims G2 glycans to G1 and G0), are viable at 30°C and 37°C
(Fanchiotti et al., 1998); (4) an alg6∆/gpt1∆ double mutant
strain (Alg6p is the enzyme that adds glucose to the lipid-linked
glycan) is viable at 30°C.
Nevertheless, strains #4 and #11, lacking the highly
conserved central domain, exhibited reduced growth rates at
37°C. Because deleted-Cnx1p and mini-Cnx1p are correctly
targeted to the ER (Fig. 2), the reduced growth rates of these
cells at elevated temperature could be due in part to the
observed decrease in Cnx1p levels (see Fig. 4). This is
supported by the fact that the presence of 0.4 M glycerol or
sorbitol corrected both the growth rates and the levels of miniCnx1p and deleted-Cnx1p. The stabilization of mutant Cnx1
proteins could be the result of general changes in chaperone
levels and protein degradation activities induced by osmotic
changes produced by these polyols (Kültz et al., 1997;
Fernández et al., 1997). Another non-exclusive possibility
could be that, in addition, glycerol might act as a chemical
chaperone as previously reported in vitro (Gekko and
Timasheff, 1981) and in vivo (Sato et al., 1996).
In spite of the fact that cells without the central region are
viable under standard conditions, this domain appears to be
required when cells are cultured under high ER stress such as
the combination of DTT and temperature. Our results are
congruent with those of Fanchiotti et al. (1998), who showed
that gpt1∆/alg6∆ double-mutant cells do not grow at 37°C, and
proposed that the interaction between monoglucosylated
folding intermediates and calnexin is essential under
conditions of extreme ER stress.
A possible role of the central domain in cell-wall
biosynthesis
Cells deleted of the central domain (strains #4 and #11)
Cells without the calnexin/calreticulin central region are viable 4459
exhibited anomalous, round-shaped morphology at 37°C, that
was suppressed by culturing the cells in the presence of 1.2 M
sorbitol or glycerol. Moreover, these cnx1 mutants exhibited
increased sensitivity to cell-wall lytic enzymes and to
hygromycin B, suggesting that these cells are defective in the
biosynthesis of some cell-wall component(s), and that Cnx1p
could be involved in this process. Interestingly, in
Saccharomyces cerevisiae, the ER glucosidase I (CWH41),
glucosidase II (GLS2) and Kar2p/BiP were shown to be
involved in β-1-6 glucan synthesis (Simons et al., 1998;
Shahinian et al., 1998), one of the cell wall components. Thus
it is tempting to speculate that in yeasts the calnexin cycle may
play a major role in the biosynthesis of cell wall components,
and a secondary one in the folding of glycoproteins, whereas
in mammalian cells, which obviously lack a cell wall, the
calnexin cycle evolved to be specialized in the folding of
glycoproteins.
The central domain is not required for Ca2+
homeostasis at 30°C
The highly conserved central domain of calnexin/calreticulin
has been shown to bind Ca2+ (Wada et al., 1991; Tjoelker et
al., 1994), and it was also proposed that calnexin could
participate in the retention of the ER soluble proteins by
anchoring a protein-calcium gel via its transmembrane domain
(Wada et al., 1991). The cells deleted of the central domain
were viable in the presence of 10 mM Ca2+ at 30°C. However,
the requirement for the Cnx1p central domain was manifested
under ER-stress, thus arguing that the ensemble of ER-resident
proteins do not provide sufficient Ca2+ buffering capacity to
ensure viability under these conditions.
The essential function(s) of Cnx1p is found within
its 123 C-terminal residues
We have shown that the essential function(s) of Cnx1p can be
delimited to the 123 C-terminal amino acids. This segment spans
the last 52 aa of the lumenal domain, the 23-aa transmembrane
domain and the 48-aa cytosolic domain. When the mini-Cnx1p
and lumenal-Cnx1p molecules are compared, the overlapping
region corresponds to the C-terminal 52 aa of the lumenal
domain. Moreover, lumenal versions of Cnx1p deleted of the 52
aa failed to support viability (constructs #17 and #18). Therefore,
the essential function(s) of Cnx1p is likely to be located within
this 52-aa stretch of the lumenal domain. Although this region
displays the same basic organization as other calnexin
molecules, no significant conservation at the level of aa sequence
can be observed among different species. Our results may
explain the observation that, in spite of the high degree of
sequence conservation of the central domain and the similarities
in the structural organization between mammalian and fission
yeast calnexin, the canine homologue failed to rescue the lethal
phenotype of cnx1∆ S. pombe cells (Parlati et al., 1995). This
lack of complementation might be due to the poor similarity of
sequence between these two proteins within the 52-aa segment.
We have shown that the 52-aa stretch of Cnx1p is sufficient
for the formation of a complex that includes BiP. Therefore, it
is tempting to speculate that one of the crucial functions of
Cnx1p may reside in the formation of this complex (perhaps
along with other chaperones) that might be implicated in protein
folding. Alternatively, this 52-aa stretch of the lumenal domain
could still contain enough chaperone activity by itself to support
viability of S. pombe. Further experiments are required to
discriminate between these possibilities, and to explore whether
this region encodes a yet to be defined function.
The viability of a calnexin-less mammalian cell line has been
interpreted as the result of a compensation by its soluble
homologue calreticulin (Scott and Dawson, 1995). However,
since glucose-trimming inhibitors do not completely obliterate
the interaction of glycoproteins with calnexin/calreticulin, nor
do they totally inhibit protein secretion, it may be suggested that
lectin-independent glycoprotein folding pathways must exist in
the ER of higher eukaryotes. Such a pathway might involve
protein-protein interactions with calnexin, as it has been
previously proposed, or with the assistance of other chaperones
like BiP, akin to S. pombe (Williams, 1995; Jannatipour et al.,
1998). In this regard, it should be noted that secretion of the
fungal glycoprotein cellulase I is not diminished in S. pombe
cells deleted of the calnexin central domain (our unpublished
results). Complexes containing calnexin and BiP seem to exist
in S. cerevisiae, since kar2/BiP mutants are synthetic with the
deletion of CNE1/calnexin (Brodsky et al., 1999; Simons et al.,
1998). Furthermore, calreticulin was shown to form a stable
complex with BiP in tobacco (Crofts et al., 1998), and
mammalian calnexin interacts with BiP in the absence of
translation (Tatu and Helenius, 1997).
Future endeavors should focus on verifying whether the
central domain of Cnx1p displays lectin activity, and the nature
of a putative chaperone function encoded by the 52-aa Cterminal region of the Cnx1p lumenal domain.
We wish to express our appreciation to Dr Kathy Gould for
providing the his3 strains and plasmids, and to Drs Guy Boileau,
Michel Bouvier and Nabil Seidah for critical reading of the
manuscript. We thank Mehrdad Jannatipour and Chai Ezerzer for their
help with some of the constructions. Also we thank Dr Robert Nabi
for helpful assistance with the microscopy. This work was supported
by grants from the Medical Research Council of Canada (L.A.R.).
J.A. is a Wellcome Trust Senior Fellow, M.C. is a Doctoral Fellow of
the Cystic Fibrosis Foundation of Canada.
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