(Received for publication, August 17, 1994; and in revised form, October 18, 1994)
From the
We have cloned a Schizosaccharomyces pombe gene, here designated cnx1, encoding the homologue of the endoplasmic reticulum molecular chaperone calnexin. Disruption of the cnx1 gene was lethal, demonstrating that it has an essential cellular function. Transcription of cnx1 mRNA is initiated at multiple sites, and it can be induced by various stress treatments that lead to the accumulation of unfolded and/or misfolded proteins in the endoplasmic reticulum. The encoded Cnx1p protein more closely resembles its plant and animal calnexin homologues than that of Saccharomyces cerevisiae. Cnx1p is acidic and migrates aberrantly on SDS-polyacrylamide gel electrophoresis, similar to its mammalian counterparts. Cnx1p contains the hallmark KPEDWD motifs that are found in all members of the calnexin/calreticulin family of proteins. Using an in vitro translation-processing system, we have shown that Cnx1p has the characteristic type I topology of calnexin proteins. Unlike its higher eukaryotic homologues, Cnx1p has a site for N-glycosylation that was modified in an in vitro translation-processing assay.
Calreticulin and calnexin are two endoplasmic reticulum (ER) ()proteins that have attracted considerable attention in
recent years. Calnexin is a type I membrane protein whose lumenal
domain shares considerable sequence similarity with calreticulin,
including three copies of the characteristic KPEDWD motif (for reviews
see Refs. 1, 2). Calnexin is capable of binding Ca
and it was proposed to be involved in the retention of soluble ER
proteins in a Ca
-dependent manner(3) .
Initial reports have identified calnexin as a protein involved in the
assembly of class I histocompatibility molecules (p88; 4) and the
T-cell receptor (IP90; 5, 6) providing an indication of its cellular
function. Further studies have demonstrated that calnexin is a major
molecular chaperone interacting with numerous newly synthesized
glycoproteins transiting through the
ER(7, 8, 9) . It has been proposed that
calnexin is part of the ER quality control machinery, binding folding
intermediates through their oligosaccharide moieties until these
substrates achieve proper folding or until misfolded proteins are
degraded(1, 2, 10) . The isolation and
sequencing of cDNA and genomic clones of mammalian, Xenopus
laevis, plant, and helminth species, have revealed that the
general structural organization of calnexin has been conserved through
evolution(1, 3, 6, 11, 12, 13, 14) .
However, Saccharomyces cerevisiae appears to be an exception,
as calnexin from this yeast contains a single copy of the KPEDWD repeat
and does not posses a cytosolic domain(15) . The cytosolic,
C-terminal domain of mammalian calnexin is phosphorylated at
serine residues by casein kinase II. It was proposed that
phosphorylation of calnexin could be involved in modulation of its
function(3, 12) .
Calreticulin is a major
calcium-binding, soluble intraluminal protein (reviewed in (16) ). Complementary DNAs encoding calreticulin have been
cloned from numerous organisms (16-23, and references therein).
The encoded polypeptides show a remarkable degree of conservation,
especially in the central portion, or P-domain, which is
relatively rich in prolines and contains three KPEDWD
repeats(16, 24) . An ever growing body of intriguing
observations indicates calreticulin may be involved in several cellular
processes(25, 26) . For instance, Dedhar and
collaborators (27) presented evidence that calreticulin binds
to the highly conserved peptide KXGFFKR present in the
cytoplasmic domain -subunit of integrin receptors, and they have
further shown that this interaction occurs also in vivo(28) . The observation that the almost identical peptide
KXFFKR is found in the DNA-binding domain of all steroid
receptors led to the demonstration that calreticulin interacts in
vitro with the glucocorticoid receptor and the androgen receptor (29, 30) . Moreover, it was shown that overexpression
of calreticulin can inhibit the transcriptional activity of the
glucocorticoid, the androgen and the retinoic receptors, as well as
retinoic acid-induced neuronal
differentiation(29, 30) .
To better understand the cellular functions of the calreticulin/calnexin family of proteins, we set out to clone the genes encoding these proteins in the fission yeast Schizosaccharomyces pombe. As its distant relative the budding yeast S. cerevisiae, S. pombe is suitable for analysis by classical and reverse genetic approaches(31, 32) . However, in several molecular, functional and morphological aspects, S. pombe appears more similar to higher eukaryotes than the budding yeast. For instance, S. pombe genes are more homologous to those of higher eukaryotes(31, 32) , the cell-cycle control and cell division are akin to those of higher eukaryotes, and S. pombe has well defined ER and Golgi structures which are easily identifiable by EM(33, 34) . In addition, glycoproteins of the fission yeast acquire terminal galactose residues like higher eukaryotes do(35, 36) . These features make S. pombe an ideal model organism for the study of gene function and cellular processes of higher eukaryotes.
In this study, we describe the isolation of the gene encoding the S. pombe calnexin homologue. The S. pombe calnexin species possesses the characteristic type I topology, and its amino acid sequence is more closely related to its plant and mammalian counterparts than to the S. cerevisiae homologue. Unlike the species from higher eukaryotes, the fission yeast calnexin contains a site that can be glycosylated in an in vitro translation-processing system. We demonstrate that disruption of the S. pombe calnexin gene is lethal and that its expression is induced by several types of stress.
Figure 4: N-glycosylation and topology of Cnx1p. Synthetic mRNAs of different lengths were used in an in vitro translation-processing system to elucidate the topology of Cnx1p. Unique internal restriction sites used to synthesize RNA are as follows: CI, ClaI; EI, EcoRI; NI, NcoI; BI, BamHI. Panels corresponding to the four versions of Cnx1p are labeled a-d. The predicted signal peptide region of Cnx1p is indicated as SP. The N-glycosylation site is shown as a diamond. Conditions used in each assay 1-7 were as described under ``Materials and Methods'' and are indicated under each lane.
Figure 2: The nucleotide and deduced amino acid sequence of the S. pombe cnx1 gene. TATA box sequences are shown as solid boxes. The putative heat-shock element and unfolded protein response element are shown as open boxes at positions -132 to -119 and -444 to -427, respectively. The predicted site for signal sequence cleavage is indicated as solid, downward triangle. The putative N-linked glycosylation site is shown as open box at amino acid positions 418-420. The potential membrane spanning region of Cnx1p is underlined. Putative sites for phosphorylation by protein kinase C are shown in bold letters. Putative polyadenylation sites are underlined with solid lines. Beginning and end of the cDNA sequences are shown by arrows. Transcription initiation sites are shown in circles. Solid circles denote sites that are preferentially utilized as compared to sites indicated with open circles.
Figure 6:
Gene disruption of S. pombe cnx1.
A 333 bp stretch was deleted form the cnx1 gene and replaced
with the ura4 marker gene. A linear fragment containing the ura4 interrupted cnx1 sequences was transformed into
the diploid SP629 in order to disrupt, by homologous recombination, one
of the copies of the cnx1 gene. Ura transformant strains were analyzed by Southern blotting; clone
SP5 had the expected genotype. A, Southern blot of genomic DNA
digested with EcoRI-XbaI and EcoRI-BglII and probed with an EcoRI-ClaI fragment (cnx1 coordinates
455-1072). DNA from wild type strain SP629 contains two hybridizing
fragments of 0.5 and 2.6 kb upon digestion with EcoRI-XbaI (lane 1) and one hybridizing
fragment of 1.3 kb upon digestion with EcoRI-BglII (lane 3). Strain SP5 contains an additional hybridizing band
of 1.9 kb upon digestion with EcoRI-XbaI (lane
2) and an additional hybridizing band of 2.7 kb upon digestion
with EcoRI-BglII (lane 4), indicating
homologous integration and disruption of one copy of the cnx1 gene. B, random spore analysis of the diploid strain SP5
containing one copy of cnx1 disrupted by ura4.
Approximately 200 spores were counted, washed, and plated on non
selective media. Colonies were picked and examined for growth in the
presence (B1) and absence (B2) of uracil. None of the
spores examined grew in the absence of uracil indicating that cnx1 is an essential gene. Only one of the plates examined is shown
here. The diploid SP5 strain was used in the lower right of the plate
as control. C, physical map of the wild type and disrupted
copies of cnx1 in SP5. The hatched boxed denotes the cnx1 coding sequence. The black box and arrow indicate, respectively, the ura4 gene and its direction
of transcription. Only relevant restriction sites are indicated: B, BglII; EI, EcoRI; EV, EcoV; C, ClaI; X, XhoI.
Figure 3: Alignment of amino acid sequences of calnexins from human (H), A. thaliana (A), S. pombe (P), S. cerevisiae (Y), and human calreticulin (C). Identical residues are denoted by colons (:), and spaces introduced for sequence alignment are designated by dashes (-). The KPEDWD repeats are shown in solid boxes, whereas less conserved versions of this motif (five matches of a total of 7 residues) are denoted with open circles. Open boxes A-D represent conserved regions among calnexins from different species and human calreticulin, according to the nomenclature in (3) . Amino acids corresponding to predicted transmembrane domains are underlined. The calreticulin amino acid sequences corresponding to probes 126, 2-18, and 2-20 are shown by single-lined, doubled-lined, and dashed arrows, respectively. Computer analyses were done with the GCG package(62) .
Figure 1: Cloning of S. pombe cnx1.A, S. pombe genomic DNA was digested with EcoRI, XbaI, and HindIII (lanes E, X, and H, respectively) and subject to Southern analysis under conditions of reduced stringency of hybridization as described under ``Materials and Methods.'' Probes used were as follows: panel 1, 126 bp; panel 2, 2-18; panel 3, 2-20; panels 4 and 5, 503-bp EcoRI-XbaI fragment from P1 clone (see below). Panel 5 represents a shorter exposure time of panel 4. An EcoRI-XbaI restriction fragment from clone P1 (503 bp; coordinates 569-1072; Fig. 2) was used in panels 4 and 5. Lane M represents molecular mass markers in kb. B, schematic representation of the clones P1 and P6. Relevant restriction enzyme sites are abbreviated as follows: EI, EcoRI; X, XbaI; H, HindIII; B, BglII.
A feature common to both calreticulins
and calnexins is the presence of KPEDWD sequence repeats which are
generally followed by Glu or Asp residues and sometimes Lys or Arg. The S. pombe calnexin contains three copies of these repeats as
well as one less conserved copy, whereas the S. cerevisiae species contains only one copy of the KPEDWD repeat (see Fig. 3). Unlike its animal and plant counterparts, Cnx1p does
not contain 2 basic residues at position 3 and
4 (or
5) form its C terminus, which constitutes a motif found in membrane proteins
retained in the ER; however, it contains a lysine at position
4(45, 46) . A computer search for
putative modifications revealed several potential sites for
phosphorylation by casein kinase II (19-SLAD-23, 26-SEQE-29,
77-TVEE-80, 144-THGE-147, 409-SIED-412, 433-SKQE-436, and 481-TIIE-484)
and protein kinase C (51-SER-53, 184-SEK-186, 540-TEK-542, and
555-TAK-557). Another feature of the amino acid sequence is the
presence of a site for potential N-linked glycosylation at
position 418-NETF-421 (see below and Fig. 2).
Calnexins from other species were reported to have a type I topology. As mentioned earlier, Cnx1p contains a stretch of 23 relatively hydrophobic amino acids (residues 490-512) with potential to be a membrane-spanning domain, and therefore it was expected that the S. pombe species could also be a type I protein. As a first step to establish the topology of Cnx1p, we investigated whether it was a membrane-integral protein by performing carbonate treatment (41) on the four translation products described above. In this procedure, peripheral proteins are found in the supernatant of carbonate-treated membranes, whereas integral proteins remain in the pellet. When lanes 4 in Fig. 4are examined, it is possible to observe that approximately 50% of the full-length polypeptide (560 aa; Fig. 4a) is found in the membrane pellet, while the shorter polypeptides, which lack the putative transmembrane domain, are found in the supernatant (Fig. 4, lanes 5). The fact that the full-length (560 aa) protein was found distributed between both pellet and supernatant could be due to incomplete precipitation during centrifugation and/or because inefficient integration of the protein in a heterologous in vitro system. These results validate that Cnx1p is an integral membrane protein. To determine the membrane orientation of Cnx1p, trypsin protection assays were performed on in vitro translated-processed products (Fig. 4, lanes 6). As control, this experiment was also carried out in the presence of Triton X-100 to dissolve the microsomes (Fig. 4, lanes 7). Based on the amino acid sequence of Cnx1p, it can be predicted that with a type I orientation, the 48 C-terminal amino acids would be exposed to the cytosolic side of the ER membranes. When the full-length translation product (560 aa) is subjected to trypsin digestion we observed an increase in the polypeptide mobility consistent with the removal of the 48 amino acids at the C-terminal end of Cnx1p. In the presence of detergent, the polypeptides were completely digested with the exception of the processed product of the 151 aa precursor (Fig. 4, lanes 7). The resistance of the latter to trypsin digestion could be due to its folding into a compact structure. We conclude from these experiments that the cnx1 gene product is a type I membrane protein.
Figure 5:
Expression of cnx1 mRNA. A, Northern blot analysis of cnx1 mRNA. Total RNA
from S. pombe cultures under different stress conditions was
analyzed by Northern blotting as described under ``Materials and
Methods.'' A 618-bp EcoRI-ClaI fragment from cnx1 gene (coordinates 455-1072) was used as probe (indicated
as cnx1). Lane 1, control cells grown at 30 °C
for 2 h; lane 2, 5 min shift from 30 to 39 °C; lanes
3-5, shift from 30 to 39 °C for 10, 30, or 60 min,
respectively; lane 6, 2 h in 10 µg/ml tunicamycin at 30
°C; lane 7, 2 h in 10 µM A23187 at 30 °C; lane 8, 2 h in 15 mM -mercaptoethanol at 30
°C; lanes 9 and 10, 2 h in presence or absence of
10 mM 2-deoxyglucose at 30 °C, respectively. For
quantitation purposes, the relative intensity of each cnx1 mRNA band was determined by soft-LASER densitometric scanning and
compared to the values obtained when samples were hybridized with an
actin probe (indicated as act1; (63) ). Note that
while ethidium bromide staining of the gel showed equivalent loading of
total RNA samples (not shown), the three bands corresponding to the
1240, 1650, and 1850 nucleotide long S. pombe actin mRNAs
varied with the treatments. B, mapping of transcription
initiation sites. Primer extension was performed as described under
``Materials and Methods'' on the same RNA samples as in A.
To
identify the cnx1 promoter, we analyzed the sequences upstream
of the cnx1 coding region searching for known regulatory
sequences. At positions 212 to
207
and
584 to
579, we identified two
stretches perfectly matching the consensus for TATA boxes (see Fig. 2; 54). The stretch GTTCCGGAACCTTC (positions
132 to
119; see Fig. 2)
closely resembles the consensus for the so-called heat-shock regulatory
element found in the promoters of heat-induced genes of different
organisms, including S. cerevisiae and S.
pombe(33, 48, 49, 50, 53) .
The unfolded protein response element is another regulatory sequence,
which is distinct from the heat-shock regulatory element, and it also
found in the promoter of several ER-protein genes (such as GRP78, KAR2, and EUG1), whose expression is induced upon
accumulation of unfolded proteins in this
compartment(33, 48, 49, 50, 53, 55, 56) .
In the cnx1-promoter region we found the stretch
TTCAAAGACTACGAGTATAGC (positions
444 to
427; see Fig. 2), that shows similarity with
the consensus unfolded protein response element, and that resembles
more closely to the sequence TTCAAAGGCACGCGTGTCC which comprises the
unfolded protein response element of the S. cerevisiae EUG1 gene (56; identities are in underlines).
To further define the cnx1 promoter as well as to gain further insight into the
regulation of the cnx1 mRNA expression, we performed primer
extension experiments with RNA extracted from cells that were exposed
to different stress conditions. In these experiments, samples of RNA,
isolated as for Northern blot, were subjected to primer extension using
an end-labeled oligonucleotide (P1CARC2: 5`-CGAACATATAAAGAGCAC-3`)
which anneals to mRNA at position +53 to +36. As shown in Fig. 5B, transcription of the cnx1 mRNA starts
at multiple sites. Consistent with the results obtained by Northern
blotting we noted that several different treatments induce the
expression of cnx1. Moreover, these primer-extension
experiments revealed that specific sites are preferentially utilized
under different stress conditions. For example, the intensity of the
bands corresponding to the 28 and
54 sites increased with the length of the heat shock
treatment, whereas the utilization of other sites, under the same
conditions, followed a more complex pattern (invariant, increase, and
decrease; see Fig. 5B, lanes 1-5). In
addition, transcription from start site at
76 becomes
apparent upon heat shock and treatment with calcium ionophore A23187
(see Fig. 5B, lanes 4, 5, and 7).
We report here the isolation of genomic and cDNA clones encoding the S. pombe homologue of the ER chaperone calnexin. As observed previously for other fission yeast gene products(31, 32) , the amino acid sequence of S. pombe calnexin, here designated Cnx1p, is more closely related to plant and animal homologues (identities between 37.3-40.1%) than to the CNE1 protein, the S. cerevisiae counterpart (35.2% identity). Furthermore, Cnx1p shares additional features with its higher eukaryotic homologues. Cnx1p contains three KPEDWD repeats (found also in calreticulins), whereas the S. cerevisiae protein contains a single copy of this motif. Akin to mammalian calnexin but unlike to the arabidopsis protein, Cnx1p moves on SDS-PAGE as a 90 kDa band although its predicted molecular mass is 63.4 kDa, the aberrant migration probably due to its acidic composition (calculated pI = 4.25). As we have shown, Cnx1p has a type I topology, with a 48 amino acid cytoplasmic domain which is similar in length to the arabidopsis protein; however, both are shorter than in the mammalian species. In contrast, according to structural predictions, the cytosolic domain would be absent in the S. cerevisiae protein. The cytosolic domain of mammalian calnexin has been shown to be phosphorylated by casein kinase II, and this phosphorylation was proposed to be involved in the regulation of calnexin function(3, 12) . In this respect, two potential sites for phosphorylation by protein kinase C are found in the cytosolic region of Cnx1p. It was of interest to find that unlike its higher homologues, the Cnx1p protein contains a site for N-glycosylation that was functional in a heterologous, in vitro translation-processing system. The S. cerevisiae protein contains several potential sites for glycosylation; however, no evidence is available if these sites are exposed.
Our studies have shown that disruption of the cnx1 gene has a lethal phenotype in S. pombe, thereby demonstrating that calnexin has an essential function in the vegetative growth of this yeast, probably playing a key role as a component of the machinery controlling correct protein folding in the ER. Moreover, these results strongly indicate that no other functional homologues of calnexin are present in this fission yeast. In addition, Southern blot analysis using as probe the cnx1 gene also indicated that no other genes, with similar sequence, can be detected in this organism. As mentioned above, calreticulin has been found in numerous animal species and recently also in barley, and these proteins show a remarkable degree of conservation. Since in evolutionary terms, yeasts are more closely related to animals than to plants, we expected to find a calreticulin homologue in S. pombe(57) . Therefore, it was rather surprising that we were not able to isolate a gene encoding the S. pombe calreticulin, especially considering that our cloning approach consisted in the screening of libraries by low stringency hybridization, using fragments of a cDNA encoding human calreticulin as probes. The simplest explanation of this result is that the gene encoding the S. pombe calreticulin has a sequence with limited similarity to its homologues from other species. This seems improbable, however, considering the striking sequence conservation among the calreticulins. Thus, it is possible to envision that the Cnx1p protein performs the function of both calreticulin and calnexin in S. pombe.
As other chaperones of the ER, the
expression of cnx1 was induced when cells were subjected to a
variety of stresses causing the accumulation of misfolded and
aggregated proteins in the ER, such as heat shock as well as treatments
with calcium ionophore A23187, -mercaptoethanol, and
2-deoxyglucose. In the promoter region of cnx1, we noted
sequences resembling regulatory boxes, such as the heat-shock
regulatory element and the unfolded protein response element, that were
previously identified in the promoters of other stress-induced genes (33, 49, 50, 53) and are likely to
be involved in the control of cnx1 expression. Additionally,
the stress treatments used in this study differentially affected the
utilization of certain among the multiple cnx1 transcription-initiation sites, probably reflecting the binding of
the stress-related transcription factors to different sites. A
fascinating question is how the signal indicating accumulation of
unfolded proteins in the ER is transmitted to the nucleus to induce
there the expression of ER chaperone genes. In S. cerevisiae,
the gene ERN1/IRE1 has been identified as being
required for this signal transduction
pathway(51, 52) . ERN1/IRE1 encodes
a type I integral protein of the ER, whose luminal N-terminal portion
is glycosylated and whose C-terminal region contains a
cdc2
/CDC28-related kinase activity. It was proposed (51, 52) that accumulation of misfolded proteins
causes a ligand-mediated dimerization of Ern1p/Ire1p, which in turn
activates its cytosolic kinase domain and that initiates the signal
transduction cascade. A similar mechanism would also be expected to
occur in S. pombe and other organisms. However, additional
signaling pathways/factors should be present, since cnx1 is
induced by 2-deoxyglucose but not by tunicamycin, whereas the
expression of S. pombe and S. cerevisiae BiP is
induced by both inhibitors(33, 49, 50) . In
this context, it should be noted that these inhibitors differ in their
mode of action, tunicamycin inhibits the synthesis of the
dolichyl-PP-GlcNAc
Man
Glc
precursor,
and, consequently, no oligosaccharide is transferred onto the
asparagine side chain of the target proteins(58) . On the other
hand, 2-deoxyglucose could be incorporated into glycoproteins and
probably inhibit the deglucosylation-reglucosylation (trimming) of
newly synthesized glycoproteins(58, 59, 60) .
Therefore, the signaling pathway inducing cnx1 expression
responds to the accumulation of misfolded-partially folded glycosylated
proteins, as those resulting from treatments such as heat-shock,
-mercaptoethanol, and 2-deoxyglucose. In contrast, no signal for
the induction of cnx1 expression is produced when
non-glycosylated, malfolded proteins are accumulated in the ER, as in
the case of treatment with tunicamycin. Mammalian calnexin has been
shown to be a chaperone with selectivity for glycosylated proteins, as
this molecular chaperone does not bind nascent proteins when cells are
treated with tunicamycin nor non-glycosylated proteins such as serum
albumin(1, 2, 8, 9, 10) .
Based on the observation that glucosidase inhibitors also obliterate
the interaction of nascent proteins with
calnexin(8, 55) , Helenius and collaborators have
proposed a model in which newly synthesized glycoproteins must be
mono-glucosylated in order to bind to
calnexin(2, 9, 10) . As observed by Parodi et al.(61) , these mono-glucosylated glycoproteins are
produced during the deglucosylation-reglucosylation trimming cycle,
carried out on partially folded glycoproteins by the glucosydases I and
II, and UDP-glucose:glycoprotein glucosyltransferase. Thereby,
mono-glucosylation would be an indication of incomplete folding (61) and thus according to Helenius' model, partially
trimmed glycoproteins could be recognized and bound by
calnexin(2, 9, 10) . Although it remains to
be proven, Cnx1p probably has the same substrate selectivity as its
mammalian counterparts. Thus the protein involved in the first step of
the transducing pathway that signals accumulation of unfolded proteins
and that induces the expression of the cnx1 gene would have
the same specificity as Cnx1p. It is therefore tempting to speculate
that this sensor protein could be Cnx1p itself and that it would
communicate the first signal via its cytosolic domain. Furthermore, the
state of phosphorylation of the Cnx1p cytosolic domain could modulate
the recruitment of factor(s) involved in this transduction pathway.
The availability of a genetic system for the S. pombe calnexin homologue Cnx1p opens new avenues of research to perform structure-function studies on calnexin and to elucidate its functional relationship with calreticulin, as well as to delineate the mechanisms controlling the unfolded protein response in the fission yeast.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U13389[GenBank].