(Received for publication, August 3, 1994; and in revised form, October 26, 1994)
From the
We have used a polymerase chain reaction strategy to identify in
the yeast Saccharomyces cerevisiae genes of the mammalian
calnexin/calreticulin family, and we have identified and isolated a
single gene, CNE1. The protein predicted from the CNE1 DNA sequence shares some of the motifs with calnexin and
calreticulin, and it is 24% identical and 31% similar at the amino acid
level with mammalian calnexin. On the basis of its solubility in
detergents and its lack of extraction from membranes by 2.5 M urea, high salt, and sodium carbonate at pH 11.5, we have
established that Cne1p is an integral membrane protein. However, unlike
calnexins, the predicted carboxyl-terminal membrane-spanning domain of
Cne1p terminates directly. Furthermore, based on its changed mobility
from 76 to 60 kDa after endoglycosidase H digestion Cne1p was shown to
be N-glycosylated. Localization of the Cne1p protein by
differential and analytical subcellular fractionation as well as by
confocal immunofluorescence microscopy showed that it was exclusively
located in the endoplasmic reticulum (ER), despite the lack of known ER
retention motifs. Although six Ca-binding proteins
were detected in the ER fractions, they were all soluble proteins, and
Ca
binding activity has not been detected for Cne1p.
Disruption of the CNE1 gene did not lead to inviable cells or
to gross effects on the levels of secreted proteins such as
-pheromone or acid phosphatase. However, in CNE1 disrupted cells, there was an increase of cell-surface expression
of an ER retained temperature-sensitive mutant of the
-pheromone
receptor, ste2-3p, and also an increase in the secretion of
heterologously expressed mammalian
-antitrypsin.
Hence, Cne1p appears to function as a constituent of the S.
cerevisiae ER protein quality control apparatus.
Calnexin is an integral membrane calcium-binding phosphoprotein
found in the ER ()of mammalian
cells(1, 2) . Closely related DNA sequences have been
found in plants and nematodes(3, 4) . A function for
calnexin as a molecular chaperone has been identified(1) . It
associates transiently with several membrane glycoproteins during their
maturation in the endoplasmic reticulum including MHC class I heavy
chain(5 8), MHC Class II(9) , the T cell receptor, membrane
Ig(10) , the viral membrane glycoproteins influenza HA and the
``G'' protein of vesicular stomatitis virus (11) as
well as the cystic fibrosis transmembrane conductance
regulator(12) , and integrin(13) . In addition,
calnexin associates transiently with the normal folding intermediates
of soluble monomeric glycoproteins including transferrin,
-antitrypsin, complement C3,
apoB-100(14, 15) , as well as the major secreted
glycoprotein of Maden-Darby canine kidney cells, gp80(16) .
A second related function has been proposed for mammalian calnexin
as a constituent of a protein quality control apparatus in the ER
recognizing and retaining some mutant proteins and components of
unassembled complexes. For example, when soluble secretory
glycoproteins are synthesized in the presence of the proline analog
azetidine 2-carboxylic acid, they are retained in the ER and remain
associated with calnexin for a prolonged period(14) .
Similarly, mutant proteins such as vesicular stomatitis virus G
glycoprotein ts045 are retained in the ER by their association
with calnexin(11) . Components of unassembled complexes are
also retained in the ER in association with calnexin for example, the
MHC class I heavy chain synthesized in the absence of
-microglobulin(5, 17) , and the T
cell receptor synthesized in the absence of the
-chain(18, 19) . Thus, calnexin has the
properties expected of a component of such a quality control mechanism.
Recently, the sequence of a gene in Saccharomyces cerevisiae with similarity to mammalian calnexin has been reported(20) . The isolation of a calnexin homolog from yeast will help elucidate the molecular mechanisms whereby calnexin carries out its roles as a molecular chaperone and the retention of proteins in the ER membrane. We have identified by a PCR strategy a candidate calnexin gene in S. cerevisiae CNE1. We have characterized and localized the Cne1p protein and have determined by gene disruption some of its functions.
Figure 1: The amino acid alignments of S. cerevisiae CNE1, canine calnexin and mouse calreticulin, and the derived PCR cloning strategy. Panel A, the central domains of calnexin and calreticulin are aligned between amino acids 254 389 and 185 281, respectively. Amino acid sequences used to design sense and antisense oligonucleotides are indicated in bold type. Panel B, amino acid sequences and corresponding nucleotide sequences (PCR S, sense; PCR A, antisense) used as primers for PCR amplification of yeast (Y = C or T; R = A or G; n = A, C, G, T). Panel C, amino acid alignment of S. cerevisiae CNE1, dog calnexin, and mouse calreticulin. Amino acids conserved in at least two sequences are shaded.
We used a specific PCR approach to clone genes with sequence
similarity to mammalian calnexin and calreticulin from S.
cerevisiae. Degenerate oligonucleotide primers were designed which
corresponded to the amino acid sequence motifs shared between mammalian
calnexin and calreticulin (Fig. 1, A and B).
Using S. cerevisiae DNA as a template, an amplified DNA
fragment of approximately 300 bp was identified, cloned, and sequenced.
The sequence corresponded most closely to that of mammalian calnexin
(nucleotides 1073-1424) (38%) and of mammalian calreticulin
(24%). This DNA fragment was then used to probe a S. cerevisiae genomic library in the yeast vector YEp24. Two independent clones
with an overlapping common region were isolated from 4 10
colonies screened. The yeast DNA insert was subcloned on the
basis of its hybridization with the DNA probe, and its nucleotide
sequence was determined. The DNA sequence predicts a protein of 502
amino acids and was found to be identical to a previously reported gene
sequence, CNE1(20) . The overall sequence identity to
canine calnexin was 24% and mouse calreticulin was 21% (Fig. 1C). The predicted protein (Fig. 2B) contains a signal sequence, N-linked
glycosylation sites, and a carboxyl-terminal transmembrane
domain(39, 40, 41) . Unlike calnexin, the
predicted Cne1p sequence did not contain a carboxyl-terminal cytosolic
domain, and unlike calreticulin it did not have a carboxyl-terminal ER
retention motif (HDEL in yeast)(2) . Thus, on the basis of
overall predicted structure, the sequence we identified did not closely
resemble either known calreticulin or known calnexin sequences.
Figure 2: Hydrophobicity plot and topology of Cne1p. Hydrophobicity plot (A) and predicted topology (B) of Cne1p showing the five predicted sites of N-linked glycosylation and the single transmembrane domain at the extreme carboxyl terminus. The predicted signal sequence cleavage is at residue threonine 20 (T20).
Figure 3:
Cne1p is an integral membrane
glycoprotein. Panel A, spheroplasts were prepared and
extracted with SDS (0.1%), sodium deoxycholate (1%), 0.1 M sodium carbonate, pH 11.5, Triton X-100, 0.5 M NaCl (high
salt), 2.5 M urea or Tris-buffered saline, pH 7.5 (mock),
followed by centrifugation (30 min at 100,000 g to
give a pellet (P) and supernatant (S) fraction.
Molecular mass markers are indicated on the left. Panel
B, a total particulate fraction of homogenized spheroplasts was
digested with Endo-H giving a change in mobility of calnexin from a
doublet at
76 60 kDa. Molecular mass markers are indicated on the right.
The identification of Cne1p as a doublet
at a molecular mass of approximately 76 kDa on SDS-polyacrylamide gels
is higher than that expected from the predicted sequence. In order to
determine if the protein was N-glycosylated, solubilized
membranes were digested with Endo-H and analyzed by SDS-PAGE. This
treatment resulted in an increased mobility of the protein with an
apparent molecular mass of 60 kDa (Fig. 3B). This
change corresponds to that predicted if all five potential sites of
glycosylation were modified by the addition of core sugars (3 kDa
for each site). However, the predicted molecular mass of the
non-glycosylated protein is 56 kDa.
Figure 4: Comparison of the distribution of the ER marker enzyme NADPH cytochrome c reductase and Cne1p. A, differential centrifugation of S. cerevisiae homogenates into nuclear (N), large granule (ML), microsomal (P), and cytosolic (S) fractions with the distribution of NADPH cytochrome c reductase expressed as a de Duve plot(47) . B, the distribution of S. cerevisiae Cne1p in the same fractions (30 µg of protein was applied to each lane except for P, to which 60 µg of protein was applied and detected by immunoblotting with anti-Cne1p antiserum). The ML fraction contains the highest specific activity of NADPH cytochrome c reductase (panel A) as well as calnexin (panel B).
Figure 5: Isopycnic sucrose density gradient centrifugation analysis of the distribution of Kar2p and Cne1p in the parent ML fraction. ML fractions were centrifuged on linear sucrose gradients as described under ``Experimental Procedures,'' and equal volumes of each fraction were examined for their content of Kar2p and Cne1p determined by immunoblotting with their respective antibodies. The median density of the Kar2p containing compartment was 1.1951 g/ml and that for calnexin was 1.1955 g/ml.
Figure 6: Sucrose density gradient analysis. The distribution of marker enzymes for the Golgi marker enzyme GDPase, the plasma membrane marker ATPase, the mitochondrial marker monoamine oxidase, the ER markers NADPH cytochrome c reductase, the ER luminal protein Kar2p, and the membrane protein Cne1p as determined by analysis of sucrose density gradient. The quantitative distribution of enzyme activities was evaluated as described under ``Experimental Procedures'' and that of Kar2p and calnexin by densitometric evaluation of the data of Fig. 5. The median densities for the distribution of the respective constituents are indicated.
Further examination was carried out by epifluorescence (Fig. 7, A-C) and confocal immunofluorescence microscopy (Fig. 7D). Cne1p (Fig. 7C) was co-localized to a compartment identical to that for the ER luminal protein Kar2p (Fig. 7B); i.e. perinuclear and in filamentous structures extending into the cytosol. DAPI staining of the nuclei is shown in Fig. 7A. Cells were analyzed by confocal microscopy (Fig. 7D) with a strong perinuclear staining pattern observed for Cne1p. In Fig. 7, AC, a sandwich protocol was used (42) whereby rhodamine fluorescence is specific for Cne1p, likewise fluorescein isothiocyanate fluorescence is specific for Kar2p distribution.
Figure 7: Double immunofluorescence of Cne1p and Kar2p in S. cerevisiae by epifluorescence and confocal microscopy. Field showing nuclear staining with DAPI (A). Same field showing Kar2p distribution (B) and Cne1p distribution (C) by epifluorescence microscopy. ER localization of Cne1p by confocal immunofluorescence microscopy (D). The bar represents 2 µm.
Figure 8:
Identification of Ca- binding
proteins in S. cerevisiae ER. Integral membrane proteins (100
µg) from dog ER (lane 1) and from S. cerevisiae ER (50 µg of protein) (lane 2) as well as from
detergent (lane 3) and aqueous (lane 4) phases of
Triton X-114-extracted S.cerevisae ER (100 µg of
protein) were electrophoresed on SDS-PAGE and transferred to
nitrocellulose membrane. In the aqueous phase, six polypeptides of
molecular masses 26, 35, 50, 59, 66, and 72 kDa were identified as
Ca-binding proteins of S. cerevisiae ER. Integral
membrane proteins of 90 and 35 kDa corresponding to mammalian calnexin
and SSR
were identified in the Triton X-114 phase of dog
pancreatic ER. Molecular mass markers as indicated on the left.
Figure 9:
Gene disruption of S. cerevisiae CNE1 and evaluation by Northern blot and Western blot. Schematic
representation of plasmid pFP10.12 containing the entire CNE1 gene and pFP10.13 containing cne1::LEU2.
The CNE1 open reading frame is shaded in black. Restriction sites referred to in the text are shown. Panel A, total RNA from cells containing wild type copy and
cne1::LEU2 was prepared (48) and probed with
labeled DNA containing the entire CNE1 gene. 20 µg of
total RNA was loaded per lane and transferred to nylon membrane. Lane 1(-),
cne1::LEU2 spore
disruptant and lane 2 (+) wild type spore for CNE1.
CNE1 RNA is not detected in
cne1::LEU2 disrupted cells. Panel B, immunoblot detection of S.
cerevisiae Cne1p. Cytosol (S) and total particulate (P) fractions from yeast cell lysates from wild type CNE1 (lanes 1 and 2) or
cne1::LEU2 strains (lanes 3 and 4) were analyzed by
immunoblotting with anti-Cne1p antisera. 20 µg of protein were
applied to each lane. Molecular mass markers are indicated on the left.
Figure 10:
Acid phosphatase secretion. Acid
phosphatase content was evaluated in CNE1-deleted strains
transformed with a calnexin GAL promoter construct. Cells were
grown in sucrose to an OD of 0.1 and then induced 2% with
galactose or repressed with 2% glucose. Sucrose was supplemented to 4%
final concentration. Aliquots were taken at the indicated times for
acid phosphatase as described under ``Experimental
Procedures.''
Figure 11:
Halo assay for -pheromone
production. Wild type strain (W303-1b pVT) (A), CNE1-deleted strain (W303-1b
cne1::LEU2 pVT) (B), or CNE1 overexpressing strain (W303-1b
cne1::LEU2 pVT-CNE1) (C) were spotted on a lawn of a-mating
type cells (strain M200-6C, as described under
``Experimental Procedures''). Agar plates were incubated at
30 °C for 2 days to allow haloes to
develop.
The soluble glycoprotein -antitrypsin is a
substrate for mammalian calnexin(14, 15) , and mutant
-antitrypsin has been shown to be retained by calnexin
prior to its degradation or accumulation in the ER(15) . When
heterologously expressed in yeast, both wild type and Z mutant
-antitrypsin are retained in the ER with the mutant
form being degraded therein(43) . Hence, we were interested to
determine the role of Cne1p in the retention of wild type and Z mutants
of mammalian
-antitrypsin. The amount of secreted
-antitrypsin was tested in wild type and
calnexin-disrupted strains by growing the appropriate strain on agar
plates overlaid with nitrocellulose and immunoblotting with antiserum
to
-antitrypsin. Both wild type (pVT-AlPi) and the Z
mutant (pVT-AlPz) of
-antitrypsin were secreted to a
higher extent in CNE1 disrupted cells than in wild type cells (Fig. 12). Quantitation of the blots showed a 2 2.6-fold
increase in secretion from calnexin disrupted cells (Table 1).
Figure 12:
Effect of Cne1p on the secretion of
-antitrypsin. Wild type
-antitrypsin (pVT-AlPi), Z mutant
-antitrypsin (pVT-AlPz), or vector alone (pVT) were transformed
into W303-1a (CNE1) or W303-1a
cne1::LEU2 (
cne1::LEU2)
cells. Equal numbers of cells were spotted onto agar plates and
overlaid with nitrocellulose membrane and incubated overnight at 30
°C. The nitrocellulose membrane was washed, immunoblotted with
anti-
-antitrypsin antisera, and revealed by the
alkaline phosphatase method (see ``Experimental
Procedures'').
The evaluation of a possible retention function for CNE1 was extended to an endogenous yeast seven transmembrane
glycoprotein, the -pheromone receptor, Ste2p. This protein is
normally present and functional in the plasma membrane of S.
cerevisiae, but the ste2-3ts mutant has been shown
to be intracellularly retained (
)at restrictive temperature
(37 °C) resulting in a 100-fold decrease in mating frequency. To
determine if Cne1p plays a role in the intracellular retention of ste2-3ts, we evaluated its function at the cell surface
with a quantitative mating assay. At the non-permissive temperature,
the relative mating efficiency was 5-fold greater in CNE1-deleted strains indicating increased transport and/or
function of ste2-3ts protein at the plasma membrane (Table 2).
In mammalian cells calnexin has been shown to have a central role in the retention of incompletely folded glycoproteins in the ER and in the assembly of multisubunit cell surface receptors (see (1) ). The presence of a calnexin homolog would be of considerable interest as its function could be studied using the range of tools available in this organism. An important question is whether CNE1 is the calnexin or calreticulin homolog in yeast. We have addressed this question in three ways: by a comparison of the sequences, by an analysis of the protein, and by the phenotype of cne1-deleted cells.
The PCR strategy that we employed was expected to generate from yeast DNA sequences which corresponded to calreticulin as well as calnexin. Although 11 separately cloned 250-350-bp products of the PCR reaction were sequenced, only the yeast CNE1 sequence was detected as an open reading frame (5 out 11 clones). All other clones sequenced did not have an open reading frame and did not contain internal similarities to calnexin or calreticulin. This PCR-generated sequence was used as a probe to clone the complete CNE1 gene from a yeast plasmid library. Of the two different plasmids recovered, both contained the same CNE1 gene. Using the complete CNE1 sequence as a probe, we further determined if there were related sequences in the yeast genome using the lambda clone grid filters. Using hybridization at low stringency on these filters and on a Southern blot of DNA from a CNE1 disrupted strain, we were unable to detect any related sequences. Thus by hybridization criteria there do not appear to be genes in yeast which are more closely related to CNE1. The CNE1 gene we mapped by this technique is located on the left arm of chromosome I, distal to genes CDC24 and CDC19 and to other known mapped genes (44) .
Mammalian calnexin and calreticulin have the motifs of KPEDWDE repeated three times. Only one related motif was found in S. cerevisiae CNE1 at residues 255 261 consisting of KPHDWDD. Mammalian calnexin also reveals three repeats of GXW. Only two were found in CNE1. In the plant Arabidopsis thaliana, a calnexin gene has been identified with greater sequence similarity to mammalian calnexin than that of S. cerevisiae(3) . All three KPEDWDE motifs are retained as well as the three GXW motifs and a cytosolic tail albeit without sequence identity to that of mammalian calnexins. In addition, the overall organization of the Cne1p terminates in a hydrophobic sequence and lacks the carboxyl-terminal cytosolic domain found in other calnexins. We also confirmed that there is not a motif for an RNA splice site present which could account for an alternative CNE1 sequence.
The sequence of the predicted S.
cerevisiae Cne1p protein predicts an NH-terminal
hydrophobic signal sequence, 5 N-linked glycosylation sites,
and a carboxyl-terminal hyrophobic potentially membrane spanning
sequence. We confirmed the localization of Cne1p in the yeast ER by
differential and analytical subcellular fractionation and by
epifluorescent and confocal immunofluorescence microscopy which showed
a co-localization of Cne1p and the ER luminal protein Kar2p. We
confirmed that Cne1p is an integral membrane protein as it could not be
extracted from membranes by treatment with 2.5 M urea, high
salt, and sodium carbonate at pH 11.5. This is a property that Cne1p
shares with mammalian calnexin which is also an integral membrane
protein, whereas calreticulin is a soluble ER luminal protein. We also
confirmed that Cne1p has N-linked glycosylation as predicted
from the sequence. After Endo-H treatment the relatively tight mobility
of Cne1p in SDS-PAGE was altered by about 18 kDa, indicating that all
potential N-glycosylation sites are utilized(45) .
An ER membrane protein such as Cne1p (depicted in Fig. 2A) is unusual because only 1 amino acid is
predicted to be cytosolically exposed. Since we have demonstrated
localization of Cne1p in the yeast ER there is a question of how it is
retained. We have confirmed that S. cerevisiae Cne1p was not
GPI linked since no incorporation of [H]inositol
was detected nor was the protein susceptible to digestion by
PI-specific phospholipase C. In mammalian calnexin the cytosolically
oriented sequence RKPRRE has been shown to act as retention and/or
retrieval sequences, maintaining this type I integral membrane protein
in the ER(18) . The lack of a cytosolic tail for S.
cerevisiae Cne1p but its localization to the yeast ER implies that
retention is effected by association with an unknown resident membrane
or luminal protein and not by the cytosolic proteins interacting with a
retention motif(46) .
Mammalian calnexin has been shown to be one of two major calcium-binding integral membrane proteins of the ER(2) . Similar experiments with yeast ER membranes showed that there do not appear to be any abundant calcium-binding proteins present in the ER membrane (Fig. 8), although we did detect yeast ER lumenal calcium-binding proteins. Indeed this is the first demonstration of calcium-binding proteins in the ER of S. cerevisiae. Confirmation of the inability of yeast Cne1p to bind calcium in vitro was obtained with isolated E. coli produced GST::Cne1p fusion protein (not shown). Calcium has been demonstrated to be essential for the binding of mammalian calnexin with its protein substrates(2, 14) . Although Cne1p has sequence similarity with mammalian calnexin, it is atypical in that it is N-glycosylated, it is an integral ER membrane protein but does not have a recognizable retention mechanism, and unlike mammalian calnexin it is not a calcium-binding protein.
Calnexin genes from
different organisms show a considerable conservation in their sequence
suggesting that the function of the protein is similar and that the
preservation of the sequence is important for that function. Mammalian
calnexin has been identified as a molecular chaperone for newly
synthesized soluble and membrane-bound glycoproteins of the secretory
apparatus(1) . Mammalian calnexin has also been identified as
responsible for the ER retention of soluble and membrane-bound proteins
prior to their exit from the ER. These functions suggested that there
would be an essential phenotype for yeast cells which lack calnexin.
However, yeast strains carrying a deletion of the CNE1 gene
were viable and grew at normal rates, and we were unable to identify
any effect on the secretion of the glycoproteins -pheromone or
acid phosphatase. From the results with some mammalian secretory
proteins, there is evidence that they bypass the participation of
calnexin in their folding(14) . This observation has been
attributed to alternative, or back up, mechanisms for protein folding
in the mammalian ER other than the calnexin pathway (1) .
We
did observe an effect on the retention of heterologously expressed
-antitrypsin in S. cerevisiae as well as
function of a temperature-sensitive mutant ste2,3 ts of the
-pheromone receptor in CNE1-disrupted cells. The effect
on ste2,3 ts could be due to an effect of Cne1p on its
intracellular trafficking or on its function at the plasma membrane.
The latter explanation is less likely since Cne1p is clearly localized
in the ER. Although these effects are small they suggest that the Cne1p
is a constituent of the yeast quality control apparatus participating
in the retention of heterologously expressed or incorrectly folded
proteins.
There remains the question of whether the CNE1 gene we have identified and its gene product, Cne1p, we have characterized represents the yeast calnexin homolog or whether there is another closer relative of mammalian calnexin or calreticulin in the yeast genome? We obviously cannot totally exclude this possibility, but the genetic methods currently available in this organism provide an opportunity to identify genes whose function are synergistic with CNE1.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L11012[GenBank].