©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Saccharomyces cerevisiae CNE1 Encodes an Endoplasmic Reticulum (ER) Membrane Protein with Sequence Similarity to Calnexin and Calreticulin and Functions as a Constituent of the ER Quality Control Apparatus (*)

(Received for publication, August 3, 1994; and in revised form, October 26, 1994)

Francesco Parlati (1) (3) Michel Dominguez (2) John J. M. Bergeron (2) David Y. Thomas (1) (3) (2)(§)

From the  (1)Department of Biology and (2)Department of Anatomy and Cell Biology, McGill University, Montreal H3A 2B2, Quebec and the (3)Genetics Group, Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec H4P 2R2, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 alpha-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 alpha-pheromone receptor, ste2-3p, and also an increase in the secretion of heterologously expressed mammalian alpha(1)-antitrypsin. Hence, Cne1p appears to function as a constituent of the S. cerevisiae ER protein quality control apparatus.


INTRODUCTION

Calnexin is an integral membrane calcium-binding phosphoprotein found in the ER (^1)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, alpha(1)-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 beta(2)-microglobulin(5, 17) , and the T cell receptor synthesized in the absence of the alpha-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.


EXPERIMENTAL PROCEDURES

Strains and Media

The S. cerevisiae diploid strain W303D (MATa/alphaade2-1/ade2-1 can1-100/can1-100 ura3-1/ura3-1 leu2-3, 112/leu2-3, 112 trp1-1/trp1-1 his3-11, 15/his 3-11, 15), W303-1a (MATaade2-1 can1-100 ura3-1 leu2-3, 112 trp1-1 his3-11, 15), W303-1b (MAT alpha ade2-1 can1-100 ura3-1 leu2-3, 112 trp1-1 his3-11, 15), DC 17alpha (MAT alpha his1), and M200-6C (MATasst1 sst2) strains were grown at 30 °C in YPD medium containing 1% yeast extract (Difco), 2% bacto-peptone (Difco), and 2% dextrose (BDH) or synthetic media (SC) with the appropriate amino acid supplements and either 2% glucose or 2% sucrose. The Escherichia coli strain MC1061 was used(21) . Yeast synthetic media were as in Sherman et al.(22).

PCR Amplification

To identify and isolate genes similar to calnexin from S. cerevisiae genomic DNA, degenerate oligonucleotides for the sequences KPEDWDE and YKG^K/(E)WKP with all possible codons at each position were synthesized using a BioSearch series 8000 DNA synthesizer (see Fig. 1B). Amplification was performed using a Perkin-Elmer Cetus thermocycler(23) . Samples were then electrophoresed on a 2% agarose gel and visualized by ethidium bromide staining. The band migrating at approximately 300 bp was purified by electroelution and cloned into the SmaI site of plasmid pTZ19R and sequenced by the dideoxy protocol using T7 DNA Polymerase (Pharmacia Biotech Inc.).


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.



Cloning of Calnexin in S. cerevisiae

In order to clone the entire sequence of the gene we identified, YEp24 genomic S. cerevisiae DNA libraries were screened using the isolated calnexin PCR fragment as a probe labeled by nick translation(21) . Two independent clones were isolated and mapped using restriction enzyme analysis. By Southern analysis, a 3.8-kb SphI fragment was found to hybridize to the PCR probe and was subcloned into the SphI site of pTZ19R (Pharmacia) to generate plasmid pFP10.1. Based on sequence information provided by the PCR fragment, oligonucleotides were synthesized and used to sequence the gene as described previously(21) .

Antibody Production

Polyclonal antibodies recognizing calnexin were obtained by immunizing rabbits with GST::Cne1p fusion proteins expressed in E. coli. The fusion was made by inserting a BamHI-SphI fragment (CNE1) into pGex-2T(24) . GST-calnexin was expressed by isopropyltho-beta-D-galactoside induction and purified(24) .

Membrane Extraction and Endo-H Digestion

Extracts of post-nuclear supernatants were mixed with 1 volume of 1 M NaCl, 0.2 M sodium carbonate, pH 11.5, 2.5 M urea, 2% Triton X-100, 0.2% Triton X-100, 2% deoxycholate, or 0.2% SDS and were subsequently analyzed as described previously(25) . Cne1p antiserum was used at 1:2000 dilution. Endo-H digestions were performed by incubating 50 µg of ML fraction proteins in 100 mM sodium acetate, pH 4.9, 150 mM NaCl, 10 mM dithiothreitol, 1% Triton X-100 + inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 mg/ml pepstatin, 1 mg/ml leupeptin, and 1 mg/ml aprotinin) and incubating with 2 µg of Endo-H for 16 h at 37 °C.

Yeast Fractionation

S. cerevisiae strain W303-1a was grown at 30 °C in YPD medium to a density 2-4 OD/ml cells were harvested by centrifugation and washed in water. Spheroplasts (100 OD/ml) were generated by a 60-min incubation at 30 °C in 0.7 M sorbitol, 1.5% peptone, 0.75% yeast extract, 0.5% glucose, 10 mM Tris, 1 mM dithiothreitol, and Zymolyase T100 1 mg/g wet weight yeast and homogenized with a Potter-Elvejhem homogenizer in 0.1 M sorbitol, 20 mM HEPES, 50 mM potassium acetate, pH 7.4, 1 mM phenylmethylsulfonyl fluoride, 5 mg/ml aprotinin. The homogenate was then subjected to differential centrifugation at 4 °C. Three different fractions, i.e. Nuclear(N), large granule (ML) and microsomal (P), and a final supernatant (S) were separated by successive centrifugation at a square angle velocity of 8.2 times 10^5, 1.8 times 10^9, and 1.2 times 10 rad^2 s. For isopycnic sucrose gradient centrifugation, the large granule (ML) fraction was loaded on a sucrose density gradient (0.5 2.3 M sucrose, 20 mM HEPES, pH 7.4) and centrifuged for 8 h at 7.6 times 10 rad^2 s (SW40 Beckman Instruments). Fractions were collected and analyzed for activity of the marker enzymes, ATPase(26) , NADPH cytochrome c reductase(27) , GDPase(28) , and monoamine oxidase(29) . Kar2p and Cne1p were detected by immunoblot and subsequently quantitated by densitometry. Anti- Kar2p and anti-Cne1p antisera were used at 1:2000 and 1:1000 dilution, respectively.

Ca Overlay

Samples were electrophoresed by SDS-PAGE and evaluated for Ca overlay exactly as described by Wada et al.(2) . Yeast ER fractions were recovered from analytical isopycnic gradients of ML fractions at densities greater than 1.151 g/ml ( > 1.151 g/ml). Membrane and soluble proteins were separated by Triton X-114 extraction as described by Bordier(30) . Control experiments were carried out with dog pancreatic ER membranes also extracted with Triton X-114 exactly as described by Wada et al.(2) .

Immunofluorescence

Staining of S. cerevisiae was performed essentially as described previously (31) with the following incubations: 1) anti-Cne1p antisera (1:1000) for 60 min, 2) rhodamine-conjugated Fab (1:50 Jackson Immunochemicals) for 45 min, 3) anti-Kar2p antisera (1:2000) for 60 min, 4) fluorescein isothiocyanate-conjugated IgG (1:50, Jackson Immunochemicals) and DAPI (2 mg/ml, Sigma) for 45 min. Cells were viewed using epifluorescence (Aristoplan, Leitz) and by confocal microscopy (Molecular Dynamics).

Disruption of the Yeast CNE1 Gene

Plasmid pFP10.1 was digested with SphI and religated to clone the insert in the opposite orientation, creating plasmid pFP10.11. A 1-kb EcoRI fragment was removed from pFP-10.11. This effectively removes the multiple cloning site to create the plasmid pFP-10.12. A 750-bp BamHI-PstI internal to CNE1 was replaced with a 2-kilobase pair BamHI-PstI fragment containing the LEU2 gene from pJJ250(32) . The resulting plasmid, pFP10.13, was cut with ScaI and SphI to linearize the plasmid and transformed into the leu2 diploid yeast strain W303D(33) . Transformants were selected on SC glucose minus leucine plates. Disruption of the CNE1 gene was confirmed by Southern blots. For further genetic analysis, diploids were sporulated and tetrad dissection was performed by standard procedures, and the presence of the disruption in parent cells and spores was confirmed by Southern blot analysis.

Acid Phosphatase Assay

Plasmid pRS306-CNE1 was constructed by inserting the ScaI-HpaI fragment containing the open reading frame of CNE1 into the BamHI site (3` to the Gal promoter) of vector pRS306 Gal (34) and transformed into strain W303-1b Deltacne1::LEU2. This strain was subsequently grown at 30 °C in SC sucrose-uracil (22) to OD of 1. Cultures were then divided in three and glucose or galactose (2% w/v final concentration) or sucrose (4% w/v final concentration) added. Cell surface acid phosphatase activity was determined as described (35) .

Heterologous Expression of alpha(1)-Antitrypsin

The cDNAs for wild type and Z mutant alpha(1)-antitrypsin were cloned into the PvuII site of pVT-101U(36) . Plasmids pVT-AlPi (wild type) and pVT-AlPz (mutant) were transformed into W303-1band W303-1b Deltacne1::LEU2, and these were grown overnight in SC glucose-uracil at 30 °C. Equal numbers of cells were spotted onto SC glucose-uracil plates and overlaid with a nitrocellulose filter (BA85 Schleicher and Schuell). Plates were incubated at 30 °C overnight, and nitrocellulose was subsequently washed to remove yeast cells and immunoblotted with alpha(1)-antitrypsin antiserum at 1:1000 dilution (Calbiochem). For detection, either a secondary antibody linked to alkaline phosphatase or Protein A linked to I was used.

Halo Assay for alpha-Pheromone Production

20-ml cultures of wild type strain (W303-1b pVT), calnexin-deleted strain (W303-1b Deltacne1::LEU2 pVT), or calnexin overproducing strain (W303-1b Deltacne1::LEU2 pVT-CNE1) were grown in SC glucose-uracil to OD of 1 and centrifuged at 1000 times g for 5 min. Cells were resuspended in 250 µl of water, and 5 µl was spotted onto a lawn of M200-6C cells on YPD agar. Agar plates were incubated at 30 °C for 48 h.

Quantitative Mating Assay

Assays were performed as described(37) . A 3.0-kilobase pair EcoRI-SphI fragment containing calnexin was cloned into the EcoRI site of pAD13, a low copy number plasmid(38) . Mating efficiency for strains DJ 283-7-1a (Mataste2-3ts can1ts bar1-1 ade2his4lys 2leu2 trp1ura3 cry1 SUP4-3ts Deltacne1::LEU2) transformed with pAD13 or pAD13-CNE1 were measured at 23 and 37 °C using tester strain DC17alpha. Strains were grown to an OD of 1 at either 23 or 37 °C and then mixed with confluent DC 17alpha for 3 h at 23 of 37 °C. Mating efficiency is defined as the number of diploids formed per input haploid. Relative mating efficiency was standardized for each experiment. The mating efficiency of DJ 283-7-1a pAD13, with DC 17alpha at 23 °C was set at 100.


RESULTS

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 times 10^4 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).



Identification of Cne1p as an Integral Membrane Protein

Antibodies were raised to Cne1p which was expressed in E. coli as a fusion protein with GST and purified by affinity chromatography on glutathione beads(24) . This antiserum recognized a protein in yeast of 76 kDa which was present in a particulate cell fraction. To determine if Cne1p is an integral membrane protein, membrane preparations were solubilized in SDS, sodium deoxycholate, or Triton X-100. No significant extraction of calnexin was observed with either sodium carbonate at pH 11.5, 0.5 M NaCl or 2.5 M urea (Fig. 3A). By these criteria, the properties Cne1p correspond to those expected of an integral membrane protein.


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 times 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.

Subcellular Localization of Cne1p

We determined the subcellular location of Cne1p by differential and analytical subcellular fractionation as well as by fluorescence microscopy. Differential centrifugation identified most calnexin in the large granule (ML) fraction of S. cerevisiae homogenates. The ML fraction was enriched in NADPH cytochrome c reductase activity as determined by de Duve plots which reveal the quantitative distribution of this marker enzyme for the ER (Fig. 4). Analytical centrifugation was then carried out with the ML fraction. Density gradient centrifugation revealed a similar distribution of the ER luminal protein Kar2p and Cne1p (Fig. 5). This distribution corresponded to median densities of 1.195 g/cc for both proteins (Fig. 6) which was also that of NADPH cytochrome c reductase (1.195 g/ml). However, these distributions were clearly different than those of the Golgi marker enzyme GDPase (median density 1.138 g/ml), the plasma membrane marker ATPase (median density 1.156 g/ml) and the mitochondrial marker monoamine oxidase (median density 1.177 g/ml). Cne1p is not localized to the vacuole since the antibodies for carboxypeptidase Y revealed (as determined by Western blotting 1:3000 dilution) it to be principally in the N fraction, with very little in the ML or P fractions (data not shown). Hence, the distribution of Cne1p corresponded most closely to that of the ER luminal protein Kar2p.


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.



Cne1p Is Not a Prominent Ca-binding Protein of S. cerevisiae ER

We have previously demonstrated that mammalian calnexin and associated SSRalpha are the major integral membrane proteins of the ER which bind Ca in an overlay assay. As shown in Fig. 8, two integral membrane of dog pancreatic ER corresponding to canine calnexin- (90 kDa) and SSRalpha- (35 kDa) bound Ca. An ER fraction from S. cerevisiae was isolated as pooled fractions 9-18 from Fig. 6. Separation into peripheral and integral membrane proteins by the method of Bordier (30) revealed that the six major Ca-binding proteins of the yeast ER fractionated into the aqueous phase. These proteins most likely correspond to lumenal ER proteins. Ca binding to an integral membrane protein of the expected mobility of Cne1p was not detected. This conclusion was supported by further experiments using a GST::Cne1p fusion protein, expressed and purified in E. coli. This protein did not reveal detectable Ca binding by the Ca overlay protocol, although control proteins (parvalbumin, calmodulin) were reactive (data not shown). This is the first report identifying Ca-binding proteins in S. cerevisiae ER although Cne1p is not one of them.


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 SSRalpha were identified in the Triton X-114 phase of dog pancreatic ER. Molecular mass markers as indicated on the left.



Deletion of the CNE1 Gene

To determine the phenotype of CNE1, the CNE1 gene was deleted by inserting the LEU2 gene into an internal deletion of CNE1 creating plasmid pFP 10.13 (Fig. 9). The plasmid was linearized, transformed into strain W303D, and LEU diploids were selected. The transformed diploid was then sporulated and seven asci were dissected. For every tetrad, all four spores were viable showing that the gene is not essential for viability. CNE1 RNA was not detected in the LEU2 spore (Fig. 9A) and neither was Cne1p as determined by immunoblots of particulate and soluble fractions isolated from the CNE1 deleted strain (Fig. 9B). The 30 kDa band as compared to wild type protein found in lane 1 represents a fragment of Cne1p which was sometimes observed. The protein was not detected by double immune epifluorescence or confocal immunofluorescence examination of S. cerevisiae cne1 deleted strains with Cne1p specific antisera (not shown).


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 Deltacne1::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 Deltacne1::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(-), Deltacne1::LEU2 spore disruptant and lane 2 (+) wild type spore for CNE1. CNE1 RNA is not detected in Deltacne1::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 Deltacne1::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.



Cne1p and Secretion

To test if Cne1p is a molecular chaperone for glycoproteins(1) , the secretion of the glycoproteins acid phosphatase (Fig. 10) and alpha-pheromone (Fig. 11) were determined in CNE1-deleted strain. The levels of secreted alpha-pheromone in wild type, deleted, or overexpressing CNE1 strains are identical, as determined by halo assay. Likewise when CNE1 expression is induced or repressed, levels of cell surface acid phosphatase remain constant.


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 alpha-pheromone production. Wild type strain (W303-1b pVT) (A), CNE1-deleted strain (W303-1b Deltacne1::LEU2 pVT) (B), or CNE1 overexpressing strain (W303-1b Deltacne1::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 alpha(1)-antitrypsin is a substrate for mammalian calnexin(14, 15) , and mutant alpha(1)-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 alpha(1)-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 alpha(1)-antitrypsin. The amount of secreted alpha(1)-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 alpha(1)-antitrypsin. Both wild type (pVT-AlPi) and the Z mutant (pVT-AlPz) of alpha(1)-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 alpha(1)-antitrypsin. Wild type alpha(1)-antitrypsin (pVT-AlPi), Z mutant alpha(1)-antitrypsin (pVT-AlPz), or vector alone (pVT) were transformed into W303-1a (CNE1) or W303-1a Deltacne1::LEU2 (Deltacne1::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-alpha(1)-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 alpha-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 (^2)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).




DISCUSSION

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(2)-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 [^3H]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 alpha-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 alpha(1)-antitrypsin in S. cerevisiae as well as function of a temperature-sensitive mutant ste2,3 ts of the alpha-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.


FOOTNOTES

*
This research was partially supported by a grant from the Medical Research Council of Canada (to J. J. M. B. and D. Y. T.). This is Publication No. 38503 from the National Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L11012[GenBank].

§
Supported by the Natural Science and Engineering Research Council of Canada. To whom correspondence should be addressed: National Research Council Canada, Biotechnology Research Institute, 6100 Royalmount Ave. Montreal, Quebec H4P 2R2.

(^1)
The abbreviations used are: ER, endoplasmic reticulum; MHC, major histocompatibility complex; HA, influenza virus hemagglutinin; DAPI, 4`,6-diamidino-2-phenyl-indole; GST, glutathione S-transferase; Endo-H, endoglycosidase H; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase(s).

(^2)
D. Jeness, personal communication.


ACKNOWLEDGEMENTS

-We thank Dr. Duane Jeness (University of Massachusetts) for the ste2-3 yeast strain, Dr. Robert Monette for his assistance with the confocal microscope, and Dr. Roland Brousseau and Alberto Mazza for oligonucleotide synthesis. We also thank Dr. Yves Bourbonnais and Dr. Malcolm Whiteway for their critical comments on the manuscript. We thank Pam Cameron for the Ca overlay.


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