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

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 Ca2+-binding proteins were detected in the ER fractions, they were all soluble proteins, and Ca2+ 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 α-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 α-pheromone receptor, ste2-3p, and also an increase in the secretion of heterologously expressed mammalian α1-antitrypsin. Hence, Cne1p appears to function as a constituent of the S. cerevisiae ER protein quality control apparatus. 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 Ca2+-binding proteins were detected in the ER fractions, they were all soluble proteins, and Ca2+ 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 α-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 α-pheromone receptor, ste2-3p, and also an increase in the secretion of heterologously expressed mammalian α1-antitrypsin. Hence, Cne1p appears to function as a constituent of the S. cerevisiae ER protein quality control apparatus. INTRODUCTIONCalnexin is an integral membrane calcium-binding phosphoprotein found in the ER ( 1The abbreviations used are: ERendoplasmic reticulumMHCmajor histocompatibility complexHAinfluenza virus hemagglutininDAPI4′,6-diamidino-2-phenyl-indoleGSTglutathione S-transferaseEndo-Hendoglycosidase HPAGEpolyacrylamide gel electrophoresisPCRpolymerase chain reactionbpbase pair(s)kbkilobase(s).)of mammalian cells(1.Bergeron J.J.M. Brenner M.B. Thomas D.Y. Williams D.B. Trends Biochem. Sci. 1994; 19: 124-128Abstract Full Text PDF PubMed Scopus (455) Google Scholar, 2.Wada I. Rindress D. Cameron P.H. Ou W.-J. Doherty J.J. Louvard D. Bell A.W. Dignard D. Thomas D.Y. Bergeron J.J.M. J. Biol. Chem. 1959; 266 (1991, 9-19610)Google Scholar). Closely related DNA sequences have been found in plants and nematodes(3.Huang L. Franklin A.E. Hoffman N.E. J. Biol. Chem. 1993; 268: 6560-6566Abstract Full Text PDF PubMed Google Scholar, 4.Sulston J. Du Z. Thomas K. Wilson R. Hillier L. Staden. R. Halloran N. Green P. Thierry-Mieg J. Qiu L. Dear S. Coulson A. Craxton M. Durbin R. Berks M. Metzstein M. Hawkins T. Ainscough R. Waterson R. Nature. 1992; 356: 37-41Crossref PubMed Scopus (421) Google Scholar). A function for calnexin as a molecular chaperone has been identified(1.Bergeron J.J.M. Brenner M.B. Thomas D.Y. Williams D.B. Trends Biochem. Sci. 1994; 19: 124-128Abstract Full Text PDF PubMed Scopus (455) Google Scholar). 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.Anderson K.S. Cresswell P. EMBO J. 1994; 13: 675-682Crossref PubMed Scopus (108) Google Scholar), the T cell receptor, membrane Ig(10.Hochstenbach F. David V. Watkins S. Brenner M.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4734-4738Crossref PubMed Scopus (213) Google Scholar), the viral membrane glycoproteins influenza HA and the “G” protein of vesicular stomatitis virus (11.Hammond C. Braakman I. Helenius A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 913-917Crossref PubMed Scopus (708) Google Scholar) as well as the cystic fibrosis transmembrane conductance regulator(12.Pind S. Riordan J. Williams D.B. J. Biol. Chem. 1994; 269: 12784-12788Abstract Full Text PDF PubMed Google Scholar), and integrin(13.Lenter M. Vestweber D. J. Biol. Chem. 1994; 269: 12263-12268Abstract Full Text PDF PubMed Google Scholar). In addition, calnexin associates transiently with the normal folding intermediates of soluble monomeric glycoproteins including transferrin, α1-antitrypsin, complement C3, apoB-100(14.Ou W-J. Cameron P.M. Thomas D.Y. Bergeron J.J.M. Nature. 1993; 364: 771-776Crossref PubMed Scopus (483) Google Scholar, 15.Le A. Steiner J.L. Ferrell G.A. Shaker J.C. Sifers R.N. J. Biol. Chem. 1994; 269: 7514-7519Abstract Full Text PDF PubMed Google Scholar), as well as the major secreted glycoprotein of Maden-Darby canine kidney cells, gp80(16.Wada I. Ou W.-J. Liu M.C. Scheele G. J. Biol. Chem. 1994; 269: 7464-7472Abstract Full Text PDF PubMed Google Scholar).A second related function has been proposed for mammalian calnexin as a constituent of a protein quality control apparatus in the ER and some mutant proteins and of soluble glycoproteins in the of the they retained in the ER and with calnexin for a W-J. Cameron P.M. Thomas D.Y. Bergeron J.J.M. Nature. 1993; 364: 771-776Crossref PubMed Scopus (483) Google Scholar). mutant proteins such as vesicular stomatitis virus glycoprotein retained in the ER by their with C. Braakman I. Helenius A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 913-917Crossref PubMed Scopus (708) Google Scholar). of also retained in the ER in with calnexin for the MHC class I heavy chain in the of Williams D.B. J. Biol. PubMed Scopus Google Scholar, Williams D.B. 1994; PubMed Scopus Google Scholar), and the T cell in the of the V. F. S. Brenner M.B. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar, S. Brenner M.B. 1994; PubMed Scopus Google Scholar). calnexin has the of a of such a quality control the sequence of a gene in Saccharomyces cerevisiae with to mammalian calnexin has been C. N. T. A. 1993; PubMed Scopus Google Scholar). The of a calnexin from yeast the molecular calnexin its as a molecular chaperone and the retention of proteins in the ER We have identified by a strategy a calnexin gene in S. cerevisiae CNE1. We have and the Cne1p protein and have by gene some of its and S. cerevisiae and were at in yeast and or with the amino acid and or The was T. J. A Scholar). were as in identify and genes similar to calnexin from S. cerevisiae for the sequences and with all at were a DNA was a P. J. Biol. PubMed Scopus Google Scholar). were on a gel and by The at was by and the of and by the DNA of in S. to the sequence of the gene we S. cerevisiae DNA were the isolated calnexin as a by T. J. A Scholar). were isolated and a was found to to the and was the of to on sequence by the were and used to sequence the gene as T. J. A calnexin were by with proteins expressed in The was by a D.B. K.S. PubMed Scopus Google Scholar). was expressed by and D.B. K.S. PubMed Scopus Google and of were with of M M sodium pH 11.5, 2.5 M urea, or and were as D. J. R. Biol. 1992; PubMed Scopus Google Scholar). Cne1p was used at were by of proteins in sodium pH and and with of for at cerevisiae was at in to a cells were by and in were by a at in M yeast and yeast and with a in M pH The was to differential at fractions, and and a were by at a of and the was on a M pH and for at were and for activity of the J. Biol. Chem. Full Text PDF PubMed Google Scholar), S. J. Biochem. PubMed Scopus Google Scholar), C. P. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), and PubMed Scopus Google Scholar). and Cne1p were detected by and by and were used at and were by and for as by I. Rindress D. Cameron P.H. Ou W.-J. Doherty J.J. Louvard D. Bell A.W. Dignard D. Thomas D.Y. Bergeron J.J.M. J. Biol. Chem. 1959; 266 (1991, 9-19610)Google Scholar). ER were from analytical of at and soluble proteins were by extraction as by C. J. Biol. Chem. Full Text PDF PubMed Google Scholar). were with ER membranes also with as by I. Rindress D. Cameron P.H. Ou W.-J. Doherty J.J. Louvard D. Bell A.W. Dignard D. Thomas D.Y. Bergeron J.J.M. J. Biol. Chem. 1959; 266 (1991, 9-19610)Google of S. cerevisiae was as PubMed Scopus Google Scholar) with the for 60 for for 60 and for were and by confocal microscopy of the CNE1 was with and to the in the A was from the to the A to CNE1 was with a the gene from L. PubMed Scopus Google Scholar). The was with and to the and the yeast K. A. J. PubMed Google Scholar). were on Disruption of the CNE1 gene was by were and was by and the of the in cells and was by was by the the of CNE1 the to the of P. PubMed Google Scholar) and was at in F. in Scholar) to of were in and or or acid activity was as A. J. PubMed Google of for and mutant were the of T. Dignard D. Thomas D.Y. PubMed Scopus Google Scholar). and were and were in at of cells were and with a and were at and was to yeast cells and with at a to or A to was for of or calnexin were in to of and at for were in of and was a of cells on were at for were as PubMed Scopus Google Scholar). A calnexin was the of a C. S. M. Biol. 1992; PubMed Scopus Google Scholar). for with or were at and were to an of at or and with for at of is as the of was for The of with at was at used a to genes with sequence to mammalian calnexin and from S. were to the amino acid sequence motifs mammalian calnexin and A and S. cerevisiae DNA as a an DNA of was and The sequence to that of mammalian calnexin and of mammalian DNA was used to a S. cerevisiae in the yeast with an were isolated from The yeast DNA was on the basis of its with the DNA and its sequence was The DNA sequence a protein of amino and was found to be identical to a gene C. N. T. A. 1993; PubMed Scopus Google Scholar). The sequence to canine calnexin was 24% and was The predicted protein a and a carboxyl-terminal transmembrane J. J. Biol. PubMed Scopus Google Scholar, G. PubMed Scopus Google Scholar, P. J. Biochem. PubMed Scopus Google Scholar). the predicted Cne1p sequence did not a carboxyl-terminal and unlike it did not have a carboxyl-terminal ER retention in I. Rindress D. Cameron P.H. Ou W.-J. Doherty J.J. Louvard D. Bell A.W. Dignard D. Thomas D.Y. Bergeron J.J.M. J. Biol. Chem. 1959; 266 (1991, 9-19610)Google Scholar). on the basis of predicted the sequence we identified did not known or known calnexin and of Cne1p. and predicted of Cne1p the predicted of and the single transmembrane domain at the The predicted sequence is at of Cne1p as an were to Cne1p was expressed in as a protein with and by on D.B. K.S. PubMed Scopus Google Scholar). a protein in yeast of 76 kDa was in a cell Cne1p is an integral membrane membrane were in sodium or extraction of calnexin was with sodium carbonate at pH 11.5, M or 2.5 M the Cne1p to of an integral membrane is an integral membrane were and with sodium M sodium pH 11.5, M 2.5 M or pH by at to a and on the a of was with a in mobility of calnexin from a at 60 on the of Cne1p as a at a molecular of 76 kDa on is that from the predicted In to the protein was membranes were with and by in an mobility of the protein with an molecular of 60 kDa to that predicted all of were by the of ( kDa for However, the predicted molecular of the protein is Localization of the subcellular of Cne1p by differential and analytical subcellular fractionation as well as by identified calnexin in the of S. cerevisiae The was in activity as by the of for the ER was with the a similar of the ER protein and Cne1p to of for proteins was also that of However, were of the the membrane and the Cne1p is not to the the for by it to be in the with in the or not Hence, the of Cne1p to that of the ER protein of the of the ER and Cne1p. differential of S. cerevisiae and with the of expressed as a C. PubMed Scopus Google Scholar). the of S. cerevisiae Cne1p in the of protein was to for to 60 of protein was and detected by with The the activity of as well as calnexin of the of and Cne1p in the were on as and of were for their of and Cne1p by with their The of the was and that for calnexin was The of for the the membrane the the ER the ER protein and the membrane protein Cne1p as by of The of was as and that of and calnexin by of the of The for the of the was by and confocal immunofluorescence microscopy Cne1p was to a identical to that for the ER protein and in the of the is shown in were by confocal microscopy with a for Cne1p. In a was used M. J. Biol. PubMed Scopus Google Scholar) is for is for immunofluorescence of Cne1p and in S. cerevisiae by and confocal with and Cne1p by ER of Cne1p by confocal immunofluorescence microscopy The a of S. cerevisiae have that mammalian calnexin and the major integral membrane proteins of the ER in an shown in integral membrane of ER to canine and ER from S. cerevisiae was isolated as from and integral membrane proteins by the of C. J. Biol. Chem. Full Text PDF PubMed Google Scholar) that the six major proteins of the yeast ER the proteins to ER binding to an integral membrane protein of the mobility of Cne1p was not was by a expressed and in protein did not binding by the control proteins were not is the Ca2+-binding proteins in S. cerevisiae ER Cne1p is not of of binding proteins in S. cerevisiae membrane proteins from ER and from S. cerevisiae ER of as well as from and of ER of were on and to In the six of molecular and kDa were identified as proteins of S. cerevisiae membrane proteins of and kDa to mammalian calnexin and were identified in the of as on the of the CNE1 the of the CNE1 gene was by the gene an of CNE1 The was and were The was and were all were that the gene is not for CNE1 was not detected in the and was Cne1p as by of and soluble isolated from the CNE1 The kDa as to protein found in a of Cne1p was The protein was not detected by or confocal immunofluorescence of S. cerevisiae with Cne1p of S. cerevisiae CNE1 and by and of the CNE1 gene and The CNE1 is in to in the from cells and was PubMed Scopus Google Scholar) and with DNA the CNE1 of was and to and for CNE1. CNE1 is not detected in disrupted of S. cerevisiae Cne1p. and from yeast cell from CNE1 and or and were by with of protein were to on the and Cne1p is a molecular chaperone for J.J.M. Brenner M.B. Thomas D.Y. Williams D.B. Trends Biochem. Sci. 1994; 19: 124-128Abstract Full Text PDF PubMed Scopus (455) Google Scholar), the secretion of the glycoproteins acid and α-pheromone were in The levels of secreted α-pheromone in or CNE1 as by CNE1 expression is or levels of cell acid was in with a calnexin were in to an of and with or with was to were at the for acid as for α-pheromone or CNE1 were on a of cells as were at for to to soluble glycoprotein is a for mammalian W-J. Cameron P.M. Thomas D.Y. Bergeron J.J.M. Nature. 1993; 364: 771-776Crossref PubMed Scopus (483) Google Scholar, 15.Le A. Steiner J.L. Ferrell G.A. Shaker J.C. Sifers R.N. J. Biol. Chem. 1994; 269: 7514-7519Abstract Full Text PDF PubMed Google Scholar), and mutant has been shown to be retained by calnexin to its or in the A. Steiner J.L. Ferrell G.A. Shaker J.C. Sifers R.N. J. Biol. Chem. 1994; 269: 7514-7519Abstract Full Text PDF PubMed Google Scholar). heterologously expressed in and mutant retained in the ER with the mutant Biol. 1993; PubMed Scopus Google Scholar). Hence, we were to the of Cne1p in the retention of and of mammalian α1-antitrypsin. The of secreted was in and by the on with and with to α1-antitrypsin. and the mutant of were secreted to a in CNE1 disrupted cells in cells of the showed a increase in secretion from calnexin disrupted cells of Cne1p on the secretion of α1-antitrypsin. mutant or were or of cells were and with membrane and at The membrane was with and by the in a The of a retention function for CNE1 was to an yeast transmembrane the α-pheromone receptor, protein is and in the membrane of S. the mutant has been shown to be retained ( in a in Cne1p a in the retention of we its function at the cell with a the the was in function of protein at the membrane in a mammalian cells calnexin has been shown to have a in the retention of glycoproteins in the ER and in the of cell J.J.M. Brenner M.B. Thomas D.Y. Williams D.B. Trends Biochem. Sci. 1994; 19: 124-128Abstract Full Text PDF PubMed Scopus (455) Google The of a calnexin be of as its function be the of in is CNE1 is the calnexin or in We have in by a of the by an of the and by the of strategy that we was to from yeast DNA sequences to as well as calnexin. Although of the reaction were the yeast CNE1 sequence was detected as an did not have an and did not to calnexin or sequence was used as a to the CNE1 gene from a yeast the the CNE1 the CNE1 sequence as a we there were related sequences in the yeast the at on and on a of DNA from a CNE1 disrupted we were to related by there not to be genes in yeast related to CNE1. The CNE1 gene we by is located on the of to genes and and to known genes L. A. 1993; PubMed Google calnexin and have the motifs of related was found in S. cerevisiae CNE1 at of calnexin also of were found in CNE1. In the a calnexin gene has been identified with sequence to mammalian calnexin that of S. L. Franklin A.E. Hoffman N.E. J. Biol. Chem. 1993; 268: 6560-6566Abstract Full Text PDF PubMed Google Scholar). motifs retained as well as the motifs and a sequence to that of mammalian In addition, the of the Cne1p terminates in a sequence and the carboxyl-terminal domain found in We also that there is not a for an for an CNE1 sequence of the predicted S. cerevisiae Cne1p protein an and a carboxyl-terminal membrane We the of Cne1p in the yeast ER by differential and analytical subcellular fractionation and by and confocal immunofluorescence microscopy showed a of Cne1p and the ER protein We that Cne1p is an integral membrane protein as it not be from membranes by with 2.5 M urea, high salt, and sodium carbonate at pH is a that Cne1p shares with mammalian calnexin is also an integral membrane is a soluble ER protein. We also that Cne1p has as predicted from the the mobility of Cne1p in was by that all A. P. J. 1993; PubMed Scopus Google ER membrane protein such as Cne1p in is amino acid is predicted to be we have of Cne1p in the yeast ER there is a of it is We have that S. cerevisiae Cne1p was not of was detected was the protein to digestion by C. In mammalian calnexin the sequence has been shown to as retention I integral membrane protein in the V. F. S. Brenner M.B. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). The lack of a for S. cerevisiae Cne1p its to the yeast ER that retention is by with an membrane or protein and not by the proteins with a retention T. J. Biol. 1993; PubMed Scopus Google calnexin has been shown to be of major calcium-binding integral membrane proteins of the I. Rindress D. Cameron P.H. Ou W.-J. Doherty J.J. Louvard D. Bell A.W. Dignard D. Thomas D.Y. Bergeron J.J.M. J. Biol. Chem. 1959; 266 (1991, 9-19610)Google Scholar). with yeast ER membranes showed that there not to be calcium-binding proteins in the ER membrane 8), we did yeast ER calcium-binding is the of calcium-binding proteins in the ER of S. of the of yeast Cne1p to in was with isolated protein has been to be for the binding of mammalian calnexin with its protein I. Rindress D. Cameron P.H. Ou W.-J. Doherty J.J. Louvard D. Bell A.W. Dignard D. Thomas D.Y. Bergeron J.J.M. J. Biol. Chem. 1959; 266 (1991, 9-19610)Google Scholar, W-J. Cameron P.M. Thomas D.Y. Bergeron J.J.M. Nature. 1993; 364: 771-776Crossref PubMed Scopus (483) Google Scholar). Although Cne1p has sequence with mammalian it is in that it is it is an integral ER membrane protein not have a retention and unlike mammalian calnexin it is not a calcium-binding genes from a in their sequence that the function of the protein is similar and that the of the sequence is for that calnexin has been identified as a molecular chaperone for soluble and glycoproteins of the J.J.M. Brenner M.B. Thomas D.Y. Williams D.B. Trends Biochem. Sci. 1994; 19: 124-128Abstract Full Text PDF PubMed Scopus (455) Google Scholar). calnexin has also been identified as for the ER retention of soluble and proteins to their from the that there be an for yeast cells lack calnexin. However, yeast a of the CNE1 gene were and at normal and we were to identify on the secretion of the glycoproteins α-pheromone or acid phosphatase. the with some mammalian proteins, there is that they the of calnexin in their W-J. Cameron P.M. Thomas D.Y. Bergeron J.J.M. Nature. 1993; 364: 771-776Crossref PubMed Scopus (483) Google Scholar). has been to or for protein folding in the mammalian ER the calnexin J.J.M. Brenner M.B. Thomas D.Y. Williams D.B. Trends Biochem. Sci. 1994; 19: 124-128Abstract Full Text PDF PubMed Scopus (455) Google did an on the retention of heterologously expressed in S. cerevisiae as well as function of a temperature-sensitive mutant of the α-pheromone in The on be to an of Cne1p on its or on its function at the The is Cne1p is in the Although effects they that the Cne1p is a constituent of the yeast quality control apparatus in the retention of heterologously expressed or the of the CNE1 gene we have identified and its gene we have the yeast calnexin or there is of mammalian calnexin or in the yeast We the in an to identify genes function with CNE1. INTRODUCTIONCalnexin is an integral membrane calcium-binding phosphoprotein found in the ER ( 1The abbreviations used are: ERendoplasmic reticulumMHCmajor histocompatibility complexHAinfluenza virus hemagglutininDAPI4′,6-diamidino-2-phenyl-indoleGSTglutathione S-transferaseEndo-Hendoglycosidase HPAGEpolyacrylamide gel electrophoresisPCRpolymerase chain reactionbpbase pair(s)kbkilobase(s).)of mammalian cells(1.Bergeron J.J.M. Brenner M.B. Thomas D.Y. Williams D.B. Trends Biochem. Sci. 1994; 19: 124-128Abstract Full Text PDF PubMed Scopus (455) Google Scholar, 2.Wada I. Rindress D. Cameron P.H. Ou W.-J. Doherty J.J. Louvard D. Bell A.W. Dignard D. Thomas D.Y. Bergeron J.J.M. J. Biol. Chem. 1959; 266 (1991, 9-19610)Google Scholar). Closely related DNA sequences have been found in plants and nematodes(3.Huang L. Franklin A.E. Hoffman N.E. J. Biol. Chem. 1993; 268: 6560-6566Abstract Full Text PDF PubMed Google Scholar, 4.Sulston J. Du Z. Thomas K. Wilson R. Hillier L. Staden. R. Halloran N. Green P. Thierry-Mieg J. Qiu L. Dear S. Coulson A. Craxton M. Durbin R. Berks M. Metzstein M. Hawkins T. Ainscough R. Waterson R. Nature. 1992; 356: 37-41Crossref PubMed Scopus (421) Google Scholar). A function for calnexin as a molecular chaperone has been identified(1.Bergeron J.J.M. Brenner M.B. Thomas D.Y. Williams D.B. Trends Biochem. Sci. 1994; 19: 124-128Abstract Full Text PDF PubMed Scopus (455) Google Scholar). 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.Anderson K.S. Cresswell P. EMBO J. 1994; 13: 675-682Crossref PubMed Scopus (108) Google Scholar), the T cell receptor, membrane Ig(10.Hochstenbach F. David V. Watkins S. Brenner M.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4734-4738Crossref PubMed Scopus (213) Google Scholar), the viral membrane glycoproteins influenza HA and the “G” protein of vesicular stomatitis virus (11.Hammond C. Braakman I. Helenius A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 913-917Crossref PubMed Scopus (708) Google Scholar) as well as the cystic fibrosis transmembrane conductance regulator(12.Pind S. Riordan J. Williams D.B. J. Biol. Chem. 1994; 269: 12784-12788Abstract Full Text PDF PubMed Google Scholar), and integrin(13.Lenter M. Vestweber D. J. Biol. Chem. 1994; 269: 12263-12268Abstract Full Text PDF PubMed Google Scholar). In addition, calnexin associates transiently with the normal folding intermediates of soluble monomeric glycoproteins including transferrin, α1-antitrypsin, complement C3, apoB-100(14.Ou W-J. Cameron P.M. Thomas D.Y. Bergeron J.J.M. Nature. 1993; 364: 771-776Crossref PubMed Scopus (483) Google Scholar, 15.Le A. Steiner J.L. Ferrell G.A. Shaker J.C. Sifers R.N. J. Biol. Chem. 1994; 269: 7514-7519Abstract Full Text PDF PubMed Google Scholar), as well as the major secreted glycoprotein of Maden-Darby canine kidney cells, gp80(16.Wada I. Ou W.-J. Liu M.C. Scheele G. J. Biol. Chem. 1994; 269: 7464-7472Abstract Full Text PDF PubMed Google Scholar).A second related function has been proposed for mammalian calnexin as a constituent of a protein quality control apparatus in the ER and some mutant proteins and of soluble glycoproteins in the of the they retained in the ER and with calnexin for a W-J. Cameron P.M. Thomas D.Y. Bergeron J.J.M. Nature. 1993; 364: 771-776Crossref PubMed Scopus (483) Google Scholar). mutant proteins such as vesicular stomatitis virus glycoprotein retained in the ER by their with C. Braakman I. Helenius A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 913-917Crossref PubMed Scopus (708) Google Scholar). of also retained in the ER in with calnexin for the MHC class I heavy chain in the of Williams D.B. J. Biol. PubMed Scopus Google Scholar, Williams D.B. 1994; PubMed Scopus Google Scholar), and the T cell in the of the V. F. S. Brenner M.B. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar, S. Brenner M.B. 1994; PubMed Scopus Google Scholar). calnexin has the of a of such a quality control the sequence of a gene in Saccharomyces cerevisiae with to mammalian calnexin has been C. N. T. A. 1993; PubMed Scopus Google Scholar). The of a calnexin from yeast the molecular calnexin its as a molecular chaperone and the retention of proteins in the ER We have identified by a strategy a calnexin gene in S. cerevisiae CNE1. We have and the Cne1p protein and have by gene some of its