The native route of synthesis of most proteins of therapeutic interest is via secretion; growth factors, hormones, thrombolytic and clotting factors, and antibodies are all secreted proteins. The eucaryotic secretory pathway mediates the folding, assembly, glycosylation, proteolytic processing, and conformational proofreading of secreted polypeptides. Thus, eucaryotic secretion expression systems represent an efficient means of producing proteins with high fidelity to the native form. Unfortunately, the specific productivity of mammalian, insect, and yeast secretion systems is generally 1-2 orders of magnitude lower than that of bacterial systems (Hodgson, 1993). The rate-limiting step in eucaryotic protein secretion is generally transport from the endoplasmic reticulum to the Golgi apparatus (Lodish et al., 1983; Shuster, 1991). Unfolded proteins are prevented from leaving the ER ()by a proofreading mechanism (reviewed by Helenius(1994)), so it is generally the rate and efficiency of protein folding and assembly in the ER that determines the overall rate of transport through the secretory pathway. The lumen of the ER is an environment adapted to the assistance of folding by the presence of a high concentration of protein folding chaperones (BiP, GRP94, and calnexin) and foldases (protein disulfide isomerase, FKBP, ERp72, and cyclophilin) (Helenius et al., 1992; Gething and Sambrook, 1992). Previous attempts to enhance secretory capacity have focused on manipulating levels of lumenal chaperones and foldases (Dorner et al., 1988, 1992; Robinson et al., 1994; Schultz et al., 1994; Hsu et al., 1994).

Perhaps the most thoroughly studied ER-resident chaperone is BiP (or heavy chain binding protein). Initially discovered in a complex with unassembled IgG heavy chains (Haas and Wabl, 1984), BiP was soon identified as an Hsp70 chaperone that is present in the endoplasmic reticulum of all eucaryotes. The KAR2 gene for the S. cerevisiae BiP homolog was identified separately by homology to mouse BiP (Normington et al., 1989) and by functional and sequence similarity of BiP to a karyogamy gene (Rose et al., 1989). BiP is induced by stressors that cause the accumulation of unfolded proteins in the lumen of the ER and is often present in stable complexes with misfolded or unassembled proteins while interacting only transiently with efficiently folded proteins (Blount and Merlie, 1991; Bole et al., 1986; Dorner et al., 1987; Gething et al., 1986; Hendershot, 1990; Ng et al., 1992). BiP seems to be most involved in the early stages of protein conformational maturation in the ER (Hammond and Helenius, 1994; Melnick et al., 1994; Kim and Arvan, 1995; Simons, et al., 1995) and has been demonstrated to play a key role in membrane translocation (Vogel et al., 1990; Sanders et al., 1992; Brodsky and Schekman, 1993; Panzner et al., 1995).

It might be expected that increasing BiP levels would increase the protein folding capacity of the ER, because chaperones act essentially as detergents to solubilize folding intermediates and circumvent kinetic trapping of aggregates (reviewed by Hartl et al. (1994)). BiP expression is induced by increased protein flux into the ER, consistent with this hypothesis (Dorner et al., 1989; Tokunaga et al., 1992; Robinson and Wittrup, 1995; Watowich et al., 1991). Furthermore, stressors that cause the accumulation of unfolded protein in the ER also induce BiP expression (Kozutsumi et al., 1988; Rose et al., 1989; Normington et al., 1989; Kohno et al., 1993). However, the capability to bind to unfolded polypeptides might instead be interpreted as serving to retain unfolded proteins in the ER, such that BiP acts as an element of the proofreading apparatus (Dorner and Kaufman, 1994).

Dorner, Kaufman, and colleagues have provided evidence that the chaperone BiP can serve a proofreading role in heterologous secretion. Reduction of BiP levels by antisense transcript expression increases secretion of a mutant tissue plasminogen activator lacking glycosylation sites (Dorner et al., 1988), and overexpression of BiP decreases secretion of factor VIII and von Willebrand factor (Dorner et al., 1992). These effects are protein-specific; a negative function for BiP in secretion appears to correlate with the detection of stable BiP-protein complexes (Dorner et al., 1992) and a high ATP requirement for secretion (Dorner et al., 1990). Dorner and Kaufman conclude that BiP does not serve a positive role in assisting folding and secretion but instead serves to bind and retain misfolded proteins (Dorner and Kaufman, 1994).

We were interested in determining the role of BiP in heterologous protein secretion in Saccharomyces cerevisiae. Towards this end, we constructed a yeast strain wherein the levels of BiP protein are tunable by a copper-inducible promoter. We examined the secretion of three proteins in this strain: human granulocyte colony-stimulating factor (GCSF), Schizosaccharomyces pombe acid phosphatase (PHO), and bovine pancreatic trypsin inhibitor (BPTI). We find that reduction of BiP below normal levels progressively inhibits secretion of all three proteins, whereas 5-fold overexpression of BiP from a strong glycolytic promoter essentially does not affect secretion. These data are consistent with a model wherein BiP functions to promote folding and secretion of proteins by competing with unfavorable aggregation rather than act predominantly in a negative proofreading capacity (Robinson and Wittrup, 1993). We discuss our results in light of a growing body of evidence that BiP functions primarily at the earliest stages of protein folding and secretion and attempt to reconcile the apparent contradiction between Dorner and Kaufman's data and our own in terms of structural differences among the proteins studied.

EXPERIMENTAL PROCEDURES

Strains and Plasmids

The plasmid pMR1341 (gift of M. Rose) was used to construct the plasmid pCUPKAR2, in which Kar2 is under the control of the inducible promoter CUP1, derived from the yeast metallothionein gene (Butt and Ecker, 1987). The CUP1 promoter was amplified from the plasmid pYR-CUP (gift of J. Sambrook) by polymerase chain reaction. Unique restriction sites were incorporated to facilitate insertion into the pMR1341 vector. The forward primer 5`-CTCGACGTCGGATCCCATTACCG-3` introduced an AatII site to the 5` end of the CUP1 promoter, and the reverse primer 5`-GGGTCGACGTACAGTTTGTTTTTC-3` introduced a SalI site to the 3` end of the CUP1 promoter. The cloned PCR product was subcloned in place of the SalI-AatII GAL1,10 promoter of pMR1341, to create pCUPKAR2.

The plasmid pMR1341 was also used in the construction of pGAPDH-KAR2, in which Kar2 is under control of the strong constitutive promoter glyceraldehyde phosphate dehydrogenase (GAPDH). To facilitate cloning, an internal SalI site was first removed from the GAPDH promoter in the vector pUC119G6 (GCSF) (gift of S. Elliot, Amgen) by a SalI digest followed by Klenow treatment. The product was purified using DNA Clean-Up (Promega), and blunt ends were religated with high concentration DNA Ligase (Boehringer Mannheim). The ligation mixture was transformed into HB101 (BRL), and product plasmid was screened by an EcoRI-BamHI digest. The promoter was then amplified by ``hot start'' polymerase chain reaction (Chou et al., 1992), and unique restriction sites were introduced to permit insertion into the pMR1341 vector. The forward primer 5`-GGCGGACGTCAAGGTCGAGTTTATCAT-3` introduced an AatII site to the 5` end of the GAPDH promoter, and the reverse primer 5`-CTCGTCGACCGTCGAAACTAAGT-3` introduced a SalI site to the 3` end. The initial three cycles were run at an annealing temperature of 37 °C because of the AT-rich primers, followed by thirty cycles at an annealing temperature of 55 °C to improve specificity for the full-length product. The product was purified using DNA Clean-Up (Promega), digested with AatII and SalI and subcloned in place of the SalI-AatII GAL1,10 promoter of pMR1341. The plasmid pGAPDH-KAR2 was screened on LB-AMP (50 µg/ml) and identified by restriction digests with PstI and EcoRI.

A low copy expression plasmid for human granulocyte colony-stimulating factor (pCEN-GCSF) was constructed by subcloning the BamHI + EcoRI fragment of the plasmid pUC119G6 (gift of S. Elliott, Amgen) containing the GAPDH promoter, -factor signal sequence and leader, and human GCSF coding sequence, and -factor transcriptional terminator into the polylinker site in the pRS314 centromere shuttle vector (Sikorski and Hieter, 1989) bearing the TRP1 selectable marker.

A low copy expression plasmid for S. pombe acid phosphatase (pCEN-PHO) was constructed by inserting a partial BamHI fragment from the plasmid pYEFPHO (gift of S. Elliot, Amgen) containing the -factor promoter and leader and S. pombe acid phosphatase coding sequence into the polylinker BamHI site of the vector pNN342 (Elledge and Davis 1988) bearing the TRP1 selectable marker. The BPTI expression plasmid was constructed as described previously (Wittrup et al., 1995).

Media and Growth Culture Conditions

Single transformant colonies of JBY100 and a control strain JBY200 (see Table 1) were grown to saturation in 5 ml of synthetic galactose medium supplemented with 2 SCAA. 250-ml baffled Erlenmeyer flasks containing 25 ml of synthetic glucose + 2 SCAA medium at indicated concentrations of copper sulfate were inoculated with JBY100 or JBY200 culture to an A of 0.1. In order to maintain the cultures in exponential growth phase, all cultures were serially diluted to A = 0.1 when the A reached approximately 1.0. Samples were taken after 22 h (approximately 6-7 doublings), at which point intracellular levels of BiP and the levels of secreted protein were measured.

BiP Immunoassay

Acid Phosphatase Assay

GCSF Immunoassay

BPTI Activity Assay

KAR2 mRNA Quantification

RESULTS

Construction of a Yeast Strain with Variable BiP Levels

Because BiP expression is induced by secretion of heterologous proteins (Dorner et al., 1989; Tokunaga et al., 1992; Robinson and Wittrup, 1995), it is necessary to delete the chromosomal KAR2 gene coding for yeast BiP in order to remove native BiP regulatory effects and explore the consequences of low BiP levels in the context of heterologous protein expression. Because KAR2 is an essential gene in yeast, BiP expression in the deletion strain was provided from a galactose-inducible plasmid-borne KAR2 gene, which expresses 10-12-fold excess of BiP protein when grown on galactose (Robinson and Wittrup, 1995). A CUP1-KAR2 expression cassette on plasmid pCUPKAR2 was transformed into this strain to provide copper controlled BiP levels on glucose by varying the concentration of CuSO in the growth medium. This strain is subsequently referred to as JBY100 (Table 1).

A stationary phase culture of galactose-grown JBY100 + pNN342 was split to inoculate nine independent cultures in glucose medium containing 0-1.5 mM CuSO (pNN342 is a control CEN TRP1 plasmid lacking a heterologous protein expression cassette). Cultures were serially transferred to maintain exponential growth. Growth rates were similar for 30 h following transfer to glucose at CuSO concentrations of 30 µM and above, whereas growth was slightly slowed at 0 and 15 µM CuSO (data not shown.) In cultures of JBY001 (lacking the pCUPKAR2 plasmid), BiP levels drop to less than 1% of normal 20 h following transfer to glucose medium (data not shown). Thus, BiP levels sampled 22 h following transfer to glucose medium are entirely due to expression from the copper-inducible KAR2 gene. Western blots with -Kar2p antisera of cell extracts taken 22 h following transfer to glucose show a smooth increase in BiP levels with copper concentration (Fig. 1A). Densitometric quantitation of several exposures of this blot show that BiP levels are controlled across a 33-fold range by varying copper concentration (Fig. 1B). BiP protein levels are linearly related to KAR2 mRNA levels, as measured by ribonuclease protection assay (Fig. 1C). To control for artifactual physiological effects of copper on BiP expression, control cultures of JBY200 (BJ5464 + pLac33)+ pNN342 were grown at 0, 0.5, 1.0, and 1.5 mM CuSO. No effect on native KAR2 expression was observed at any of the CuSO concentrations used, although cultures at 1.5 mM exhibited a slightly reduced growth rate (data not shown).

Figure 1: A, BiP protein levels as a function of added CuSO concentration in JBY100, an S. cerevisiae strain constructed for copper-regulated BiP expression (Table 1). Cell extracts were performed on samples taken after 30 h of growth on glucose and analyzed by Western blot with -BiP antibody (as described under ``Experimental Procedures''). B, densitometric quantitation of a series of exposures of the Western blot shown in A (as described under ``Experimental Procedures''). Error in quantitation is typically less than 10%. C, BiP mRNA transcribed from the CUP1 promoter is linearly related to BiP protein present in JBY100 (open circles). JBY100 (open circles) was grown as described in A. BiP/Kar2p protein was determined by Western blot analysis as described for A and B. Total mRNA was purified, and KAR2 and DPM1 mRNA levels were quantified by ribonuclease protection assay (Ambion) as described under ``Experimental Procedures.'' The closed circle represents JBY200 possessing the wild-type chromosomal KAR2 gene.

Effect of Varying BiP Levels on Heterologous Protein Secretion

Secretion of GCSF was examined at varying BiP levels. Cultures of JBY100 transformed with a low copy GCSF expression plasmid (pCEN-GCSF) were grown and sampled as described for the experiment represented in Fig. 1. GCSF secreted to the growth medium was measured by immunoassay, and BiP levels were quantitated by densitometry of Western blots as described under ``Experimental Procedures.'' GCSF secretion was normalized to cell density to eliminate any effects due to differential cell growth. The addition of 0, 0.5, 1.0, or 1.5 mM CuSO had no detectable effect on growth, BiP expression, or GCSF secretion in control JBY200 + pCEN-GCSF cells. Maximal GCSF secretion is attained at BiP levels equivalent to those found in the JBY200 control strain (Fig. 2). Reduction of BiP below this level steadily reduces GCSF secretion to 10% of the maximal secretion at the lowest BiP level attained. A similar experiment was performed with S. pombe acid phosphatase with similar results: decreasing levels of BiP decreases specific secretory productivity (Fig. 3). BPTI secretion depends on BiP levels in a similar fashion (Fig. 4).

Figure 2: Human GCSF secretion decreases when BiP levels in JBY100 fall below those in control JBY200 cells. Cultures of JBY100 (open circles) and JBY200 (closed circle) were transformed with a low copy GCSF expression vector and then grown and sampled as described in the legend to Fig. 1. Secreted GCSF was measured in cellular supernatants by enzyme-linked immunosorbent assay assay (Quantikine GCSF kit, R& Systems) as described under ``Experimental Procedures'' and normalized by cellular growth. BiP protein levels were analyzed by quantitative Western blot as described in the legend to Fig. 1and under ``Experimental Procedures.'' BiP and GCSF were measured in four independent control JBY200+pCEN-GCSF cultures (closed circle), and error bars encompass clonal variability as well as measurement precision. A duplicate experiment showed similar trends in GCSF secretion.

Figure 3: S. pombe acid phosphatase secretion decreases when BiP levels in JBY100 fall below those in control JBY200 cells. Cultures of JBY100 (open circles) and JBY200 (closed circles) were transformed with a low copy PHO expression vector and then grown and sampled as shown in Fig. 1. Secreted acid phosphatase was determined by a spectrophotometric assay as described under ``Experimental Procedures.''

Figure 4: BPTI secretion decreases when BiP levels in JBY100 fall below those in control JBY200 cells. Cultures of JBY100 (open circles) and JBY200 (closed circles) were transformed with a low copy BPTI expression vector and then grown and sampled as shown in Fig. 1. Supernatant was assayed for BPTI levels by inhibition of trypsin as described under ``Experimental Procedures.''

It should be noted that in cells overexpressing any of the three heterologous proteins the maximum level of CUP-driven BiP expression is 2.5-fold that of cells possessing the native chromosomal KAR2 gene, although in cells not secreting foreign proteins the expression level is similar from the two promoters. This is likely a result of a previously observed tendency for levels of ER chaperones and foldases to decrease with prolonged constitutive secretion of heterologous proteins (Robinson et al., 1994; Robinson and Wittrup, 1995).

BiP Overexpression Does Not Significantly Affect Heterologous Secretion

DISCUSSION

We have constructed a strain of S. cerevisiae in which levels of the ER chaperone BiP can be continuously varied over a 33-fold range by use of the CUP1 copper-inducible promoter. Using this strain, we have examined the relationship between BiP levels and secretion of three heterologous proteins: GCSF, PHO, and BPTI. For all three proteins, secretion increases with increasing BiP levels up to a saturating point beyond which further increases in BiP provide no significant benefit. Furthermore, maximal secretion is obtained at BiP levels equivalent to wild-type levels from an unmodified strain.

Our results indicate a positive role for BiP in heterologous protein secretion, which differs from the findings of Dorner and co-workers (Dorner and Kaufman, 1994; Dorner et al., 1988, 1992). It is unlikely that this discrepancy is due to fundamental differences between the role of BiP in yeast and in mammalian cells, because expression of mammalian BiP has been shown to complement the kar2-1 mutation in yeast (Normington et al., 1989). Further evidence for the similarity of the yeast and mammalian lumenal environment is that expression of mammalian protein disulfide isomerase complements depletion of yeast protein disulfide isomerase, another essential gene product involved in protein folding in the ER (Gunther et al., 1993).

A more plausible explanation for the discrepancy between our results and those of Dorner and co-workers lies in the nature of the proteins studied. In fact, BiP may have multiple functions in the ER: promoting translocation, solubilizing folding precursors, stabilizing unassembled subunits, direct proofreading, retention, degradation, and perhaps other as yet unexplored functions. The particular facet of BiP function observed could depend on the structural characteristics of the substrate polypeptide studied. The proteins for which BiP has been shown to impede secretion in Chinese hamster ovary cells are either mutant (tissue plasminogen activator with three N-linked glycosylation sites deleted) or very large proteins (factor VIII is 250 kDa; von Willebrand Factor is 300 kDa) (Dorner et al., 1988, 1992). Furthermore, the processing and assembly of fVIII and vWf are complex; in mammalian cells, fVIII and vWf form a stabilizing complex together, and vWf is packaged in secretory granules for induced secretion (Voorberg et al., 1993). By contrast, the proteins we have examined are considerably smaller (GCSF, 19 kDa; PHO, 49 kDa; BPTI, 6.5 kDa) and perhaps more typical of a majority of proteins of pharmaceutical interest. It should also be noted that BiP overexpression in Chinese hamster ovary cells increases secretion of macrophage colony-stimulating factor (46 kDa) an average of 25-fold, although this effect was attributed to unexpected increases in macrophage colony-stimulating factor mRNA (Dorner et al., 1992).

Convergent lines of evidence indicate that BiP acts early in the secretory process, as the nascent polypeptide chain is translocated across the ER membrane into the lumen. Depletion of BiP from yeast or the shift to nonpermissive temperatures of certain temperature sensitive alleles of KAR2 result in a translocation block (Vogel et al., 1990), and BiP can be cross-linked to trapped translocation intermediates in contact with the Sec61p translocon component (Sanders et al., 1992). Genetic interactions between SEC63 and KAR2 imply an interaction similar to that found between the cytoplasmic bacterial chaperones DnaJ and DnaK (Scidmore et al., 1993), and the SEC63 gene product, possessing homology to DnaJ in a lumenal domain, is required for translocation in yeast (Feldheim et al., 1992). Furthermore, a Sec63p-BiP complex is a necessary component in reconstituted translocation reactions (Brodsky and Schekman, 1993; Panzner et al., 1995). It appears that the mitochondrial hsp70 plays a similar role in mitochondrial membrane translocation (Ungermann et al., 1994). Secreted proteins have been shown to undergo sequential interaction first with BiP and then with other lumenal chaperones. IgG polypeptides form transient complexes first with BiP and then with GRP94 (Melnick et al., 1994), whereas VSV G protein has been shown to bind first to BiP and then calnexin during folding (Hammond and Helenius, 1994). Thyroglobulin also interacts sequentially with BiP and calnexin while it folds in the ER (Kim and Arvan, 1995).

The function of BiP in translocation might involve direct binding of the nascent chain or alternatively cycling or assembly of translocon components. A proposed mechanism for translocation termed the Brownian ratchet has been proposed, wherein the driving force for membrane translocation is random thermal motion biased toward the lumenal direction by events on the lumenal side that sterically prevent backwards motion out of the ER (Simon et al., 1992). Binding by BiP could serve in this role. Given that BiP binds to relatively short polypeptide stretches (7 amino acids) (Flynn et al., 1991) in an extended conformation (Landry et al., 1992), BiP could bind nascent polypeptide chains as they emerge in the lumen. An alternative model is that BiP serves as a ``translocation motor'' that generates force to pull polypeptides into the ER (Glick, 1995). If the primary role for BiP is in membrane translocation, the lumenal requirement for BiP would be stoichiometrically related to the number of translocation sites in the ER membrane rather than the nature or quantity of proteins secreted. Our data are consistent with this interpretation, because the relationship between secretion and BiP levels is similar for all three proteins examined, despite substantial structural differences among these proteins.

The role of BiP in protein folding and secretion has previously been examined via a mathematical model that incorporates in vitro binding constants reported in the literature (Robinson and Wittrup, 1993) The predictions of this model are qualitatively similar to the results shown in Fig. 2Fig. 3Fig. 4. The model predicts that unless binding by BiP blocks folding to the extent that a polypeptide's tendency to aggregate is unchanged after release from BiP, secretion increases with increasing BiP to a plateau level. Thus, if a protein is less likely to aggregate after BiP release because some degree of folding took place while in complex with BiP, then decreasing BiP levels are predicted to decrease secretion efficiency, as our data indicate. The model also predicts the saturation effect observed, that increasing BiP levels beyond a certain point provides no incremental improvements in secretion.

Experimental evidence for a positive role for BiP in protein folding has also been obtained. Carboxypeptidase Y is bound by yeast BiP ATPase mutants at the restrictive temperature, essentially preventing the folding of carboxypeptidase Y and enhancing the tendency of the reduced form to aggregate (Simons et al., 1995). This result suggests that BiP plays an active role in carboxypeptidase Y folding and maturation.

From a practical perspective, it is unfortunate that manipulation of BiP levels does not improve heterologous protein secretion relative to unmodified strains. By contrast, overexpression of protein disulfide isomerase has been shown to result in substantial improvements in secretion of heavily disulfide bonded proteins (Schultz et al., 1994; Robinson et al., 1994). It is possible that limiting levels of Sec63p or an as yet unidentified ER GrpE homolog constrain the beneficial effects of BiP overexpression. However, it is also possible that chaperones such as GRP94 and calnexin that act later in the conformational maturation of proteins will play a more significant role in determining the capacity of the ER lumen to solubilize and assist in the folding of secreted proteins.

FOOTNOTES

*

This research was supported by Grant NSF BCS 92-13895 from the National Science Foundation (to K. D. W.). 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.

§

Supported by a Clare Booth Luce Fellowship. Present address: Dept. of Biology, Massachusetts Institute of Technology, Cambridge, MA.

¶

Present address: Dept. of Chemical Engineering, University of Texas, Austin, TX.

**

To whom correspondence should be addressed: Tel.: 217-333-2631; Fax: 217-244-8068; wittrup{at}aries.scs.uiuc.edu.

()

The abbreviations used are: ER, endoplasmic reticulum; GCSF, granulocyte colony-stimulating factor; PHO, S. pombe acid phosphatase; BPTI, bovine pancreatic trypsin inhibitor; GAPDH, glyceraldehyde phosphate dehydrogenase.

ACKNOWLEDGEMENTS

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