Biofechnol. frog. 1995, 11, 171-177
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Constitutive Overexpression of Secreted Heterologous Proteins Decreases Extractable BiP and Protein Disulfide Isomerase Levels in Saccharomyces cerevisiae Anne Skaja Robinson and K. Dane Wittrup" Department of Chemical Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801
High-level gene expression does not always lead to corresponding high-level secretion of heterologous proteins in yeast. The rate-limiting step in many cases h a s been shown to exit from the endoplasmic reticulum (ER). Within t h e ER, the correct folding of secreted proteins is required for export competence; hence, t h e cellular proteins involved in these events a r e likely to be important for efficient secretion. We have found t h a t the extractable levels of two ER-resident proteins involved in folding-heavy chain binding protein (BiP) a n d protein disulfide isomerase (PD1)-are significantly reduced by prolonged constitutive overexpression of h u m a n granulocyte colony stimulating factor (GCSF), h u m a n erythropoietin, or Schizosaccharomyces pombe acid phosphatase. However, the rate of BiP synthesis measured in pulse-chase radiolabeling experiments is not reduced by GCSF overexpression, a n d galactose-directed transcription of the BiP gene does not restore normal BiP protein levels once they have been depleted. The observed loss of lumenal resident proteins, either by proteolysis or irreversible aggregation, is expected to contribute significantly to t h e inefficiency of foreign protein secretion in yeast.
Introduction The yeast Saccharomyces cereuisiae has proven to be a useful organism for the expression of eukaryotic proteins for both basic research studies and pharmaceutical applications (Emr, 1990; Hodgson, 1993; Romanos et al., 1992). Human insulin and hepatitis B subunit vaccine are currently produced commercially in yeast (FDA Consumer, 1986; Diers, 1993). However, high-level transcription of foreign genes often does not produce high-level protein secretion in yeast (Shuster, 1991; Smith et al., 1985). Many studies of the secretion of both native and foreign proteins have shown that transit from the endoplasmic reticulum (ER) to the Golgi is the rate-limiting step (Lodish et al., 1983; Shuster, 1991). For example, expression of the hepatitis B large surface protein in S. cereuisiae leads to retention in the ER and causes enlargement of the ER cisternae (Biemans et al., 1991). Immunolocalization of secreted foreign or mutant proteins in yeast cells (Bielefeld & Hollenberg, 1989) and CHO cells (Gennaro et al., 1991) shows enlarged ER cisternae, also indicating that exit from the ER is ratelimiting. ER retention may be due to interactions with heavy chain binding protein (BiP) (Dorner et al., 19871, a member of the hsp7O family that was originally found in association with unassembled IgM heavy chains produced in mammalian cells (Haas & Wabl, 1984). BiP-heavy chain complexes are very stable and can be dissociated with the addition of ATP in vitro or by the coexpression of IgM light chains within the cells (Bole et al., 1989; Hendershot, 1990; Hendershot et al., 1988; Kassenbrock et al., 1988). ER-retained T-cell antigen receptor a-chain (TCR-a) is also found in a stable complex with BiP (Suzuki et al., 1991).
* Corresponding author: (telephone) 217-333-2631; (FAX) 217244-8068;
[email protected].
Aside from these stable interactions with misfolded or unassembled proteins, transient association of BiP with newly translocated proteins has also been demonstrated in several instances (Blount & Merlie, 1991; Bole et al., 1986; Dorner et al., 1987; Gething et al., 1986; Hendershot, 1990; Ng et al., 1992). For example, BiP forms a transient association with incompletely disulfide-bonded mutants of vesicular stomatitis virus G protein within mammalian cells, although more severe mutants form a stable interaction (Machamer et al., 1990). Transient association of BiP with tissue plasminogen activator has been correlated with efficient secretion in mammalian cells, while mutants with reduced secretion show increased association with BiP (Dorner et al., 1987). Overall, the evidence points to a transient association of the chaperone with normal proteins and a more stable interaction with mutant o r misfolded forms of a protein. As a result, BiP may play a dual role in solubilizing folding precursors and preventing the transport of unfolded and unassembled proteins. Kar2, the S. cerevisiae BiP homolog, was identified separately by homology to mouse BiP (Normington et al., 1989) and by function and sequence similarity of BiP to a karyogamy gene, Kar2 (Rose et al., 1989). Cross-linking studies with model translocation substrates suggest that yeast BiP plays a n active role in the earliest steps of membrane translocation (Sanders et al., 1992). Yeast BiP is a n essential gene, and temperature sensitive alleles produce a translocation block a t the restrictive temperature (Vogel et al., 1991). Previous work has shown that BiP message levels are induced shortly after heat shock (Normington et al., 1989; Rose et al., 19891, inhibition of glycosylation with tunicamycin (Dorner et al., 1987), or induction of foreign or mutant protein production (Kozutsumi et al., 1988; Tokunaga et al., 1992; Watowich et al., 1991). Another major lumenal resident protein is protein disulfide isomerase (PDI), which catalyzes disulfide bond formation and exchange within the oxidizing environ-
8756-7938/95/3011-0171$09.00/0 0 1995 American Chemical Society and American Institute of Chemical Engineers
172 Table 1. Characteristics of Foreign Proteins in Study
ex ression protein" pyasmid oligomer N-glycosylated size (aa) + 193 EPO pGPDaFEPO* pKEhFScEP@ 207 GCSF pYE3aG4GCSFb PHO pYEaFPHOb + + 435 GCSF-PHO pGPDP/Db + + 548 a All are expressed as prepro a-factor fusions. EPO, erythropoietin; GCSF, granulocyte colony stimulating factor; PHO, S. pombe acid phosphatase. Glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter. Galactose-regulated hybrid (GAL1,lOGPD) promoter. ment of the ER. PDI has been localized through microsome studies and marker enzyme analysis to both the smooth and rough ER (Freedman, 1984). In yeast, a protein originally identified as a glycosylation site binding protein (GSBP) was found to be identical to PDI purified from yeast (LaMantia et al., 1991; Mizunaga et al., 1990). Yeast PDI copurifies with the microsomal fraction and possesses a carboxyl-terminal HDEL, the ER retention signal in yeast. Yeast PDI is essential for spore germination (LaMantia et al., 1991) and is important but not strictly required for vegetative growth (Natalia et al., 1993). We have found t h a t PDI overexpression can significantly enhance heterologous protein secretion in yeast (Robinson et al., 1994). A number of foreign proteins were selected for this study on the basis of their different physical properties and secretion characteristics and the possibility of different interactions with BiP and PDI (Table 1). Human granulocyte colony stimulating factor (GCSF), a cytokine that regulates the proliferation and differentiation of progenitor cells into granulocytes (Nagata et al., 19861, is a monomer and has no N-linked glycosylation sites. Human erythropoietin (EPO) is a small monomeric glycoprotein (193 amino acids) involved in stimulating red blood cell production and has clinical value in the treatment of anemia (Browne et al., 1986). EPO is poorly secreted from S. cerevisiae and has three potential N-linked glycosylation sites (Elliott et al., 1989). Schizosaccharomyces pombe acid phosphatase (PHO) is a large enzyme (435 amino acids) that breaks down organic phosphates. PHO is highly glycosylated, and the major active form of the enzyme is a tetramer (Dibenedetto & Teller, 1981). A fusion between GCSF and acid phosphatase (GCSF-PHO), which contains the N-terminal one-third of the GCSF protein, was also studied. We examine here the effect of foreign protein secretion on BiP (Kar2p) and PDI protein levels in Saccharomyces cerevisiae. We find that prolonged constitutive expression of foreign secreted proteins reduces soluble BiP and PDI to levels undetectable by Western analysis. This is the first report of lowered ER chaperone and foldase levels as a consequence of heterologous protein secretion, and it has important implications for attempts to improve yeast expressiodsecretion systems.
Materials and Methods Strains and Plasmids. Cultures of YPH5OO (a ura352 lys2-801a ade2-101 trpl -A63 his3-A200 leu2-Al) a n d BJ5464 (a ura3-52 trpl leu2-A 1 his3-A200 pep4::HIS3 prbl-A1.6R can1 GAL) were obtained from the Yeast Genetic Stock Center (Berkeley, CA). All transformations were performed by electroporation using a Bio-Rad gene pulser (Becker & Guarente, 1991). Transformants were selected on synthetic complete (SC) medium (Sherman et al., 1986) buffered with sodium citrate buffer (50 mM, pH 4.5). Transformants were grown in liquid SC
Biofechnol. Prog., 1995, Vol. 11, No. 2 medium and stored as frozen stocks in 20% glycerol at -70 "C. Stocks were revived on SC plates, and individual colonies were grown overnight in culture tubes prior to inoculation. Samples were taken from cells grown in 50 mL of medium in 250 mL baffled Erlenmeyer flasks a t 30 "C. Plasmids for foreign protein expression (gift of S. Elliott, Amgen) are listed in Table 1. Small-scale plasmid DNA preparations were performed using Magic Minipreps (Promega). All proteins were directed for secretion by the a-factor signal sequence (the prepro leader region) and are multicopy 2p plasmids with TRPl as the selectable marker (Bitter et al., 1987). Inducible expression of erythropoietin (in Figure 2) was controlled by a GAL1GAL10 intergenic region placed in the glyceraldehyde3-phosphate dehydrogenase promoter (GPD(G)) (Bitter & Egan, 1988). This hybrid promoter allows for increased expression on galactose, although it is not completely repressed in glucose. Cells were grown overnight on glucose and then transferred as a 0.5% inoculum to medium lacking glucose and containing galactose (20 g/L). Samples in midexponential growth were taken a t 1.0 OD~OO. Plasmids for the overexpression of Kar2, pGalKar2HIS and pGalKar2-LEU, were subcloned from the URA3based plasmid pMR1341 (gift of M. Rose, Princeton University). The uracil marker was removed by digestion with XmaIII and SmaI and replaced with a synthetic double-stranded oligonucleotide cassette (Illinois Genetic Engineering Facility) with the sequences 5'-GGCCGATGAGCTCGGATCCGCATGCATCGATCCC-3' and 3'-CTACTCGAGCCTAGGCGTACGTAGCTAGGG-5'. This sequence contains anXmaIII-SmaI site as well as several unique sites (CZaI, SphI, BamHI, S a d , and EagI). The histidine marker (HIS31 was excised from pJJ217 (Jones & Prakash, 1990) by digestion with SphI and Sac1 and ligated into the linker of the pGalKar2 plasmid using the same restriction sites. The leucine marker (LEU2) was excised from pJJ252 (Jones & Prakash, 1990) and ligated as described above. Cells transformed with pGalKar2-HIS or pGalKar2LEU were grown on 20 g/L galactose for the induction of BiP overexpression. On glucose, expression of BiP was equal to that of wild-type cells, and on galactose, 10-fold increases in BiP protein were measured in cells transformed with these plasmids. Immunological Techniques. For the preparation of protein extracts, an adaptation of the standard glass bead lysis protocol was used (Franzusoff et al., 1991; Klekamp & Weil, 1982). Briefly, cells were collected in midexponential phase and resuspended in disruption buffer (20 mM Tris C1 (pH 7.9),10 mM MgC12,l mM EDTA, 1mM EGTA, 5% glycerol, 1 mM DTT,and 0.3 M ammonium sulfate) containing protease inhibitors (1mM PMSF, 1 pg/mL pepstatin A, 300 pglmL leupeptin, 100 pg/mL aprotinin, and 100 pglmL antipain). Cells were lysed by agitation in an equal volume of zirconium oxide beads in two 50 s cycles in a Bead-Beater (BioSpec Products) and separated by cooling for 1 min on ice. The extract was then collected and boiled for 5 min in 2% sodium dodecyl sulfate (SDS) and 80 mM dithiothreitol (DTT). After centrifugation a t 16000g for 30 min, the supernatant was collected and is referred to as the SDS-soluble extract. A second lysis technique was adapted from Hann and Walter (1991). One ODsoo milliliter of cells collected a t midexponential phase was resuspended in 100 pL of TCA buffer (20 mM Tris (pH 7.9), 50 mM ammonium acetate, 2 mM EDTA, and protease inhibitors as above). The suspension was then added to 100 pL of 20% TCA and
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Figure 1. Quantitation of intracellular BiP protein levels. TCAprecipitated protein extracts from yeast strain BJ5464 were diluted (with 1mg/mL BSA as a carrier protein), separated by 8% SDS-PAGE, and detected by ECL Western blot analysis with antisera again yeast BiP (Materials and Methods).
600 pL of zirconium oxide beads. After two 50 s cycles in a Bead-Beater and separation by cooling for 1min on ice, the extract and two 500 pL washes (1:l TCA buffer/ 20% TCA) were collected and centrifuged a t 16000g for 5 min. The pellet of this lysis was resuspended in 200 pL of TCA resuspension buffer (3% SDS, 100 mM Tris (pH 111, and 3 mM DTT) and boiled for 5 min; it is referred to a s the TCA-precipitable extract. For Western blots, equivalent total protein levels or equivalent ODs00 milliliters of extract were separated by 8% SDS-polyacrylamide gel electrophoresis. Proteins were electrophoretically transferred to nitrocellulose (Hoeffer tank electrophoresis). Anti-BiP primary IgG (Rose et al., 1989) was incubated with the nitrocellulose membrane at 1:lOOOO dilution or anti-PDI primary IgG (gift of V. Hines, Chiron) a t 1:2000 was incubated with goat anti-rabbit secondary antibody conjugated to horseradish peroxidase a t 1:2000 (Sigma). Detection of the antigen-antibody complex was performed with enhanced chemiluminescence (ECL, Amersham) and images were recorded on film (Hybond ECL, Amersham). For quantitation, several exposures of samples were scanned (Ofoto 2.0, Apple Scanner), and values in the linear range of the film were analyzed (Image 1.44, NIH). To determine the linearity of the immunoassay for BiP protein, dilutions of the extract of BJ5464 were assayed by Western analysis and quantitated as described earlier (Figure 1). For metabolic labeling of the cells, 50 pCi of [35Slmethionine (Amersham) was incubated at 30 "C with 1OD, of exponentially growing BJ5464 transformed with either a control plasmid or the expression plasmid for GCSF grown on minimal medium lacking methionine (Sherman, 1991). After 7 min, excess cold methionine was added (to 100 mM), and a cell pellet was collected by centrifugation for 2 min a t 3000g. To examine turnover of protein, the cell pellet was resuspended in 1 mL of minimal medium containing 10 mM methionine and incubated a t 30 "C for the period indicated. Cells were lysed by the SDS extraction method above. For detection of BiP protein, immunoprecipitation was performed essentially as described previously (Franzusoffet al., 1991) with 1 pL of anti-BiP primary antibody per ODsoo, and proteins were electrophoretically separated by 8% SDSPAGE. Following electrophoresis, the gel was soaked in Amplify (Amersham) for 30 min, and dried between Saran Wrap and 3MM Whatman paper (Hoeffer gel dryer). The dried gel was subsequently exposed to preflashed Hyperfilm-MP (Amersham) in a series of exposures to quantify results as before.
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