Jun 1, 2018 - AGOo 1. 1.9. 4. 5.5. 6.9. 8.1. 11.6 pelleted and resuspended in fresh YPD medium to the same density. Half of the suspension was grown at 24 ...
Vol. 269,No. 19,Issue of May 13,pp. 13887-13892, 1994 Printed in U.S.A.
JOURNAL OF BIOWGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. THE
The Role of the Carrier Protein and Disulfide Formation in the Folding of P-Lactamase Fusion Proteins in the Endoplasmic Reticulum of Yeast* (Received for publication, November 8, 1993, and in revised form, February 28, 1994)
Marjo SimonenS, Eija Jamsa, and Marja Makarow From the Institute of Biotechnology, University of Helsinki, Valimotie 7, 00380 Helsinki, Finland
We have studied the relationship between folding and secretion competence of hspl50-f?-lactamasefusion proteins in Saccharomyces cerevisiae. hspl50 is a secretory protein of yeast, and p-lactamase was chosen, since its folding can be monitored by assaying its enzymatic activity. The hspl50 pre-pro-protein consists of a signal peptide, subunit I, a repetitive region, and a unique C terminus. Fusion of 0-lactamase to the C terminus of hspl50 produced Cla-blaprotein, which was secretioncompetent but inactive. The Pst-blaprotein, where P-lactamase was fused to subunit I, was also inactive and mostly secreted, but part of it remained in the pre-Golgi compartment. When p-lactamase was fused to the C terminus of the repetitive region, the fusion protein (Kpnbla) was translocated to the endoplasmic reticulum, acquired disulfide bonds, and adopted an enzymatically active conformation. Kpn-bla was secreted to the medium without decrease of specific activity or retention in the cell. Folding of Kpn-bla to an active and transportcompetent form required co-translational disulfide formation, since treatment of cells with dithiothreitol resulted in endoplasmic reticulum-retainedinactive Kpnbla. When dithiothreitol was removed, Kpn-bla resumed transport competence but remained inactive. Reduction of prefolded Kpn-bla did not inhibit enzymatic activity or transport.The repetitive hspl50 carrier mayhave use in heterologous protein productionby conferringsecretion competence to foreign proteins in yeast. Correct foldingis a prerequisite for intracellular transport of proteins in mammalian cells. ER* proteins like BiP have been suggested to assist the folding process and to exert quality control by binding to immature and malfolded proteins (1-5). Folding studies of proteins in t h e ER of yeast have lagged behind, although yeast offers a genetic approach to characterize relevant proteins. Moreover, heterologous proteins are usually not secreted in yeast without a carrier polypeptide (6). Thus, the understanding of protein foldingand acquisition of a secretion-competent conformation in Saccharomyces cerevisiae is important for the developmentof production systems to secrete correctly folded heterologous proteins to the culture medium of yeast. The relationship of folding and transport competence has been studied in mammalian cells by altering the conformation of proteins by mutations or inhibition of glycosy-
* This work was supported by the Academy of Finland and the University of Helsinki. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. t To whom correspondence should be addressed. Tel.: 358-0-4346024; Fax: 358-0-4346028. The abbreviations used are: ER, endoplasmic reticulum; DTT, dithiothreitol; kb, kilobasecs);PAGE, polyacrylamide gelelectrophoresis; PDI, protein disulfide isomerase.
lation (2). Recently, a convenient method was introduced to manipulate proteins by reducing their disulfides by treating live cellswith DTT (7-9). We have appliedthis method to yeast cells, and shown that the disulfides of the secretory hspl50 Y (CPY) protein and the vacuolar enzyme carboxypeptidase with DTT, causing retentionof the proteins in could be reduced the ER, while the secretory apparatus remained functional. The sulfhydryls could be reoxidized by removal of DTT, resulting in resumption of transport (10). Here we studied in S. cerevisiae the relationshipof folding and secretion competence following the fate of p-lactamase fusion proteins, whose folding could be monitored by assaying their p-lactamase activity. MATERIALS AND METHODS Strains andMedia-The S. cereuisiae strains H1 (SEyalOla, MATa, ade2-101 ura3.52 leu23,112 sue269 ga121, secl8 (mBy12-6D, Mata secl8-1 trpl-289 leu23,112 ura352 his‘),sec63(RSY153, Matu sec63-1 leu23,112 ura3-521, and the transformants H337 (H1, Clabla), H335 (Hl, Kpn-bZa), H393 (secl8, Kpn-bla), H395 (H1, Pst-bla), H421 (secl8, Pst-bla), and H422 (sec63,Pst-bla),were grownat 24 “Cin YPD medium. Escherichia coli strains XL-1 Blue (Stratagene) and DH5a (Life Technologies, Inc.) were grownin LB medium supplemented with ampicillin (100 pg/ml) or tetracycline (12.5 pg/ml). Construction of the HSPl5O-bla Fusion Genes and the Integration Vector-From the genomic clone ofiYSF‘150 ( l l ) , a SalI-ClaI DNAfragment, which contains the HSP150 gene devoid of its four last codons, the plus approximately 2 kb of upstream sequence, was cloned between SalI and ClaI sites of Bluescript I1 SK- (Stratagene). The 450 bp ADCl terminator from pAAH5 (12) was ligated downstream from HSP150, between the HindIII and BamHI sites. The KpnI site of the vector was removed by digesting approximately 100 bp of the plasmid with Ba131 nuclease starting from the SalI site, and a BamHI linker was added. The bla gene without its signal sequence was synthesized with polymerase chain reaction using Pfu polymerase(Stratagene), and pUC8 as template. The 3’ primer A (5‘-GCAACCAAGCTTGAGTAAACTTGGTCTGACAG) containing a HindIII site was usedfor all constructions. The 5‘ primer B (5’-GCTTATATCGATGGTACCTGCAGTCACCCAGAAACGCTGGTG) was used for CZa-bla and Kpn-bla constructions, and the 5’ primer C (5’-GCTTATCTGCAGCTCACCCAGAAACGCTGG) forthe Pst-bla construction. The suitably digested polymerase chain reaction products were ligated between the ClaI and HindIII, KpnI and HindIII, and PstI and HindIII sites to create the Cla-bla, Kpn-bla, andPst-bla constructions, respectively,whichwere transferred as blunt-ended BamHI fragments to the ClaI site of the pKTH4542 vector. The plasmids were then linearized at the NcoI site and transferred to S. cereuisiae strains by electroporation (13). The integration vector was derived fromYEp24 (14) by removing its 2-pm replication origin. The ampicillin resistance gene, bla, was removed by digesting approximately 950 bp of the plasmid with Ba131 nuclease (Promega) starting from the AseI site. The resulting plasmid was designated pKTH4542. Standard DNA technologymethods were used(15). Metabolic Labeling, Immunoprecipitation, and Western AnalysisCells (lOs/2O0 pl) were labeled in YPD medium with 50-100pCi/ml 135Slmethionine/cysteine (SJQ.0079;1000 Ci/mmol, Amersham).The labeling and chase periods wereterminated by 10 mM NaN, (Sigma).The washed cells were lyzed, and the lysates and growth media were immunoprecipitated as described (10, 11)with anti-hspl50 against subunit 11,anti-p-lactamase, or preimmune serum, diluted 1:lOO. For Western analysis, proteins were transferred from SDS gels to nitrocellulose membrane (Hybond-C extra, Amersham), and subjected to immuno-
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FIG.1.A, schematic presentation of the products of the HSP150-bla genes. The primarytranslation products of the HSP150 gene, the bla gene, and theCla-bla, Kpn-bla, and Pst-bla genes, where bla was fused to different fragments ofHSP150, are shown. The numbers refer to the lastamino acid residue of each domain. SS, signal peptide; SUI, subunit I; SUII, subunit11; rr, repetitive region. Uppercase letters denote the amino acids of the linkers between the hspl50 and p-lactamase moieties. hspl50 has cysteines Cys3I6, Cys3", Cys4", and Cys4I3.Kpn-bla has cysteines Cys3I6 (hspl50-fragment), Cys322(linker), and CYS"~and C Y S ~(p-lactamase). ~' B, Northern analysisof chimeric mRNA molecules. RNA was isolated from strains H337 (Cla-bla, C ) , H335 (Kpn-bla, K),H395 (Pst-bla, P), and H1. Prior to RNA extraction, the cells were grown overnight a t 24 "C and incubated for 30 min a t 24 "C (HS-) or 37 "C (HS+). 10 pg of each RNA sample was subjected to Northern analysis, probing the membrane successively with HSP150 ( a ) ,bla (b), andACT1 (c) DNA. The estimated sizes (kb) of the RNA molecules are indicated. staining with antisera diluted 1:1000, using the ECLTMWestern blotting reagents (Amersham), according to manufacturer's instructions. Assay of p-Lactamase Activity-The p-lactamase activity was assayed using Nitrocefin (Glaxo), according to Ref. 16, but a t room temperature. Culture medium samples were assayed after removal of the cells by centrifugation. For the determination of p-lactamase activity of whole cells, the pelleted cells were lysed and suspended in 100 mM potassium phosphate, pH 7, to the original sample volume. To determine the p-lactamase activity entrapped in the cell wall (Nitrocefin is non-penetrable), washed cells were suspended in 1ml of the potassium phosphate buffer containing 10 mM NaN,, to the original volume. The intracellular activity was calculated by subtracting the cell wall activity from the whole cell activity. Secretory p-lactamase was not nonspecifically bound to the cells, as shown by assaying the p-lactamase activity of H1 cells incubated with Kpn-bla-containing growth medium. In Vitro Dunslation-The Pst-bla construct was placed under the control of the lac promoter in pUC18, a t the BamHI site. The plasmid was digested with PvuII, and a 1.9-kb fragment containing both the promoter and the fusion gene was isolated to servea s a template for in vitro transcriptiodtranslation, using the E. coli S30 Extract System for Linear Templates (Promega) according to manufacturer's instructions. Other Procedures-Northern analysis of total RNA was performed, as described (17). The stabilityof the transformants was studied by growing them to early, middle, and late logarithmic phase in YPD medium, followed by plating on YPD plates. The p-lactamase activity of 60 colonies from each plate was assayed by mixing them with Nitrocefin solution on microtiter plates; all produced p-lactamase activity and grew on SC-URA plates. SDS-PAGE was in reducing 8% gels unless otherwise stated. Non-reducing SDS-PAGE and in vivo protein reduction were performed a s before (10). Precipitation was with 14% trichloroacetic acid for 1 h on ice. Zymolyase lOOT was from Seikagaku; cycloheximide and D'IT were from Sigma. For scanning, an LKB Ultroscan XL laser densitometer was used. RESULTS
Fusion of the p-Lactamase Gene to Fragments of the HSP150 Gene-"he product of the HSP150 gene is composed of four
domains: (i) thesignal peptide, (ii) subunit I, (iii)the repetitive region (rr)of subunit 11, and (iv) the C-terminal region of subunit I1 (Fig. IA). The repetitive region consists of an 11-fold repeat of a 19-aminoacid peptide. The signal peptide is cleaved off in the ER, and subunit I is detached from subunit I1 a t a kex2 recognition site. The subunits remain noncovalently associated and areefficiently secreted to the culture medium (11). We wanted to study whether fusion of p-lactamase to fragments of hspl50 would allow folding of the p-lactamase portion to an enzymatically active conformation, and whether the fusion protein would be secreted to the growth medium. "he E. coli TEM p-lactamase gene, bla, lacking its signal sequence, was fused to various portions of the HSP150 gene. p-Lactamase was chosen as a reporter, because its folding can be monitored by determining its enzymatic activity (18) and because it is not secreted in S. cereuisiae in its authentic form (19, 20). The largest construct, Clu-bla, lacked thelast four codons of HSP150. Kpn-bla contained the signal sequence, and codons for SUI and the repetitive region. Pst-bla contained the signal sequence and 45 codonsof SUI. The fusions were introduced in an integrative derivative of yEp24 to strain H1, to create strains H337 (Clu-bla),H335 (Kpn-blu),and H395 (Pst-blu). Southern analysis showed that theplasmids had integrated as single copies into theURA3 locus (not shown), and integration appeared stable (see "Materials and Methods"). Dunscription of the Chimeric Genes-The chimeric genes were under the control of the heat-regulated HSP150 promoter (17). Two RNA samples were extracted: one from cells incubated at 24 "C (HS-) and theother from cellsheat-shocked for 30 min a t 37 "C (HS+).Northern analysis using an HSP150 probe (Fig. lL3,panel a ) and a bla probe (panel b ) showed that
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m c m c m c FIG.2. Secretion of the fusion proteins. Strains H337 (Cla-blu,A), H335 (Kpn-bla,B ) , and H395 (Pst-blu,C, panel a),were labeledfor 1h at 37 "C with [3sS]methionine/cysteine.Cycloheximide was addedfor a chase period of 20 min at 37 "C. The culture medium was separated from the cells, which were lysed. The culture medium (n; lanes 1 , 3 , and 5)and cell lysate (c; lanes 2 , 4 , and 6 ) samples were divided in three equal portions, which were subjectedto immunoprecipitation with anti-hspl50 (lanes 1 and Z ) , anti-p-lactamase(lanes 3 and 4 ) . and preimmune serum (lanes 5 and 6). C , panel 6 , H421 cells (lane I ) or H422 cells (lane 2 ) were preincubated for 15 min at 37.5"C and labeled with [3sSlmethionine/ cysteine for 60 and 30 min, respectively, at 37.5 "C. The lysed cells were subjected to immunoprecipitationwith anti-p-lactamase.In lane 3 , the [3sS]methionine/cysteine-labeled in vitro translation product of the Pst-bla gene was immunoprecipitated with anti-p-lactamase.Pst-bla,, secretory Pst-bla;Pst-bla,,intracellular Pst-bla.SDS-PAGE analysiswas in 8%gels (A and B ) or in 10%gels ( C ) .Molecular size markers (kDa) are indicated. the transformants synthesized novel mRNA molecules, in addition to HSP150 mRNA of 1.6 kb. The chimeric mRNAs hybridized with the bla probe (panel b),and the HSP150 probe (panel a ) ,but hybridization ofPst-bla mRNAwith theHSP150 probe was not detectable (panel a, lanes P ) .The chimeric genes were heat-regulated likeHSPISO (compare HS- to HS+). The amounts of the sampleswere controlled by hybridization with the actin geneACTl, which is not heat-regulated (panel c). Secretion of the hspl50-~-lactamaseFusion Proteins-The expression and secretion of the fusion proteins were studied by labeling them with [35S]methionine/cysteinefor 1 h at 37 "C, followed by a 20-min chase with cycloheximide to stopprotein synthesis. The culture media and cell lysates were subjectedto immunoprecipitation and SDS-PAGE analysis.Immunoprecipitation with anti-hspl50 detected from the culturemedium of H337 cells (Cla-bla)hspl50 of 150 kDa, anda new protein of about 135 kDa (Fig. 2A, lane I; m, medium). The new protein was also detected with anti-p-lactamase(lane 3 ) , and wasdesignated Cla-bla. Cla-bla was detected also in the cell lysate with both antisera (lanes 2 and 4; c, cell lysate). Preimmune serum recognized no proteins (lanes 5 and 6 ) . Immunoprecipitation of the culture medium of H335 cells (Kpn-bla),35S-labeledlike theH337 cells above, revealed a new protein of about 135 kDa which was recognized by anti-hspl50 (Fig. 2 B , lane 1 )and anti-p-lactamase (lane 3 ) . I t was designated Kpn-bla. Very little of Kpn-bla-related proteins could be detected in the lysate(lanes 2 and 41, indicating thatKpn-bla was secreted efficiently to thegrowth medium. When H395 cells (Pst-bla)were analyzed, anti-hspl50 detected only hspl50 (Fig.2C,panel a , lanes l and 2), because the antiserum was raised against subunitI1 (see Fig. lA).Anti-plactamase detected a 48-kDa protein from the culturemedium (lane 3 ) , and a 44-kDa protein from the cell lysate (lane 4 ) . According to densitometric scanning, about 35% of Pst-bla (32 and 38% in two experiments) remainedcell-associated, and the rest wassecreted. The 44-kDa protein was thepre-Golgi form, since Pst-bla labeled at 37 "C in H421 cells migrated like a 42-kDa protein (Fig. 2C, panel b, lane I). In the H421 strain (secl81, secretory proteins accumulate in the pre-Golgi comthe partment at 37 "C. When Pst-bla was arrested in cytoplasm
in the translocation-deficient H422 cells (sec63),it migrated like a 38-kDa protein (lane 2), like the in vitro transcription/ translation product of the Pst-bla gene (lane 3 ) . To compare the relative amounts of the threefusion proteins secreted to the culture medium, they were 35S-labeledand subjected to immunoprecipitation and SDS-PAGE analysis in parallel. According to scanning of the bands, similar amounts of Cla-bla (10 Met, 6 Cys) and Kpn-bla (9 Met, 4 Cys) were detected in the media (Fig. 3 A , lanes l and 2, open arrowhead), whereas the amount of Pst-bla (9 Met, 2 Cys) was roughly one-fourth of that of Cla-bla (lane 3 , black arrowhead). This was confirmed by Western analysis of culture medium samples of strains H1, H337( C ) ,H335 ( K )and H395 (PI, using anti-plactamase (Fig. 3B, panel a ) and anti-hspl50 (panel b).Thus, Kpn-bla was efficiently secreted to the culturemedium, apparently withoutsignificant retardation insecretory organelles, or in thecell wall or periplasm. Cla-bla was transport-competent, part of it remainingcell-associated. Pst-bla wastranslocated to the ER, but only small amounts were secreted. Pst-bla was evidently harmful for the cells, since the generation time of strain H395 was 3 h, 25 min, whereas thatof the other transformants and the parental strain was 2 h. p-lactamase Activity of the Fusion Proteins-To monitor the folding of the fusion proteins, their p-lactamase activities were measured. When strain H335 (Kpn-bla), wasincubated at 24 "C, 0.31 unitlml p-lactamase activity was in the culture medium after 3 h (Fig. 4C, triangles). Much less activity was found in the cell walVperiplasm (circles) and intracellularly (diamonds).When the incubation was performed at 37 "C, to enhance synthesis by heat shock, 1.62 unitdm1 p-lactamase activity was detected in the culture medium, 0.29 unitlml in the cell wall, and only traces intracellularly (Fig. 40).Very little p-lactamase activitycould be detected at either temperature in the media or cells of strains H337 (Cla-bla)(Fig. 4, A andB) or H395 (Pst-bla)(Fig. 4, E and F). To study whether the fusion proteins were enzymatically active in theER, the Kpn-bla and Pst-bla constructs were expressed in a s e d 8 mutant (strains H393 and H421, respectively). When H393 (Kpn-bla) was incubated for 90 min at 37.5 "C, p-lactamase activity accumulated insidethe cells (Fig.
Folding of p-Lactamase Fusion Proteins inthe Yeast ER
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FIG.3. Relative amounts of the fusion proteins in the culture medium.A, strains H337 (Cla-bla,C, lane I ), H335 (Kpn-bla, K,lane 2 ) ,and H395 (Pst-bla,P,lane 3 ) were labeled in parallel for 1h at 37 "C with [%lmethionine/cysteine, and the culture media were immunoprecipitated with anti-p-lactamase, followedby SDS-PAGE analysis. B, strains H1 (lanes Id), H337 ( C , lunes 4 4 ) ,H335 ( K , lanes 7-91,and H395 (P,lanes 10-12) were incubatedfor 1h at 37 "C (5x lo8 cells/ml). Then, 20-, 40-, and 8O-pl samples of the culture media were lyophilized and subjected to SDS-PAGE. The proteins were blotted and immunostained using anti-p-lactamase (panel a ) or anti-hspl50 (panel b). Open arrowhead, Cla-bla and Kpn-bla; blackarrowhead,Pst-bla; star, hspl50. Molecular size markers are on the left.
5A,triangles), and very littlewasinthe medium (closed circles). When cycloheximide was added (arrowhead), and the cells wereshifted to 24 "C, the cell-associated activity decreased (closed triangles), while the activity in the medium increased, showing that the accumulated fusion protein was secreted. Thus, the p-lactamase portion of the Kpn-bla fusion protein folded in the ER to an active conformation, and the specific activity of the fusion protein did not change significantly after exitfrom the ER. In contrast, the Pst-bla molecules accumulating in the ER in strain H421 had very little enzymatic activity (Fig. 5B 1. The Effect of Reduction of the Disulfides of Kpn-bla on Its Z'kansport Competence-Next we wantedtomanipulatethe conformation of intracellular Kpn-bla by reducing its possible disulfides (lo), to study theconsequences on transport. H335 cells were metabolically labeled in thepresence of DTT. Immunoprecipitations with anti-hspl50 (Fig. 6A, panel a ) and antibla (panel b ) showed that thefusion protein was not secreted (lanes 3 ) . Cell-associated Kpn-bla was barely visible (lanes 41, probably due to heterogeneity of the glycans. Nevertheless, it could be efficiently chased to the growth medium after the removal of DTT (lanes 5-12). Thus, under normal conditions, Kpn-bla acquired disulfides,which were reduced by DTT treatment, leading toreversible intracellular retention. When Kpn-bla was synthesizedin thepresence of DTT, P-lactamase activity could not be detected in the culture medium (Fig. 6B,panelb, closed circles) or intracellularly (open circles). Removal of DTT (arrowhead) did not increase p-lactamase activity. In vitro reduction of secreted Kpn-bla did not inactivate the enzyme (not shown). Thus, synthesis under reducing conditions produced inactive Kpn-bla, and removal of DTT resulted in resumption of transport competence of the molecules. However, the reoxidized structure wasdifferent from the native one, since it hadno enzymatic activity. Kpn-bla was thenallowed to fold in theabsence of DTT, while retaining it at 37.5 "C in the ER in strainH393 (sec18)(Fig. 5A).
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t (rnin) FIG.4.PLactamase activity of the fusion proteins. Strain H337 (A and B , Cla-bla),strain H335 (Cand D,Kpn-bla),and strainH395 (E and F , Pst-bla) were grown a t 24 "C to a density of 2.5 x lo7 celldml (A,,, = 1).The cells were pelleted, resuspended in fresh YPD medium to the same density, and divided into two portions. One flask was incubated further at 24 "C (A, C, and E), and the othera t 37 "C (B,D,and F ) . p-Lactamase activities were measuredfrom the culture media( M ) , cell walls ( C W ) ,and intracellular compartments( I C )at different time points and plotted against time.
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t (min) FIG.5.P-Lactamase activity of Kpn-bla and Pst-bla blocked in the ER. Strain H393 (A, Kpn-bla) and strain H421 ( B , Pst-bla) were grown a t 24 "C to a densityof 2.5 x 10' cells/ml. The cells were pelleted, resuspended in freshYPD medium to a density of 5 x lo7 cells/ml, and incubated a t 37.5 "C. Cycloheximide was added (arrowhead),and the cells were incubated further at 24 "C (closed symbols). In panel A, parallel cells received 20 mM DTT with cycloheximide (open symbols). At different time points, samples were taken for analysis of p-lactamase activity of the culture medium (circles) and cell lysates (triangles). p-Lactamase activity is plotted against incubation time.
Part of the cells were incubated further a t 24 "C in thepresence of cycloheximide only, which led to secretion of the molecules, as described above (arrowhead, filled symbols). Addition of DTT with cycloheximide to part of the cells had no effect on the se-
Folding of P-Lactamase Fusion Proteins in Yeast the
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tated and resolved in SDS-PAGE. Kpn-bla appeared tobe the major protein detected by Coomassie Blue staining (Fig. 7B, large arrowhead). Its amountat A,,, = 11was estimated tobe about 2 gg/ml, as compared to the staining of BSA (Fig. 7B). This may be an underestimation, since hspl50 (small arrowhead) is very poorly stained by Coomassie Blue. According to Western analysis, its amount in medium the was similar to that of Kpn-bla (Fig. 3B, panel b). DISCUSSION
We fused the TEM p-lactamase of E. coli, lacking its own signal sequence, to the C termini of various portions of the yeast secretory glycoprotein hspl50.The chimericproteins were used to study theeffect of the hspl50-carriersequence on the folding of the p-lactamase portion and theeffect of folding on the secretion competence of the fusion proteins. In thelargest chimera, Cla-bla, hspl50 lacked its 4 C-terminal amino acids. In Kpn-bla, 0-lactamase was fused to the repetitiveregion. The smallestfusion protein, Pst-bla, containedthe signal peptide and two-thirds of subunit I (seeFig. lA). The folding of the p-lactamase portion was monitored by assaying its enzymatic activity (18).The authentic structuralbla gene produces in S. cerevisiae enzymatically inactive, cell-associated pre-Plactamase (19, 20). The HSPl50-bla fusiongeneswere transcribed similarly. Their products were translocated to the ER but differed in p-lactamase activity and secretion competence. Pst-bla did not gain anenzymatically active structure in theER. Less Pst-bla was detected in the culture medium than Cla-bla or Kpn-bla, t (min) and one-third of it appeared to remain in the pre-Golgi comFIG.6. A, effect of DTT on secretion of Kpn-bla. Strain H335 (Kpn- partment. The almost entire hspl50 fragment rendered Cla-bla bla) was pulse-labeled ( p ) for 1 h at 37 "C in the absence (lanes I and secretion-competent, some remaining in theGolgi, periplasm, 2; DTT-), or presence of DTT (lanes 3 and 4; DTT+). Parallel cells, labeled in the presence of DTT, were washed and chased (c) at 37 "C or cell wall. However, Cla-bla had virtually no p-lactamase with cycloheximide in theabsence of Dl" for 30 min (lanes 5 and 6 ) .60 activity. It seems that the incomplete subunit I and the Cmin (lanes 7 and 8),90 min (lanes 9 and IO),or 120 min (lanes I 1 and terminal domain of hspl50 interfered somehow with the fold12).The growth media ( m )and cell lysates (c) were subjected to immu- ing of the p-lactamase portion, causing malfolding. noprecipitationwith anti-hspl50 (panel a ) or anti-p-lactamase (panel In contrast to Pst-bla and Cla-bla, Kpn-bla adopted a n enzyb ) and analyzed by SDS-PAGE.Small arrowhead,hspl50; large arrowmatically active conformation and acquired disulfide bonds in head, Kpn-bla. B , effect of DTT on p-lactamase activity of Kpn-bla. Strain H335 was incubated at 37 "C in the absence (panel a ) or pres- the ER. It was efficiently secreted to the culture medium, withence (panel b ) of DTT. In panel b, the cells were washed after 2 h out decrease of specific activity, or retention in the secretory (arrowhead) and incubated with cycloheximide in the absence of DTT. route or the cell walllperiplasm. Partial secretion of active, unp-Lactamase activities of the growth media (closed circles) and cell stable p-lactamase has been achieved using the invertase signal lysate (open circles)were plotted against incubation time. sequence (21),but thesecreted activitywas 1/30that of Kpn-bla. cretion or enzymatic activity of Kpn-bla (open symbols). Native We were able to distort conformation the of the fusion proteins and in vivo reduced, ER-trapped Kpn-bla migrated differently by reducing their disulfides in vivo. When disulfide formation of in non-reducing SDS-PAGE,the lattercomigrating with invitro Kpn-bla was prevented by DTT treatment of cells, the protein completely reduced ER-trapped Kpn-bla (not shown). This dem- was inactive and retained in the ER. Removal of DTT allowed onstrated that prefolded Kpn-bla was not resistant to DTT. Kpn-bla to adopt a secretion-competent structure, which was Since the reduction of prefolded Kpn-bla did not inhibit its en- different from the nativeform, since the enzymatic activitywas zymatic activity or secretion, we assume that co-translational not resumed. DTT treatment distorted the folding of Kpn-bla disulfide formation was required for the folding of the fusion severely only co-translationally. Once Kpn-bla was allowed to protein into anenzymatically activeconformation and that the fold normally, subsequent reduction did not affect its enzymatic site of retention of reduced Kpn-bla was the ER. activity or exit from the pre-Golgi compartment. The role of proContinuous Secretion of Active Kpn-bla-The above results tein folding in secretion in yeast has been observed before in the suggest that theKpn fragment of hspl50 may havebiotechno- case of human a-amylase,where substitution of Cys,,, abolished logical potential for production of heterologous proteins,which, secretion and activity (22). In thecase ofyeast acid phosphatase, without a carrier, are retained in the yeast cells. Therefore we the removal of N-glycosylation sites caused malfolding and irstudied the production of Kpn-bla more closely. When strain reversible ER retention (23). Reduction of hspl50 and carH335 (Kpn-bla) was cultivated at 24 "C, p-lactamase activityin boxypeptidase Y by DTT treatment resulted in their retention the medium increased as long as the cells were growing, reach- in theER. This must havebeen due tomalfolding and not free ing a maximum of 18 units/ml in the stationary phase (Fig. 7A, sulfbydrylsper se, since sulfhydryl-containing invertase was sepanel a, triangles). When the cells were grown to a density of creted normally in thepresence of DTT (10). Thus,yeast clearly A,,, = 1 at 24 "C and cultured further at 37 "C, the activity has a quality control apparatus, which monitors proteins and reached a maximum of 29 unitdm1 (Fig. 7A, panel b, triangles). retains them in theER, unless they display acceptable strucThe fusion protein appeared stable at both temperatures. tural features. For foreign proteins, the transport-competent Proteins from the culture medium of H335 cells, grown at conformation does not need to be the biologically active struc30 "C to different densities, were trichloroacetic acid-precipi- ture, since Cla-bla, Pst-bla, and reoxidized Kpn-bla were se-
ER
Folding of p-Lactamase Fusion Proteins Yeast the in
13892
A
b
l
..
i
i
L i 116 97.4-
66-
1
AGOo
1
1.9
4
5.5
6.9
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11.6
FIG.7. Continuous secretionof Kpn-bla.A, strain H335 was grown at 24 "Cto A,, = 1 (2.5 x lo' cells/ml) and divided in two. The cells were pelleted and resuspended in fresh YPD medium to the same density. Half of the suspension was grown a t 24 "C (panel a), and the other halfa t 37 "C (panel b ) . The p-lactamase activities (units/ml; triangles) and thecell densities (A6o,,;dots) are plotted uersus time. B , strain H335 was grown a t 30 "C. Samples (1 ml) were taken at the indicated cell densities, and the growth media were subjected to trichloroacetic acid precipitation, SDS-PAGE, and Coomassie Blue staining(lanes 1-7). Lane 8,growth medium of strain H1. Large arrowhead,Kpn-bla; small arrowhead, hspl50. Lane 9, 3.4 pg of BSA (arrow).Molecular size markers areon the left.
creted, even though they were inactive. Yeast proteins anticipated to be involved in folding of proteins include B ~ P (Kar2p1, protein disulfide isomerase (PDI), and the PDI homolog Euglp (24-28). Depletion ofpDI and ~~~l~ led to accumulation of the ER form of carboxypeptidase Y, suggesting arrest of transport. BiP has been shown to function in the translocation of polypeptides the ER membrane (29). we have demonstrated thatfully translocated ER-retained reduced pro-hspl50 is associated with Bip, but association with native hsp150 could not be detected (10).The yeast plasma membrane H+ATPasehas been found to beproperly folded and transported in the absence of functional BiP(30). Many heterologous proteins expressed in S. cereuisiae without a carrier sequence are notsecreted. The only carrier, which so far has appeared to have general applicability, is thea-factor leader (6, 31-37). Pro-a-factor is a secretory protein of yeast. a-Factor moieties are from the leader in the and are secreted to the medium (38).It is not known how the carrier sequence confers secretion competence to the heterologous protein portion. Probably a functional carrier should form a complete domain tobe recognized by the quality control machinery as properly folded and should not interfere severely with the folding Of the heterologous protein portion. The repetitive region of the hspl50 is likely to form a structural entity and may have biotechnological potential in production of heterologous proteins to the yeast culture medium, since it confers secretion competence even to mammalian proteins.* Acknowledgments-we thank A. L' Nyfors and T. for technical assistance, T. Ahola for p-lactamase assays and stability tests, and Dr. L. E l r i a i n e n for critical reading of the manuscript. REFERENCES ~ A, l (1990) ~ J~, cell i ~ ; , ~, l ,111,857-866 ~ , 2. Gething, M.-J., and Sambrook, J. (1992) Nature 3 5 5 , 3 3 4 5 3. Hurtley, S. M., Bole, D. G., Hoover-Litty, H., Helenius, A., andCopeland, C. S. (1989)J. Cell Diol. 108, 2117-2126 4. Kassenbrock, C.K., Garcia, P. D., Walter, P., and Kelly, R. B. (1988) Nature 333.90-93 5. Rothman, J. E. (1989) Cell 59, 591-601 6. Romanos, M. A., Scorer, C. A,, and Clare, J. J. (1992) Yeast 8 , 4 2 3 4 8 8 7, Alberini, c, M,, Bet, p,, ~ i l ~ k i , c,, , , and Sitia, R, (1990)N~~~~~347,485487 1. de silva, A. M., Balch,
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