Proc. Natl. Acad. Sci. USA Vol. 93, pp. 9612-9617, September 1996 Cell Biology

Split invertase polypeptides form functional complexes in the yeast periplasm in vivo OSHRAT SCHONBERGER*, CAROLINE KNOX*, EITAN BIBIt, AND OPHRY PINES*t *Department of Molecular Biology, Hebrew University-Hadassah Medical School, Jerusalem, Israel; and tDepartment of Biochemistry, Weizmann Institute of Science, Rehovot, Israel Communicated by Randy Schekman, University of California, Berkeley, CA, April 19, 1996 (received for review October 24, 1995)

ABSTRACT The assembly of functional proteins from The assembly of functional proteins from fragments in vivo fragments in vivo has been recently described for several has been recently demonstrated for several proteins. In fact, proteins, including the secreted maltose binding protein in Bibi and Kaback (6) initiated,a series of studies to exploit this Escherichia coli. Here we demonstrate for the first time that possibility. When the lactose permease of Escherichia coli was split gene products can function within the eukaryotic secre- expressed as two approximately equal-size fragments, an as- tory system. Saccharomyces cerevisiae strains able to use sociation between the two polypeptides led to a stable, cata- sucrose produce the enzyme invertase, which is targeted by a lytically active complex. To date, examples of other functional signal peptide to the central secretory pathway and the split genes, mainly of membrane, but also a few soluble periplasmic space. Using this enzyme as a model we find the cytoplasmic proteins, have been described (for example, refs. following: (i) Polypeptide fragments of invertase, each con- 7-9). More recently, independently exported protein frag- taining a signal peptide, are independently translocated into ments of the maltose binding protein have been shown to the endoplasmic reticulum (ER) are modified by glycosylation, assemble in vivo into an active complex in the periplasm of E. and travel the entire secretory pathway reaching the yeast coli (10). These studies have improved our understanding of periplasm. (ii) Simultaneous expression of independently protein folding and structure in vivo. Nevertheless, a study of translated and translocated overlapping fragments of inver- split gene product assembly in the ER, which is the folding tase leads to the formation ofan enzymatically active complex, compartment of exported proteins, has not been undertaken. whereas individually expressed fragments exhibit no activity. Here we study a model system in which independently ex- (iii) An active invertase complex is assembled in the ER, is pressed fragments of the enzyme invertase are secreted into the targeted to the yeast periplasm, and is biologically functional, ER, glycosylated, and assembled into an enzymatically active and as judged by its ability to facilitate growth on sucrose as a the the single carbon source. These observations are discussed in biologically functional complex reaching yeast periplasm, relation to protein folding and assembly in the ER and to the endmost subcellular target of the secretory pathway. trafficking of proteins through the secretory pathway. MATERIALS AND METHODS Upon entry into the endoplasmic reticulum (ER), newly Strains, Media, and Growth. The Saccharomyces cerevisiae synthesized proteins undergo rapid folding and assembly to strains used were DGY505 (MATa, his4, ura3, trpl, ade2, acquire their functional tertiary and quaternary structure. The suc2-A9; kindly provided by D. Granot, The Volcani Center, ER provides the appropriate environment and components Israel), DMM1-15A (MATa, leu2, his3, ura3, ade2; ref. 11), that are needed to facilitate the functional assembly of trans- ref. BJ2168 prcl-407, located proteins. From the ER, proteins are distributed to JTY-5186 (secl8, leu2, ura3; 11), (pep4, various destinations in the cell via the central secretory path- prb-1122, leu2, ura3, trpl; provided by Y. Ben Neriah, The way. Nascent secretory proteins are delivered to the lumen of Hebrew University, Jerusalem), 8979-3A (kar2-1, leu2, ura3, the ER, pass through the Golgi complex, and then accumulate his4, ade2, CANS; ref. 11), SEY5018 (secl, leu2, ura3, Asuc2, in specialized secretory vesicles, which fuse with the cell provided by R. Schekman), and RSY586 (kar2-159, leu2, ura3, surface, allowing exocytosis. Movement of the proteins between ade2; provided by R. Schekman). The following were obtained these compartments occurs by the budding and fusion of trans- by the indicated cross: OSH4 (kar2-159, leu2, ura3, trpl, porting vesicles (1, 2), and the current view is that secreted suc2-A9), RSY586 x DGY505; OSH5 (secl, leu2, ura3, trpl, proteins will follow this pathway unless retained or diverted by suc2-A9), SEY5018 x DGY505; OSH7 (secl8, leu2, ura3, trpl, specific signals in their sequence or structure. Since proteins must suc2-A9), JTY5186 x DGY505; and OSH9 (kar2-1, leu2, ura3, fold and assemble correctly to leave the ER (3), secretion out of trpl, ade2, his3, suc2-A9), 8979-3A x DGY505. The growth the cell is an indication of its ability to fold in the ER. medium (SD) used contained 0.67% (wt/vol) yeast nitrogen Our current understanding of protein folding and assembly base without amino acids (Difco) and 0.5-2.0% (wt/vol) has been enhanced by the classical approach of protein frag- glucose, galactose, or sucrose. ment assembly in vitro, which allows the examination of Preparation of Cell Extracts and Cell Fractionation. Extracts intermediates in the folding process. For example, in vitro were prepared by growing 10 ml of yeast culture to 3.0 optical complementation ofvarious combinations of overlapping frag- density units (at-600 nm) on SD medium at 25°C, harvesting the ments of staphylococcal nuclease and cytochrome c are the cells by centrifugation, and suspending them in 800 ,ul of TE well-known models of this approach (4, 5). Many experiments buffer (10 mM Tris HCl buffer, pH 8.0/1 mM EDTA) containing of this sort, involving functional complementation in vitro, 1 mM phenylmethylsulfonyl fluoride (PMSF). Cells were broken have been performed either with fragments produced by by vigorous mixing with glass beads for 1.5 min followed by limited proteolysis, by chemical cleavage, or by using incom- centrifugation at 4°C for 4 min. The supernatant fraction ob- plete polypeptide chains expressed via genetic manipulation. tained was used for all assays and analyses.

The publication costs of this article were defrayed in part by page charge Abbreviations: ER, endoplasmic reticulum; endo H, endoglycosidase payment. This article must therefore be hereby marked "advertisement" in H. accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 9612 Downloaded by guest on September 23, 2021 Cell Biology: Schonberger et al. Proc. Natl. Acad. Sci. USA 93 (1996) 9613 Subcellular fractionation using Zymolyase-20T (Siekbagaku on SDS/10% polyacrylamide gels, transferred to nitrocellulose, Kogyo, Tokyo) was performed as described (11). Protease and probed with rabbit anti-invertase antiserum (20). inhibitors were added at the following final concentrations: Electroelution. Triplicate samples of nondenatured extract a-macroglobulin, 2 units/ml; antipain, 5 ,ug/ml; pepstatin, 1 were fractionated as above for invertase activity detection, Ltg/ml; aprotinin, 5 uLg/ml; leupeptin, 2 Ig/ml; PMSF, 10 mM; Coomassie blue staining, and electroelution of protein. The and EDTA, 5 mg/ml. Membrane and soluble fractions of the activity-stained portion of the-gel was excised for electroelu- spheroplasts were prepared by centrifugation at 120,000 x g tion, which was carried out in a dialysis bag in gel running for 90 min at 4°C in a Beckman L5-50 ultracentrifuge. The buffer (without detergent) at 100 V for 5-6 h at 4°C. Elec- soluble fraction was removed, and the membrane pellet was troeluted samples were concentrated [15-ml Vivaspin contain- resuspended in 0.5 ml of the same buffer. ers (Vivascience, Lincoln, U.K.) for 20 min at 1960 x g], Enzyme Activities and Assays. Invertase activity was based denatured, and treated with endo H for 3 h at 30°C. on the determination of reducing sugars (12), and protein was determined by the method of Bradford (13). For activity in RESULTS whole cells, half of a cell suspension was used to make an extract as described above, and the remainder (whole cells) was Construction of Split Invertase Genes. We have demon- treated with 0.01% NaN3. Invertase activity ofboth extract and strated that theE. coli lipoprotein signal sequence is functional whole cells was determined by incubation in citrate buffer (pH in secretion of proteins in S. cerevisiae (11, 19, 21-23). Pro- 5) containing 1% sucrose for 2 h at 30°C and removal of cells cessing of the lipoprotein signal peptide in yeast occurs at a and debris by centrifugation, followed by determination of unique site (between Cys-21 and Ser-22), which is one residue reducing sugars. To assay invertase activity at different tem- from the signal peptidase II processing site in E. coli (11). To peratures, cultures were grown at 22°C, and samples of their allow a similar targeting of N- and C-terminal fragments of extracts were incubated in citrate buffer (pH 5) containing 8% invertase in yeast, we have used the lipoprotein signal peptide. sucrose for 2 h at 22°C, 30°C, 37°C, 50°C, or 60°C. Invertase The vectors that we have created, termed the PRS-PGK-lpp activity was determined by the Sumner method. series, harbor either the URA3 or TRP1 genes, allowing the To detect invertase activity on gels, nondenatured extracts selection of strains harboring combinations of vectors each were separated by PAGE (7% polyacrylamide, 0.1% sarcosyl) encoding a different polypeptide. These vectors each contain for 4-5 h at 4°C. The gel was incubated in 0.1 M sodium acetate the phosphoglycerate kinase (PGK) promoter and, down- (pH 4.6) containing 0.1 M sucrose at 30°C for 30 min and then stream to it, encode the lipoprotein signal peptide, followed by stained with 0.1% 2,3,5-triphenyl tetrazolium chloride in 0.5 M an EcoRI site and the entire mature invertase-encoding se- NaOH at 100°C (14). quence. When the lipoprotein signal peptide is used for DNA Manipulations. Plasmid pTinv was created by destroy- expression and secretion, 13 aa of mature lipoprotein and ing the EcoRI site of plasmid pT7-5 (15) and cloning of the linker sequences are added to the processed form of the Sall-DraI fragment of plasmid pSEY304 (16) into the Sall- expressed protein. The invertase N- or C-terminal coding SmaI sites of the pT7-5 derivative. pTinv was used for creating sequences were cloned in-frame with the signal peptide coding the following plasmids. (i) pT-EH(C135): pTinv was cleaved sequence between the EcoRI and SacI sites in this vector. with EcoRI and HpaI, treated with Klenow and ligated. (ii) Schematically presented in Fig. 1 are two N-terminal frag- pT-ES(C211): pTinv was cleaved with EcoRI and StyI, treated ments [AH(N378) and X(N258)] containing 378 and 258 aa with Klenow, and ligated. The resulting plasmid was cleaved from the invertase N terminus, respectively, and three C- again with EcoRI, treated with Klenow, and ligated. (iii) terminal fragments [EH(C135), ES(C211), and EX(C256)] pT-EX(C256): pTinv was cleaved with EcoRI and XbaI, containing 135, 211, and 256 aa from the C terminus, respec- treated with Klenow, and ligated. The resulting plasmid was tively. These fragments are expressed as hybrids with the cleaved again with EcoRI, treated with Klenow, and ligated. lipoprotein signal peptide and under the PGK promoter as (iv) pT-AH(N378): pTinv was cleaved with HpaI and AgeI, described above. In addition, for control experiments, plasmids treated with Klenow and ligated. (v) pT-X(N258): pTinv was were created encoding the mature invertase protein sequence cleaved with XbaI, treated with Klenow, and ligated. or the ES(C211) fragment both lacking signal peptides Plasmid pEF1 contains the PGK promoter on an EcoRI- [InvASP and ES(C211)ASP, respectively]. S. cerevisiae (strain BglII fragment (of plasmid pMA91; ref. 17) cloned into the DGY505) harboring a chromosomal deletion of SUC2 were EcoRI and BamHI sites of plasmid pSEY304 (16). Plasmids transformed with the various invertase plasmid constructions. pRS-PGK424 and pRS-PGK426 were created in two steps. The first was cloning of the EcoRI-XbaI small fragment of Ipp-SP Invertase pEF1 containing the PGK promoter and part of the SUC2 into Inv IPGK t-` -5 [: _ n~~~~~ 51 3aa the EcoRI and SpeI sites of pRS424 and pRS426 (18). The second step was destroying the extra SalI site of the resulting EH(C135) _i::22ZJ1 35 a a plasmids by cleavage with XhoI and EcoRI, treatment with ES(C211) _l21laa Klenow, and ligation. EX(C25 me_ m 256aa Plasmids pRS-PGK-Inv, pRS-PGK-EH(C135), pRS-PGK- 6) ES(C211), pRS-PGK-EX(C256), pRS-PGK-AH(N378), and AH(N378) pRS-PGK-X(N258) were constructed by cloning of the Sall- X(N258) _: --:: 1258aa SacI fragments of the respective pTinv derivatives into either pRS-PGK-424 and/or pRS-PGK-426. pRS-PGK-InvASP and InvASP I Ml---~ 51 2aa pRS-PGK-ES(C211)ASP were constructed by cleaving pRS- ES(C21 1)ASP - 211 aa PGK-Inv and pRS-PGK-ES(C211) with SalI and EcoRI, treat- ing with Klenow, and ligating. Plasmid YEp-GAL-ES(C211) FIG. 1. Schematic representation of the phosphoglycerate kinase was constructed by cloning the Sall-SacI fragment of pRS- (PGK) promoter-invertase split genes. Invertase sequences (open into boxes) are aligned with the unmutated mature invertase sequence of PGK-ES(C211) pRH3 (19). Inv. Solid boxes represent the lipoprotein signal peptide (21 aa), and Total Protein Extraction and Immunoblot Analysis. For stippled boxes represent mature lipoprotein and linker sequences (13 Western blot analysis, cell extracts were boiled in endoglyco- aa) that connect the signal peptide to mature invertase sequences sidase H (endo H) denaturing buffer (0.5% SDS/1% 2-mercap- (represented by open boxes). Numbers to the right of each gene toethanol) and split, and half the sample was subjected to endo indicate the number of invertase amino acids in each of the encoded H treatment for 2 h at 37°C. The samples were electrophoresed polypeptides. Downloaded by guest on September 23, 2021 9614 Cell Biology: Schonberger et al. Proc. Natl. Acad. Sci. USA 93 (1996) Expression, Glycosylation, and Secretion of Invertase Frag- ES(C211) are glycosylated provides evidence for their trans- ments. Two complementing constructs, which represent C- location into the ER. and N- terminal truncated forms of invertase [AH(N378) and To examine if invertase fragments exit the ER and follow the ES(C211), respectively], were chosen for studying the fate of general secretory pathway, subcellular fractionation experi- independently expressed invertase fragments. Expression of ments were conducted. Zymolyase was used to digest the cell invertase fragments was examined by SDS/PAGE and immu- wall in fractionation experiments (19) in which we found that noblotting of whole cellular extracts with anti-invertase anti- the split gene products are extremely sensitive to proteolysis bodies. As shown in Fig. 2, the AH(N378) deglycosylated (Fig. 3; and data not shown). The major invertase immuno- fragment appeared as a major 45-kDa band with minor lower reactive bands found in whole cell extracts are those of bands, which probably represent degradation or truncated AH(N378) and ES(C211) (Fig. 3, lane 6, bands a and b, translation products (lane 4). These forms also appear in respectively). Periplasmic fractions prepared in the presence of strains defective for vacuolar proteases such as PEP4 (data not various protease inhibitors contain only small amounts of the shown). The deglycosylated ES(C211) fragment appeared as a full-length AH(N378) polypeptide and barely detectable single 21-kDa band (lane 3). Both fragments were clearly amounts of the ES(C211) polypeptide, whereas these prepa- detected in extracts expressing the combination (lane 2), and rations contain significant amounts of invertase degradation each was significantly smaller than Inv, the unfragmented products (lanes 3-5). This degradation is probably the result of mature invertase (compare lanes 3 and 4 to lane 5). Essentially, known proteolytic activity in Zymolyase enzyme preparations no invertase immunoreactive material was detected in extracts used for the fractionation procedure (26). In fact, the sphero- of the chromosomally deleted suc2-A9 strain, DGY505, which plast fraction, in contrast to the periplasmic fraction, is pro- was used as the host strain for all our experiments (lane 1). tected by the membrane from this proteolytic activity (com- Core glycosylated forms of secretory proteins, such as pare lane 2 with lanes 3-5). Under the same conditions, the invertase, are assembled in the ER and are further modified by unfragmented invertase (Inv) is targeted to the periplasm and addition of outer chain carbohydrates during transit through is very stable, whereas the signal peptide lacking invertase the Golgi body. Thus, glycosylation serves as an indication of secretion (24, 25). Our invertase antiserum recognizes several (InvASP) is localized to the cytosol (data not shown). From nonspecific glycosylated proteins in yeast cell extracts (Fig. 2, these results, we conclude that a significant amount of the split lane 12), yet specific invertase bands are clearly detected on invertase gene products reach the yeast periplasm, yet a similar that background (compare lanes 7-11 with lane 12). Both the amount is retained within the spheroplast. It is important to AH(N378) and ES(C211) polypeptides are glycosylated as note that the localization and appearance of the AH(N378) concluded from the extensive changes in their electrophoretic polypeptide in subcellular fractionation experiments is the mobility following deglycosylation with endo H [Fig. 2; for same regardless of whether it is expressed in combination with polypeptide ES(C211) compare band d, lanes 2 and 3 to band the ES(C211) or on its own (data not shown). d', lanes 10 and 11; for polypeptide AH(N378) compare band Enzymatic Activity. Among the six possible combinations of c, lanes 2 and 4 to c', lanes 9 and 11]. In contrast, invertase pairs of ER-targeted N- and C-terminal invertase fragments, lacking a signal peptide (InvASP) is not targeted to the ER and only two exhibit enzymatic activity (Table 1). Cell extracts of not modified, and therefore it exhibits no change in its S. cerevisiae DGY505 strains expressing fragment AH(N378) electrophoretic mobility upon treatment with endo H (band b, and either fragment ES(C211) or EX(C256) exhibit detectable lane 6, and band b', lane 7). The fact that AH(N378) and enzymatic activity; however, the AH(N378)/ES(C211) com- bination displays a much higher activity than that of Zndo H + Endo - AH(N378)/EX(C256). Both cellular levels of activity and the amount of invertase polypeptides in strains expressing the AH(N378)/ES(C211) combination are much lower than the 3 corresponding levels and amounts found in strains expressing the full invertase gene (Inv). In particular, the amounts of the 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 _.. ) a' ,,...= ,, . * a ...: a _ ...I .... b : ~ bt-cd' c_-om 0 - Wd'

FIG. 3. Cellular location of invertase polypeptides in S. cerevisiae. Cultures of S. cerevisiae (DGY505) expressing the combination FIG. 2. Immunodetection of glycosylated and deglycosylated in- AH(N378)/ES(C211) were grown in glucose medium, and the cells were vertase split gene products. Strains containing a chromosomal deletion fractionated into periplasm and spheroplasts. Fractions were analyzed by of SUC2 and harboring the indicated plasmids were grown in glucose immunoblotting as in Fig. 2. Gel order was as follows: lane 1, total cell standard medium, and cell extracts were prepared. These were de- extract of the control culture (strain DGY505) that is devoid of invertase glycosylated by treatment with endo H (Endo H+) or untreated polypeptide expression; lane 2, spheroplast fraction ofcells expressing the (glycosylated, Endo H-) and subjected to SDS/PAGE and immuno- AH(N378)/ES(C211) combination; lanes 3,4 and 5, periplasmic fractions _ _,,j to blotting using anti-invertase antibodies. Gel order is according ofcells expressing the AH(N378)/ES(C211) combination prepared in the invertase polypeptides expressed, as indicated above each lane. Arrows absence of protease inhibitors (lane 3), in the presence of antipain, and letters on both sides of the gel indicate positions of deglycosylated pepstatin, aprotinin, leupeptin, and PMSF (lane 4), and in the presence and glycosylated forms of invertase polypeptides, respectively, as ofmacroglobulin (lane 5); and lane 6, total extract ofthe strain expressing follows: Inv, a and a'; InvASP, b and b'; AH(N378), c and c'; and the AH(N378)/ES(C211) combination. Arrows indicate positions of the ES(C211), d and d'. Black dots at the center of the gel indicate the AH(N378) (a) and ES(C211) (b) polypeptides. Black dots to the right of position of molecular mass markers: top, 84 kDa; middle, 48 kDa; and the gel indicate 48-kDa (top), 36-kDa (middle), and 27-kDa (bottom) bottom, 27 kDa. molecular mass markers. Downloaded by guest on September 23, 2021 Cell Biology: Schonberger et al. Proc. Natl. Acad. Sci. USA 93 (1996) 9615 Table 1. Invertase activity and growth on sucrose significant portion of the invertase fragments expressed in Invertase Growth on yeast reach the periplasm and form an active enzyme complex. Polypeptide(s) expressed activity* sucroset Growth on Sucrose. To examine if complexes of invertase fragments in the periplasm were biologically functional, cul- Inv + + tures were grown on sucrose as the single carbon source. Of the InvASP + various possible combinations of fragments, only simultaneous AH (N378) plus EH (C135) - - expression of the AH(N378)/EX(C256) or the AH(N378)/ AH (N378) plus EX (C256) + + ES(C211) pairs (which exhibit invertase activity) enable AH (N378) plus ES (C211) + + growth of yeast cells on sucrose (Table 1). Cells expressing the AH (N378) plus ES (C211) ASP - - AH(N378)/ES(C211) combination were capable of efficient X (N258) plus any fragment growth in sucrose medium, whereas cells expressing individual Any single fragment fragments or no fragments did not grow (Fig. 4). There was no *Activity was measured by the Sumner method for determination of difference in the growth of any of these strains in glucose reducing sugars. A + indicates a >100-fold higher activity than a -. medium (data not shown). The growth of the cells expressing (See text for a description of the differences in the specific activity the AH(N378)/ES(C211) combination was somewhat slower between full-length and fragmented invertases). than the of cells the unfragmented mature tA + indicates at least a 10-fold increase in culture optical density at growth expressing 600 nm after 35-40 h of growth (see Fig. 4). invertase, probably due to the much lower invertase activity of the combination in the yeast periplasm. Consistent with this ES(C211) fragment in cells expressing this combination is notion is the finding that upon transfer into sucrose medium, much lower than the corresponding level of the full invertase. yeast cells expressing mature invertase without a signal peptide By determining enzymatic activities together with quantitative (InvASP) show essentially no growth for 20 h and only very densitometric analysis of Western blots with varying amounts weak growth thereafter (Fig. 4). The level of enzymatic activity of cell extracts, the estimated specific activity of the in extracts ofcells expressing InvASP is at least 10-fold higher than AH(N378)/ES(C211) combination is -7% of that found for cells expressing the AH(N378)/ES(C211) combination, yet they the full invertase. display poor growth when compared with the latter. Since it is As expected, the expression of mature invertase lacking a without a signal peptide, the InvASP enzyme is not targeted to the signal sequence (InvASP) results in high enzymatic activity in periplasm and therefore cannot cleave extracellular sucrose. cell extracts. This activity is intracellular, whereas the activity These results clearly indicate that a complex between AH(N378) exhibited by Inv (containing a signal peptide) is located mainly and ES(C211) remains active during transit through the secretory in the periplasm (see below). In contrast to the AH(N378)/ pathway and release into the periplasm. ES(C211) combination, the expression of ES(C211)ASP to- The possibility of producing a full-length invertase from a gether with AH(N378) does not result in detectable enzymatic combination of split genes by genetic recombination can be activity, indicating that secretion of two functionally compat- ruled out, since extracts of cells, grown on sucrose, produce ible fragments of a pair into the same compartment (ER) is a distinct AH(N378) and ES(C211) polypeptides (data not prerequisite for obtaining enzymatic activity. This conclusion shown), and no protein species at the full mature invertase size is supported by the finding that the assembly is impaired in a can be detected (the pattern obtained is essentially identical to kar2-159 temperature-sensitive mutant that is defective for lane 2 of Fig. 2). protein translocation into the ER at the restrictive tempera- Association Between Fragments. In a control experiment, ture. We find that the activity of the AH(N378)/ES(C211) extracts prepared from cultures expressing either AH(N378) combination induced at the restrictive temperature in a kar2- or ES(C211) were combined, and the mixture was assayed for 159 mutant is <35% of the activity found at the permissive invertase activity. No invertase activity was detected even temperature. This kar2-159 strain, and those described below, though both the AH(N378) and ES(C211) fragments can be contain in addition to a plasmid with a constitutive promoter detected in the mixture at levels similar to those found in cells for expression of the large fragment (pRS-PGK-AH), a plas- expressing both fragments simultaneously (Fig. 2, lanes 2-4). mid with an inducible GAL10 promoter for expression of the To obtain enzymatic activity, both fragments must therefore small fragment (YEp-GAL-ES). Induction is obtained by growth in galactose medium. In contrast to the effect of 1 0 kar2-159 mutation on invertase fragment assembly, the cor- responding secl8 (allows a ER to Golgi block) or secl (allows a secretory vesicle to periplasm block) mutant strains exhibit a wild-type degree of assembly at the restrictive temperature, which E is higher than at the permissive temperature. Thus, while inver- tase fragment entry into the ER is required for assembly, transfer to the Golgi or the periplasm are not. These results indicate that Q0 the assembly of invertase fragments occurs in the ER. To allow the direct demonstration of invertase activity in the periplasm of yeast cells expressing split genes, activity was 0 S t p D a measured in whole cells vs. total cellular extracts. The rationale behind this approach is that only periplasmic (and not intra- cellular) invertase should contribute to activity measured in whole cells and that both the periplasmic and intracellular 0 20 40 60 80 fractions should contribute to the activity measured in cell Hours extracts. Invertase lacking the signal peptide (InvASP) exhibits low activity in whole cells when compared with its activity in FIG. 4. Growth of S. cerevisiae strains on sucrose as the single carbon source. Yeast cells harboring the appropriate plasmids were cell extracts, whereas invertase containing a signal peptide incubated at 25°C in sucrose (1%) medium, and cell growth was (Inv) exhibits similar levels of activity in the two. The combi- monitored by optical density at 600 nm. Symbols indicating the nation AH(N378)/ES(C211) displays a significant level of invertase polypeptides expressed by the respective strains: a, Inv; A, activity in whole cells, although this activity is only 40-60% of AH(N378)/ES(C211); 0, InvASP; *, AH(N378); 0, ES(C211); and 0, that found in cell extracts. These results indicate that a none (strain DGY505 with no plasmids). Downloaded by guest on September 23, 2021 9616 Cell Biology: Schonberger et al. Proc. Natl. Acad. Sci. USA 93 (1996) be expressed simultaneously in the same cell, indicating that a the activity at 60°C (Fig. 6). In addition, in experiments not complex is formed in vivo. shown, a complete loss of enzyme activity was observed when To examine the association between the split gene products, extracts of cells expressing the combination AH(N378)/ nondenatured extracts prepared from cells expressing the ES(C211) were exposed to low concentrations of SDS (e.g., AH(N378)/ES(C211) combination were subjected to PAGE, 0.1%), in contrast to mature unfragmented invertase, whose with the detergent sarcosyl instead of SDS. The reason for this activity is hardly effected. These results are consistent with the is that invertase activity of the combination is not destroyed in dissociation of the complex with increasing temperature or SDS the presence of sarcosyl (0.1%), whereas even very low con- concentrations and suggest diminished structural stability. centrations of SDS demolish its enzyme activity. After incu- bation of the gel in sucrose (0.1 M), invertase activity was assayed by staining with triphenyl tetrazolium chloride (14). As DISCUSSION expected, the strains expressing mature invertase (Inv; Fig. 5A, In this paper, we show that when overlapping fragments of lane 5) displayed invertase activity, whereas no activity was invertase are simultaneously expressed in yeast, they (i) are detected in strains expressing single fragments (lanes 3 and 4). independently translocated into the ER, (ii) are modified by For the combination, active enzyme was detected at a position glycosylation, and (iii) travel through the entire secretory corresponding to a high molecular weight (Fig. SA, lanes 1 and pathway reaching the yeast periplasm. The AH(N378) frag- 2). Electroelution of the active species from the gel, denatur- ment of invertase is translocated into the ER and reaches the ation and deglycosylation of the eluates followed by SDS/ periplasm, regardless ofwhether it is expressed simultaneously PAGE, and immunoblotting detected both the AH(N378) and or separately with ES(C211). This conclusion is based on the ES(C211) fragments, suggesting that they are in a high mo- finding that all the AH(N378) molecules are glycosylated and lecular weight active complex (Fig. SB, compare lane 5 to lanes that we do not detect any unmodified fragments in samples not 2-4). Coomassie blue staining of a gel containing the combi- treated with endo H. In addition, fractionation experiments of nation before and after electroelution (corresponding to sam- cells expressing the AH(N378) alone or its combination with ples in lanes 4 and 5 in Fig. 5B) is shown in Fig. SC (lanes 1 and ES(C211) show that in both cases a significant portion of these 2, respectively). polypeptides reaches the periplasm. In this regard, invertase After electroelution, the AH(N378) fragment appeared fragments behave differently than other incorrectly folded predominantly as a smaller cleaved species (Fig. SB, lane 5). In mutant, truncated, or heterologous proteins, which are re- this regard, the invertase fragments [AH(N378)/ES(C211)] tained and/or degraded in the ER (27-31). For example, a are highly susceptible to proteolytic degradation in sharp study dealing with bacterial toxoid EtxB assembly in yeast contrast to unfragmented mature invertase, which is very showed that all the toxoid oligomers are confined to the ER stable (data not shown). Indeed, this observation is consistent and essentially no oligomeric toxoid (or its activity) can be with the suggestion that the complex is comprised of two detected in the periplasm (19). It is also interesting to compare polypeptide fragments, which are more sensitive to proteolytic secretion of single invertase fragments, which does not depend degradation than the native mature protein. on assembly, with immunoglobulin assembly and secretion, in The same is true with respect to the sensitivity of the which exit of the heavy chain from the ER depends upon its complex to temperature as measured by changes in enzymatic association with the light chain (32, 33). The molecular basis activity. Extracts of cells expressing Inv, InvASP, and the for this latter phenomenon has been worked out, showing that combination AH(N378)/ES(C211) were assayed for invertase the heavy chain is complexed with the molecular chaperone activity at different temperatures (Fig. 6). Whereas Inv and BiP and is thereby retained in the ER until interaction with the InvASP activity increased with temperature up to 60°C, the light chain releases BiP and allows exit of the immunoglobulin combination lost 50% of its activity at 50°C and essentially all from the ER (32, 33). A major conclusion from this study is that simultaneously B C expressed complementing fragments of invertase are translo- A 1 2 3 4 5 1 2 300 1 2 3 4 5

.... a_ !w ._ 200 b_-0

P-o X FIG. 5. Electroelution of invertase AH(N378) and ES(C211) 100 polypeptides from a gel stained for invertase activity. (A) Extracts of cultures expressing the AH(N378)/ES(C211) combination were ana- lyzed by PAGE in the presence of sarcosyl followed by staining of the gel for invertase activity. Cell extracts from two independent cultures expressing the AH(N378)/ES(C211) combination (lanes 1 and 2) and separate cultures expressing the AH(N378) (lane 3), ES(C211) (lane 20 30 40 50 60 70 4), and Inv (lane 5) polypeptides were examined. (B) Immunoblot Temperature ( OC) analysis of cell extracts of strains expressing no invertase (lane 1), AH(N378) (lane 2), ES(C211) (lane 3), AH(N378)/ES(C211) (lane 4) FIG. 6. Effect of temperature on the enzymatic activity of the invertase polypeptides, and the sample electroeluted from positions AH(N378)/ES(C211) combination. Total cell extracts of S. cerevisiae corresponding to stained portions of gels shown in lanes 1 and 2 ofA strain expressing AH(N378)/ES(C211) (0), Inv (O), and InvASP (-) (lane 5). Arrows indicate positions of the AH(N378) (band a) and were prepared and assayed for invertase activity at 30°C, 37°C, 50°C, ES(C211) (band b) polypeptides. (C) SDS/PAGE and Coomassie blue 60°C, and 65°C as described. The data are presented as percentages of staining of cell extracts of strains expressing the AH(N378)/ES(C211) activity at 30°C for each strain, because there is a >10-fold difference combination, before (lane 1) and after (lane 2) electroelution de- between the specific activities of the full-length invertase (Inv) vs. the scribed in B. AH(N378)/ES(C211) combination. Downloaded by guest on September 23, 2021 Cell Biology: Schonberger et al. Proc. Natl. Acad. Sci. USA 93 (1996) 9617 cated into the ER and assembled into an enzymatically active We thank D. Weiss for his support and A. Taraboulos for helpful complex. Experiments analyzing secretory mutants strongly discussions and suggestions. E.B. is supported by the Leo and Julia suggest that assembly of invertase fragments occurs in the ER. Forchheimer Center for Molecular Genetics, Weizmann Institute of These results are reminiscent of those of Betton and Hofnung Science. C.K. was supported by The Golda Meir Fellowship Fund. (10) in the prokaryotic system, in which they have demon- strated translocation of maltose binding protein fragments 1. Pelham, H. R. B. & Munro, S. (1993) Cell 75, 589-592. through the bacterial plasma membrane and their functional 2. Rothman, J. E. (1994) Nature (London) 372, 55-63. 3. Helenius, A., Marquart, T. & Braakman, I. (1992) Trends Cell. assembly in the periplasm of E. coli, a compartment which is Biol. 2, 227-231. functionally analogous to the ER. However, here we show that, 4. Anfinsen, C. B., Cuatrecasas, P. & Taniuchi, H. (1971) Enzymes in the eukaryotic system (represented here by yeast), split gene 4, 177-204. products traveling through the secretory pathway form an 5. Taniuchi, H., Parr, G. R. & Juillerat, M. A. (1986) Methods active complex that reaches its final subcellular destination, Enzymol. 131, 185-217. which is outside the cell plasma membrane. One question that 6. Bibi, E. & Kaback, H. R. (1990) Proc. Natl. Acad. Sci. USA 87, remains to be resolved in the eukaryotic system is whether 4325-4329. assembly of split invertase occurs before, during, or after 7. Shiba, K. & Schimmel, P. (1992) Proc. Natl. Acad. Sci. USA 89, glycosylation. We know that separately expressed fragments 1880-1884. are glycosylated, but the question of its timing with respect to 8. Berkower, C. & Michaelis, S. (1991) EMBO J. 10, 3777-3785. assembly remains open. 9. Kaur, P. & Rosen, B. (1993) J. Bacteriol. 175, 351-357. This study opens the way for examination of cellular factors 10. Betton, J.-M. & Hofnung, M. (1994) EMBO J. 13, 1226-1234. required for the assembly of invertase fragments, a process that 11. Pines, O., Lunn, C. A. & Inouye, M. (1988) Mol. Microbiol. 2, be to In 209-217. may also related the folding of the wild-type enzyme. 12. Bernfeld, P. (1955) Methods Enzymol. 1, 149-151. this regard, BiP/KAR2 (30, 34-36) was an obvious candidate, 13. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. since it has been shown to be required in vivo for the assembly 14. Carlson, A., Osmond, B. C. & Botstein, D. (1981) Genetics 98, of multisubunit complexes, such as the bacterial EtxB toxoid in 25-40. yeast (19) and immunoglobulin assembly in higher eukaryotes 15. Tabor, S. & Richardson, C. C. (1985) Proc. Natl. Acad. Sci. USA as discussed above. For that reason we have examined a kar2-1 82, 1074-1078. temperature-sensitive mutant, which allows protein transloca- 16. Bankaitis, V. A., Johnson, L. M. & Emr, S. Z. (1986) Proc. Natl. tion into the ER and secretion at the restrictive temperature Acad. Sci. USA 83, 9075-9079. (and differs from kar2-159, which has a null phenotype). 17. Mellor, J., Dobson, M. J., Roberts, N. A., Tuite, M. F., Emtage, Surprisingly, in preliminary experiments analogous to those J. S., White, S., Lowe, P. A., Patel, T., Kingsman, A. J. & previously performed with EtxB, we find that in contrast to the Kingsman, S. M. (1983) Gene 24, 1-14. toxoid assembly that is blocked at the restrictive temperature 18. Sikorski, R. S. & Hieter, P. (1989) Genetics 122, 19-27. in the kar2-1 mutant strain, functional invertase fragment 19. Schonberger, O., Hirst, T. & Pines, 0. (1991) Mol. Microbiol. 5, assembly is not affected by the kar2-1 mutation. Since invertase 2663-2671. 20. Geller, D., Taglicht, D., Rotem, E., Tara, A., Pines, O., Michaelis, and its fragments are glycosylated, it will be intriguing to S. & Bibi, E. (1996) J. Biol. Chem. 271, 13746-13753. examine if other chaperones such as calnexin and calreticulin, 21. London, A. & Pines, 0. (1990) Mol. Microbiol. 4, 2193-2200. which interact with glycoproteins, may be involved (37, 38). 22. Pines, 0. & London, A. (1991) J. Gen. Microbiol. 137, 771-778. The oligomers of invertase fragments and EtxB in yeast also 23. Shani, 0. & Pines, 0. (1992) Mol. Microbiol. 6, 189-195. differ with respect to stability: whereas the polypeptides of the 24. Esmon, B., Novick, P. & Schekman, R. (1981) Cell 25, 451-460. AH(N378)/ES(C211) combination are extremely sensitive to 25. Haselbeck, A. & Schekman, R. (1986) Proc. Natl. Acad. Sci. USA proteolysis, EtxB oligomers expressed in yeast are resistant 83, 2017-2021. even to proteinase K treatments (19). In this regard, the 26. Jones, E. W. (1991) in Methods in Enzymology: Guide to Yeast oligomers of complexed invertase fragments are also much Genetics and Molicular Biology, eds. Guthrie, C. & Fink, G. R. more sensitive to proteolysis than the full-length unfragmented (Academic, New York), Vol. 194, pp. 428-453. invertase (Inv). These observations indicate that although the 27. Gething, M. J. & Sambrook, J. (1992) Nature (London) 355, fragments of invertase associate to obtain enzymatic activity, 33-45. the compactness of the complex's structure is distinct from that 28. Hurtley, S. M., Bole, D. G., Hoover-Litty, H., Helenius, A. & also Copeland, C. S. (1989) J. Cell. Biol. 108, 2117-2126. of the native enzyme. This conclusion is supported by the 29. Dorner, A. J., Bole, D. G. & Kaufman, R. J. (1987) J. Cell. Biol. marked temperature and SDS sensitivity of the AH(N378)/ 105, 2665-2674. ES(C211) complex vs. that of the unfragmented invertase. 30. Kozutsumi, Y., Segal, M., Normington, K., Gething, M. J. & Native invertase has been reported to exist in vivo as oligomers Sambrook, J. (1988) Nature (London) 332, 462-464. (dimers, tetramers, and octamers; refs. 39 and 40). It will be 31. Machamer, C. E., Doms, R. W., Bole, D. G., Helenius, A. & interesting to determine whether analogous oligomeric com- Rose, J. K. (1990) J. Biol. Chem. 265, 6879-6883. plexes are formed with split invertase fragments. In this regard, 32. Haas, I. G. & Wabl, M. (1983) Nature (London) 306, 387-389. expression of the AH(N378) fragment separately caused its 33. Hendershot, L., Bole, D., Kohler, G. & Kearney, J. F. (1987) J. accumulation in a high molecular weight material (data not Cell. Biol. 104, 761-767. shown), similar to the AH(N378)/ES(C211) combination (Fig. 34. Rose, M. D., Misra, L. M. & Vogel, J. P. (1989) Cell 57, 1211- 5). This is in contrast to ES(C211), which does not appear in high 1221. molecular weight form when it is expressed on its own. Thus, it 35. Vogel, J. P., Misra, J. M. & Rose, M. D. (1990) J. Cell. Biol. 110, is possible that sites responsible for invertase oligomerization may 1885-1895.- in its 378-aa N-terminal 36. Normington, K., Kohno, K., Kozutsumi, Y., Gething, M. J. & reside sequence. Sambrook, J. (1989) Cell 57, 1223-1236. Of particular importance is the finding that active split inver- 37. Bergeron, J. J. M., Brenner, M. B., Thomas, D. Y. & Williams, tase complexes are actually secreted into the periplasm, enabling D. B. (1994) Trends Biochem. Sci. 19, 124-128. growth on sucrose of suc mutants. This phenotype can be 38. Hammond, C. & Helenius, A. R. I. (1994) Science 266, 456-458. exploited in the future for selecting mutants that are either 39. Esmon, P. C., Esmon, B. E., Schauer, I. E., Taylor, A. & Schek- enhanced or incapable of supporting the formation of active split man, R. (1987) J. Biol. Chem. 262, 4387-4394. invertase complexes, thus possibly leading to the identification of 40. Kern, G., Schulke, N., Schmid, F. X. & Jaenicke, R. (1992) new cellular components required for the assembly process. Protein Sci. 1, 120-131. Downloaded by guest on September 23, 2021