Contrasting secretory processing of simultaneously expressed heterologous proteins in Saccharomyces cerevisiae

Contrasting Secretory Processing ofSimultaneously Expressed HeterologousProteins in Saccharomyces cerevisiae

Andy Rakestraw,1 K. Dane Wittrup2

1Division of Biological Engineering, Massachusetts Institute of Technology,Cambridge, Massachusetts2Department of Chemical Engineering, Massachusetts Institute of Technology,Building 66-552, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139;telephone: 617-253-4578; fax: 617-258-5766; e-mail: [email protected]

Received 13 June 2005; accepted 19 October 2005

Published online 6 December 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20780

Abstract: In this study, secretory processing of cell-surface displayed Aga2p fusions to bovine pancreatictrypsin inhibitor (BPTI) and the single chain Fv (scFv)antibody fragment D1.3 are examined. BPTI is moreefficiently processed than D1.3 both when secreted andsurface-displayed, and D1.3 expression imparts a greateramount of secretory stress on the cell as assayed by areporter of the unfolded protein response (UPR). Surpris-ingly, simultaneous expression of the two proteins in thesame cell somewhat improves BPTI surface display whiledecreasing D1.3 surface display with minimal effect onUPR activation. Furthermore, co-expression leads to theaccumulationofpunctatevacuolar aggregatesofD1.3andincreased secretion of the D1.3–Aga2p fusion into thesupernatant. Overexpression of the folding chaperonesproteindisulfide isomerase (PDI) andBiP largelymitigatesthe D1.3 surface expression decrease, suggesting thatchanges invacuolar andcell surface targetingmaybedue,in part, to folding inefficiency. Titration of constitutiveUPR expression across a broad range progressivelydecreases surface display of both proteins as UPRincreases. D1.3-Aga2p traffic through the late secretorypathway appears to be strongly affected by overallsecretory load as well as folding conditions in the ER.� 2005 Wiley Periodicals, Inc.

Keywords: heterologous protein expression; unfolded-protein response (UPR); vacuolar degradation; ER foldingchaperones; yeast surface display

INTRODUCTION

The yeast Saccharomyces cerevisiae is an attractive expres-

sion host for non-glycosylated, disulfide bonded proteins due

to its eukaryotic secretion machinery, facile genetics, and its

use in well-developed industrial scale fermentation. How-

ever, heterologous proteins, although stably folded and

secreted into the supernatant, are often produced in lower

yields than in prokaryotic systems (Harrison and Keshavarz-

Moore, 1996; Olmos-Soto and Contreras-Flores, 2003;

Palva, 1982; Pluckthun, 1994; Wu et al., 2002). This low

protein output could be due to poor protein stability, poor

folding in theER, aggregation resulting fromoverexpression,

ER-associated degradation (ERAD), or vacuolar degradation

(Coughlan et al., 2004; Hayano et al., 1995; Hiller et al.,

1996; Holkeri and Makarow, 1998; Kjeldsen et al., 2002;

Robinson et al., 1994; Sagt et al., 2002; Shusta et al., 1998,

1999; Umebayashi et al., 2001; Werner et al., 1996). In

addition to its applicability in improving heterologous

protein production, understanding protein folding and

secretory traffic in yeast may give relevant insights into the

mechanisms of protein folding diseases such as cystic

fibrosis, prion-related diseases, and type-II diabetes mellitus

(Laurent, 1996; Lorenzo et al., 1994; Qu and Thomas, 1996).

Nascent, secretion-directed proteins are either co-transla-

tionally or post-translationally translocated into the ER

through the Sec61/Sec63 complex (Musch et al., 1992;

Sanders et al., 1992). Once in the ER lumen, the still unfolded

proteins interact with folding chaperones in an ATP-

dependent manner until they reach a final, stable conforma-

tion suitable for export. Folding chaperones may consist of a

variety of aggregation-inhibiting heat-shock proteins (Hsp),

most notably immunoglobulin binding protein (BiP), cha-

perones involved in disulfide bond formation and isomeriza-

tion such as protein disulfide isomerase (PDI), or

carbohydrate-binding lectins such as calnexin and calreticu-

lin (Kaiser and Shaywitz, 1997). Modulation of these

folding chaperones has been a successful strategy to improve

heterologous protein secretion for single-chain antibodies

(scFv) and human lysozyme (Hayano et al., 1995; Robinson

et al., 1994; Shusta et al., 1998). Proteins that are unable to

fold into a stable, exportable form are retrotranslocated out of

the ER through the Sec61 pore, ubiquinated by ubiquitin

ligase, and degraded by the 26S proteosome (Hiller et al.,

1996; Werner et al., 1996). This balance between protein

synthesis, protein secretion, and protein degradation is

essential for cell viability.

�2005 Wiley Periodicals, Inc.

Correspondence to: K. Dane Wittrup

Contract grant sponsors: NSF BES; NIH

Contract grant numbers: 00-02235; GM008334, CA96504

Yeast expressing heterologous proteins frequently experi-

ence a state of elevated secretory stress characterized by the

induction of the unfolded protein response (UPR) (Cudna and

Dickson, 2003; Kauffman et al., 2002). This response is

induced by the endoribonuclease activity of the ER

membrane kinase inositol requiring protein (IRE1). Under

normal conditions, the luminal portion of IRE1 is thought to

be associated with BiP. Upon levels of high secretory stress,

BiP is recruited away allowing IRE1 to homodimerize and

transphosphorylate. Activated IRE1 removes an intron near

the 30 end of the mRNA for the leucine zipper transcription

factor Hac1. The spliced Hac1 translation homodimerizes,

migrates to the nucleus, and stimulates the transcription of a

wide range of genes involved in helping the cell cope with

secretory stress (Cox and Walter, 1996). Some UPR targets

include folding chaperones, genes involved in membrane

proliferation, and genes associated with the degradation of

misfolded protein (Ng et al., 2000; Sagt et al., 2002; Travers

et al., 2000). Because of its intimate association with the

secretory stress of the cell, UPR manipulation has been an

approach used to improve the secretion of heterologous

proteins (Valkonen et al., 2003).

Proteins leaving the ER are packaged into COPII vesicles

and exported to the Golgi apparatus. In addition to being the

location for a variety of protein modifying enzymes, the

Golgi also serves as the final quality control checkpoint.

Proteins that are targeted to the vacuole or are not suitable for

export to the yeast surface are engaged by one of a large

family of vacuolar sorting proteins (VPS) (Graham and Emr,

1991). These proteins interact with the secretory cargo and

either sort them directly to the vacuole or sort them to the

vacuole through an endosomal intermediate (Bryant and

Stevens, 1998; Harsay and Schekman, 2002). Once in the

vacuole, the proteins are degraded by a host of proteases. It

has been shown that heterologous proteins are sometimes

trafficked in this way (Zhang et al., 2001). Furthermore, it has

been demonstrated that the propensity for a particular protein

to be sorted to the vacuole may depend on its stability

(Coughlan et al., 2004), and deletion of VPSs has proven to

be a productivemethod of improving the secretion of proteins

normally trafficked to the vacuole (Zhang et al., 2001).

In this study, we examine the processing of the hetero-

logous proteins bovine pancreatic trypsin inhibitor (BPTI)

and the lysozyme-binding single-chain antibody (scFv)

D1.3. BPTI is a 58 residue, single domain protein containing

three disulfide bonds and is relatively well secreted in S.

cerevisiae producing titers up to 180 mg/L with tuned

expression (Parekh and Wittrup, 1997). By contrast scFvs,

two domain proteins with two disulfide bonds, are relatively

poorly secreted at levels of 10–20 mg/L when expressed

from low copy plasmids using identical culture conditions,

promoters, and signal peptides (Shusta et al., 1998). From

these results, it is clear that the cellular capacities to secrete

BPTI and scFv differ. However, the reasons for this

difference and the relationships between secretory stress,

secretory load, the protein folding environment, and the

production of these two proteins have not been examined. In

order to understand how loading of a particular protein

affects the secretory health of the cell, the two heterologous

proteins were co-expressed in the same cell. In this manner,

the expression level of one protein can be used tomeasure the

effects of increased secretory loading due to the expression of

the other.

Because of the intrinsically delocalizing nature of

secretion, it is difficult to identify subpopulations in a

heterogeneous population of secretors, much less determine

relationships between phenotypes and secretory processing

in individual clones. In order to analyze cells individually,

BPTI and D1.3 were expressed as fusions to the yeast mating

protein agglutinin-2 (Aga2p). This fusion links to the yeast

cell wall protein agglutinin-1 (Aga1p) in the ER via two

disulfide bonds.With this construct, proteins are displayed on

the yeast surface rather than secreted into the media.

Successful processing of the protein fusions can be analyzed

by flow cytometry facilitating a quantifiable linkage between

secretory output and individual cells. Previous work and

work presented here show that cell surface display correlates

well with secretory productivity as both surface displayed

and secreted protein traverse the secretory pathway (Shusta

et al., 1999). The studies outlined here show that simulta-

neous expression has opposing effects on BPTI and D1.3,

indicating that the secretion-limiting mechanisms differ for

the two proteins and are acted upon differently by increased

secretory load. Furthermore, post-Golgi trafficking of D1.3-

Aga2 is strongly affected by BPTI co-expression in a manner

modulated by ER protein folding conditions. In addition,

overstimulation of the UPR has a negative effect on

processing of both surface displayed D1.3 and surface

displayed BPTI.

MATERIALS AND METHODS

Plasmid Construction

All plasmid amplification was carried out in DH5a E. coli

(Invitrogen, Chicago, IL). XL-1Blue SupercompetentE. coli

(Stratagene, La Jolla, CA)was used for all cloning. Low copy

plasmids carrying the secreted forms of D1.3 and BPTI,

pD1.3s and pBPTIs respectively, consist of the GAL1

promoter, synthetic prepro signal sequences, D1.3 or BPTI,

c-myc epitope tag, and the GAL4 terminator sequence. The

surface display plasmids, pD1.3Cmd and pBPTICmd,

consist of the GAL1 promoter, synthetic prepro signal

sequences, D1.3 or BPTI, c-myc epitope, the agglutinin-2

(Aga2) fusion protein, and the GAL4 transcriptional

terminator. The surface display plasmid pBPTICfd has a

flag tag epitope substituted for the c-myc tag in pBPTICmd.

The doxycycline-regulated Hac1 plasmid pCYHi-1 was

constructed by amplifying the constitutively active Hac1i

gene from pJC-835 (Cox and Walter, 1996) through PCR,

introducingBamHI andNotI restriction sites into the 50 and 30

ends, respectively. The PCR product was then subcloned into

the MCS of pCM189 (ATCC) using a BamHI/NotI digest

followed by ligation with Quick T4 Ligase (New England

Rakestraw and Wittrup: Simultaneous Processing of Heterologous Proteins 897

Biotechnology and Bioengineering. DOI 10.1002/bit

BioLabs, Beverly, MA). Plasmid and strain descriptions are

given in Table I. All PCR reactions were carried out using

Vent DNA Polymerase (New England BioLabs) in a PTC-

200 Thermocycler (MJ Research, Waltham, MA).

Strain Description

EBY100 (Invitrogen) was used for secretion of BPTI and

D1.3. JARD1.3, the integrated D1.3 display strain, was

made by amplifying pD1.3Cmd from promoter to terminator

using a 50 primer carrying 49 bp homology to the S. cerevisiae

URA3 gene and a 30 primer containing a 48 bp linker. In

addition, the KanMX gene was amplified from pFA6-

kanMX4 (Wach et al., 1994) using a 50 primer homologous

to the linker used in the first PCR reaction and a 30 primerwith

45 bp homology to theURA3 gene. Then, 10 mg of each PCRproduct was transformed into EBY100 using the lithium

acetate method outlined in Johnston et al. (2002), and

transformants were selected by growth on YPD plates (2%

glucose, 2% peptone, 1% yeast extract, 1.5% agar)

supplemented with 200 mg/mL Geneticin (GibcoBRL,

Chicago, IL) and 1 mg/mL 5-Fluoroorotic acid (PCR Incor-

porated, Gainesville, FL). JARBPTI was constructed in a

similar manner using pBPTICmd as the template for the first

PCR reaction. The co-expressing strain JAR2DB was

constructed by PCR amplification of D1.3 from pD1.3Cmd

and KanMX from pFA6-kanMX4 using the URA3 homo-

logous primers as described above. BPTI was amplified from

pBPTICfd using a 50 primer with 45 bp homologous to the S.

cerevisiae TRP1 gene and a 30 primer with a 49 bp linker

different than the linker used in theD1.3 PCR reaction above.

The Zeocin resistance gene was amplified from picZm(Invitrogen) using a 30 primer homologous to the linker and

a 50 primer homologous to the TRP1 gene. 10 mg of all four

PCR products were transformed into EBY100 and selected

using the method outlined above with an additional 0.1 mg/

mL Zeocin (Invitrogen) added to the plates. JARD1.3 and

JARDBwere analyzed for integratedD1.3 gene copy number

by PCR. For this procedure, genomic DNAwas isolated from

5� 107 cells using the Zymoprep Yeast Plasmid Miniprep

Kit (Zymo Research, Orange, CA). 1.2 mL of DNA, from

undiluted to 1,000-fold dilution, was used as a template in a

PCR utilizing one primer set that annealed to the integrated

D1.3 gene and another primer set that annealed to the yeast

glyceraldehyde 3-phosphate dehydrogenase (GPD) gene

serving as an amplification control. After PCR, the products

were separated on a 1% agarose/TAE (4 mM Tris-acetate,

1 mMEDTA) gel, treated with 50mL of a 1:5,000 dilution of

SYBR Gold (Molecular Probes, Chicago, IL) nucleic acid

stain, and imaged on a Fluor-S MultiImager (Bio-Rad,

Hercules, CA). PCR band intensities were quantified using

the Fluor-S MultiImager Quantity One 4.2.0 software, and a

linear range of product intensity versus template concentra-

tion was found for the D1.3 and GPD products. After

normalizing the D1.3 intensities to the GPD intensities in the

linear range, it was found that the amount of D1.3 product

DNA relative to GPD product DNA was the same for

JARD1.3 and JARDB. This result shows that the number of

integrated D1.3 genes in the two strains is the same.

Furthermore, it is expected that mRNA transcript levels in

the two strains will be similar upon induction because the

UPR has minimal effect on the GAL1 promoter (Travers

et al., 2000). This check was not made for the BPTI integra-

tions because no direct comparisons between JARBPTI and

JARDB are presented here. All transformations of CEN-

based plasmids were carried out using the EZ Yeast

Table I. Plasmids and strains utilized in this study.

Name Description Source/reference

Strains

EBY100 mat a, trp 1, leu2delta 1, pep4::HIS3 prb1del, URA3::galactose promoted Aga1 Shusta et al. (1999)

JARD1.3 EBY100 with integrated, surface-displayed, c-myc tagged D1.3 This work

JARBPTI EBY100 with integrated, surface-displayed, c-myc tagged BPTI This work

JAR2DB EBY100 with integrated, surface-displayed, c-myc tagged D1.3 and flag tagged BPTI This work

Plasmids

pD1.3s CEN plasmid containing secreted c-myc tagged D1.3 This work

PBPTIs CEN plasmid containing secreted c-myc tagged BPTI This work

pD1.3md CEN plasmid containing surface-displayed, c-myc tagged D1.3 This work

pBPTImd CEN plasmid containing surface-displayed, c-myc tagged BPTI This work

pBPTIfd CEN plasmid containing surface-displayed, flag tagged BPTI This work

pJC-835 CEN plasmid containing constitutively active form of Hac1i Cox and Walter (1996)

pCYHi-1 CEN plasmid containing Haci promoted from doxycycline repressible CYC promoter This work

pCM189 CEN shuttle vector containing doxycycline repressible CYC promoter ATCC

pPDI CEN plasmid with galactose-promoted PDI LaMantia and Lennarz (1993)

pgalKar2 CEN plasmid with galactose promoted Kar2 (BiP) Robinson et al. (1996)

pGPDKar2 CEN plasmid with GPD promoted Kar2 (BiP) Robinson et al. (1996)

pRS316 CEN shuttle vector bearing Ura3 nutritional marker New England BioLabs

pKT048 CEN plasmid containing UPR activated GFP reporter gene Travers et al. (2000)

picZ CEN shuttle vector containing Zeocin resistance gene Invitrogen

pFA6-kanMX4 CEN shuttle vector containing G418 resistance gene Wach et al. (1994)

898 Biotechnology and Bioengineering, Vol. 93, No. 5, April 5, 2006

DOI 10.1002/bit

Transformation Kit (Zymo Research) and 1 mg of each

plasmid. Transformants were selected on SD-CAA plates

(2% glucose, 0.67% yeast nitrogen base, 0.54% Na2HPO4,

0.86% Na2HPO4, 0.5% casein amino acids, 1.5% agar).

Western Blot of Secreted D1.3 and BPTI

Transformants carrying either pD1.3s or pBPTIs were grown

overnight in SD-CAA at 308C. Cells were then induced by

inoculating them in 5 mL SG-CAA (SD-CAA with 0%

glucose and 2% galactose) at an OD600 of 0.5 (Cary 50 Bio

UV Spectrophotometer, Varian, Walnut Creek, CA). Growth

continued in SG-CAAat 308C for 3 days before the cellswere

harvested, spun down, and the supernatant collected. 21mLof

supernatant was combined with 9 mL of SDS denaturation

buffer and glycerol loading buffer, boiled for 5 min and

loaded on an SDS 12% Tris-Glycine polyacrylamide gel

(Invitrogen). After electrophoresis, the protein was trans-

ferred onto a nitrocellulose membrane then immunolabeled

in 1 mg/mL chicken anti-c-myc antibody (Molecular Probes)

diluted in 5% milk/TBST (10 mM Tris-HCl, 150 mM NaCl,

0.05%Tween-20, pH�7.6) for 1 h at room temperature. After

three washes in TBST, the membrane was incubated

in (1:1,000) HRP-conjugated goat anti-chicken antibody

(Sigma, St. Louis,MO) for 20min at room temperature. After

four washes in TBST, the blot was developed using

SuperSignal ELISA Femto Maximum Sensitivity Substrate

(Pierce, Rockford, IL) and imaged on a Fluor-SMultiImager

(Bio-Rad). The Western blot of secreted surface displayed

protein was done similarly but with (1:1,000) 9e10 anti-myc

antibody (Covance, Denver, PA) and (1:1,000) goat anti-

mouse HRP (Sigma). Blot densities were extracted using the

Fluor-S MultiImager Quantity One 4.2.0 software.

Flow Cytometry

JARD1.3 and JARBPTI were grown in 5 mLYPD overnight

at 308C and then induced in 5 mL YPG (2% galactose, 2%

peptone, 1% yeast extract) at an OD600 of 0.5. Growth

continued overnight at 308C, then 0.2OD600 were taken from

each culture, washed in 500 mL PBS/1% BSA (0.8% NaCl,

0.02% KCl, 0.14% Na2HPO4, 0.024% KH2PO4), and

resuspended in 20 mg/mL chicken anti-c-myc antibody

diluted in 50 mL PBS/BSA. The cells were incubated on ice

for 15min, washed in 500 mL PBS/1%BSA, and resuspended

in 40 mg/mLAlexa488-conjugated goat anti-chicken antibody

(Molecular Probes). After 15 min of incubation on ice, the

cells were washed in 500 mL PBS/1%BSA, resuspended in

300 mL PBS/BSA, and analyzed on a Coulter Epics XL flow

cytometer (Coulter, Miami Lakes, FL).

For the co-expressing screens, JAR2DB was transformed

with pKT048-the UPR-sensitive GFP reporter plasmid

(Travers et al., 2000), and the empty nutritional marker

plasmid pRS316 (New England BioLabs). Two single

transformants were grown in 5 mL SD-CAA at 308Covernight then inoculated into 5 mL YPG at an OD600 of

0.5. After growing overnight at 308 to an OD600 of 4, 0.2

OD600 of cells were harvested and labeled with 80 mg/mL

anti-flag M2 antibody (Sigma) and 3 mg/mL biotinylated

lysozyme (Sigma) in 50 mLPBS/1%BSA as described above.

After washing, cells were incubated in 10 mg/mL allophy-

cocyanin-conjugated streptavidin (Molecular Probes) and

40 mg/mL phycoerythrin (PE)-conjugated goat anti-mouse

antibody (Molecular Probes) as described above. Cells were

washed then screened on a FACSCalibur Flow Cytometer

(BD Biosciences, San Jose, CA). The data were analyzed on

Cytomation Summit MoFlo Acquisition Software (Cytoma-

tion, Fort Collins, CO). Because of instability in the

integrations as checked by antibiotic sensitivity of sorted

cells, some of the cells did not display both proteins but

instead displayed BPTI or D1.3 only (data not shown). For

fluorescence quantification, gates were drawn around D1.3

and BPTI co-displaying cells, BPTI only displaying cells,

and D1.3 only displaying cells. The peaks derived from these

three gates were fit to a log-normal distribution equation

using Microsoft Excel (Microsoft, Redmond, WA), and the

average of the co-expressor peak mean normalized to the

single expessor peak mean are reported in the figure.

For the secretory stress studies, pKT048 and pRS316 were

transformed into JARD1.3, JARBPTI, and JAR2DB. Cells

were grown and induced as described above then analyzed on

an Epics XL Flow Cytometer.

For the UPR titration studies, pCYHi-1 and pKT048 were

transformed into JAR2DB. pRS316 was also transformed

into JAR2DB in place of pCYHi-1 to serve as a negative

control. Cells were inoculated into liquid SD-CAA with

100 mg/mL doxycycline hyclate (Sigma). This was enough

doxycycline to repress expression of the Hac1i gene until

induction. Cells were passaged into 5mLYPG to anOD600 of

0.5. At this point, doxycyclinewas added to cultures such that

the final doxycycline concentrations were between 0 and

10 mg/mL. Cells were grown overnight to an OD600 of �9,

and 0.2OD600 of cells were collected for analysis. These cells

were stained with either 20 mg/mL biotin-trypsin or 3 mg/mL

biotin-lysozyme for BPTI and D1.3 surface display,

respectively. The secondary reagent for both strains was

10 mg/mL streptavidin-PE (Molecular Probes). Cells were

analyzed on an Epics XL.

For the chaperone studies, pKT048 was transformed into

JAR2DB with pGal-PDI (LaMantia and Lennarz, 1993),

pGalKar2-URA, pGPDKar2 (Robinson et al., 1996), or

pRS316. Cells were grown and induced as described above.

They were then labeled for either BPTI or D1.3 surface

display as described previously and analyzed on an Epics XL

Flow Cytometer.

Fluorescence Microscopy

JAR2DB was inoculated into 5 mLYPD, grown overnight at

308C, passaged to an OD600 of 0.5 in YPG, and grown

overnight at 308C to an OD600 �3. 500 mL of formaldehyde

was added to the culture, and after 1.5 h of incubation at 308C,the cells were washed twice in 1 mL PBS/0.5% Tween-20.

Cells were incubated in 0.5 mL 50 mg/mL zymolase (Zymo



Research) diluted in PBST for 20 min at 378C then washed

in 0.5 mL PBST three times. 0.5 OD600 of cells were

resuspended in 10 mg/mL chicken anti-c-myc antibody and

20 mg/mL 13D11 anti-vacuole membrane antibody (Mole-

cular Probes) diluted in 20 mL PBS/4% BSA. After a 1 h

incubation at room temperature, cells were washed three

times in 100 mL PBS/4%BSA. Cells were then incubated in

40 mg/mL Alexa488-conjugated goat anti-chicken antibody,

10 mg/mL PE-conjugated goat anti-mouse antibody, and

1 mg/mL Hoechst dye diluted in 20 mL PBS/4% BSA. After

1 h of incubation at room temperature in the dark, cells were

washed three times and placed on L-polylysine coated

microscope slides. Uninduced cells were also fixed and

labeled to serve as a negative control. Microscopy was

performed on a Zeiss Axiovert 100TV Deconvolution

Fluorescence Microscope (Zeiss, Dublin, CA). Images were

captured on a Series 300 cooled CCD camera (Photometrics,

Tucson, AZ) and manipulated using DeltaVision softWoRx

software (Applied Precision, Issaquah, WA). This experi-

ment was repeated multiple times with similar results

each time.

RESULTS

BPTI Is More Efficiently Processed by the YeastSecretory Apparatus Than D1.3

BPTI and D1.3 were expressed in secreted form from low-

copy plasmids in the yeast strain EBY100 for 3 days at 308C.BPTI is secreted at approximately five times higher levels on

a molar basis than D1.3 (Fig. 1A). Furthermore, there was no

discernable difference in the growth rate during induction

between the two transformants.

BPTI and D1.3 were fused to the yeast mating protein

agglutinin 2 (Aga2p) and integrated into the yeast chromo-

some. Chromosomal integrations were used to eliminate

expression artifacts brought about by plasmid loss. After

expression induction late log phase cells were assayed for

surface expression via flow cytometry (Fig. 1B). The results

show that BPTI surface expression is 3.5-fold greater than

D1.3 surface expression indicating a surface-displayed D1.3

processing deficiency similar to that seen for secreted D1.3.

These results suggest that the traversal efficiency of the

secretory pathway by surface-displayed Aga2p fusion

proteins is similar to that for their secreted counterparts.

This similarity and the secretion/surface display relation-

ships found by Shusta et al. (1999) indicate that surface

display levels can be an effective screening proxy for

secretion when comparing proteins with different folding

efficiencies. Finally, labeling of surface displayed D1.3 or

BPTI with trypsin or lysozyme respectively demonstrates

that surface-displayed protein is properly folded and

functional (data not shown).

Co-Expression of D1.3 and BPTI Increases BPTISurface Display But Decreases D1.3 SurfaceDisplay

When surface-displayed D1.3 and BPTI are co-expressed in

the same cell during mid-log phase growth, flow cytometric

analysis shows that BPTI surface display is stimulated 20%

while D1.3 surface display decreases about 25% over single

protein expression alone (Fig. 2). This decrease is not due to

a shortage of the agglutinin-1 (Aga1p) fusion partner,

as additional overexpression of galactose-induced Aga1p

yielded no change in the level of surface display (data not

shown). The increase in BPTI surface expression due to D1.3

co-expression indicates that the secretory capacity for BPTI

expression is being underutilized in singly expressing BPTI

Figure 1. The cellular secretory machinery processes BPTI more

efficiently than D1.3 when expressed from low-copy plasmids as evidenced

by a larger secretory output (A). This difference is also seen when BPTI andD1.3 are integrated as fusions to the cell wall protein Aga2 and displayed on

the yeast surface (B).

Figure 2. Co-expression (dark bars) of integrated surface-displayed BPTI

and D1.3 affects the two proteins differently compared to cells expressing

integrated BPTI or D1.3 only (light bars). BPTI display is enhanced in co-

expressing cells; however, D1.3 display is negatively impacted. Expression

levels are normalized to the average single-protein expressing, cell-surface

fluorescence with one standard deviation shown.


DOI 10.1002/bit

transformants. Moreover, the increased secretory stress

imposed on the cell by D1.3 expression appears to be slightly

beneficial to BPTI processing. On the other hand, BPTI

expression has a negative impact on D1.3 expression

indicating that D1.3 secretory capacity may already have

been met or exceeded in singly expressing D1.3 transfor-

mants. BPTI co-expression reduces the D1.3 secretory

capacity further, possibly through elevated secretory stress

levels or monopolization of protein folding chaperones.

These results show that the secretory capacities for BPTI and

D1.3 are dissimilar and are affected differently by increased

flux of heterologous protein through the secretory pathway.

Co-Expression Results in Elevated D1.3-Aga2pLevels in the Vacuole and Supernatant

Accumulated D1.3 was imaged inside single expressing and

co-expressing mid-log cells by immunofluorescent micro-

scopy. Analysis of intracellular D1.3 showed that it was

largely confined to the vacuole, which suggests that the

protein was routed for degradation but was not degraded,

most likely due to the pep4/prb mutations that decreases

proteolytic activity in this organelle. Cells expressing BPTI

and D1.3 show increased levels and frequency of vacuolar

D1.3 aggregates (Fig. 3A) compared to cells expressing D1.3

alone (Fig. 3B) even though the number of D1.3 integrations

in the two strains is the same. This difference in retention

suggests that BPTI expression affects D1.3 trafficking in the

Golgi resulting in preferential routing of D1.3 to the vacuole

for degradation. Additional comparison between the two

populations shows aggravated cellular swelling in the co-

expressing cells suggesting increased cellular stress. This

swelling is further verified by an increase in the forward

scatter of the co-expressing cells as measured by flow

cytometry (data not shown). Furthermore, the co-expressing

cells exhibit a twofold decrease in specific growth rate upon

galactose induction indicating significant cellular stress. Co-

expression also causes an increase in D1.3–Aga2p fusion

found in the supernatant as analyzed by Western blot

(Fig. 3C). After normalizing for cell density, it was found

that co-expressing cells secreted approximately tenfoldmore

D1.3-Aga2p into the supernatant than single expressing cells.

Functional analysis shows that at least some of this protein is

properly folded (data not shown). The large increase in the

net amount of D1.3-Aga2 found in the vacuole and super-

natant suggests that perhaps vacuole-directed trafficking

capacity is exceeded by BPTI-Aga2p co-expression, causing

overflow of D1.3-Aga2p into the supernatant.

Kar2 and PDI Overexpression EnhanceD1.3 Surface Display

One hypothesis for theD.1.3 surface display deficiency in co-

expressing cells is that co-expression saturates available ER

folding chaperones, leaving insufficient capacity to direct the

folding of the D1.3-Aga2p/Aga1p protein fusion. With the

Figure 3. Cells co-expressing surface-displayed D1.3 and BPTI (A) showelevated levels of intracellular D1.3 (green) inside the vacuolar membrane

(red) compared to cells expressing D1.3 alone (B). Co-expressing cells alsoshow aggravated cellular swelling. Yeast nuclei are stained blue. In addition,

co-expressing cells secrete more total D1.3-Aga2p (light bars) and more

D1.3-Aga2p per cell (dark bars) than D1.3 only displaying cells (C). [Colorfigure can be seen in the online version of this article, available at

www.interscience.wiley.com.]



lack of sufficient chaperones, D1.3-Aga2p may be unable to

pair with an Aga1p partner causing the D1.3–Aga2p fusion

to be secreted rather than anchored to the cell wall. To test this

hypothesis, co-expressing cells were transformed with an

extra copy of galactose-promoted yeast PDI, galactose-

promoted yeast Kar2 (BiP), glyceraldehyde-3-phosphate

dehydrogenase (GDP)-promoted Kar2, or an empty pRS316

vector as a control. Cell surface labeling of the transformants

shows that BiP and PDI mostly compensate for the 25%

decrease in D1.3 display upon co-expression with BiP being

slightly more beneficial than PDI (Fig. 4A). On the other

hand, only a small negative effect onBPTIwas observedwith

chaperone coexpression (Fig. 4B). These results are

consistent with those showing that both BiP and PDI improve

processing of secreted D1.3 (Shusta et al., 1998) but have no

beneficial effect on the production of secreted BPTI (Parekh,

1996). Overexpression of BiP and PDI increase the secretory

capacity for D1.3-Aga2p/Aga1p by increasing the number of

folding chaperones available to process the surface-dis-

played D1.3 complex.

D1.3 and Co-Expressing Cells Exhibit ElevatedSecretory Stress

In order to quantify the degree of secretory stress imparted by

each protein, single expressing and co-expressing yeast were

transformed with a GFP reporter plasmid influenced by

secretory stress levels via a UPR-sensitive promoter

(Travers et al., 2000). Specifically, secretory stress activates

GFP transcription through activated-Hac1p binding UPR

elements (UPRE) in the promoter. Flow cytometric analysis

shows that D1.3 expression induces a 3.5-fold rise in theUPR

compared to BPTI expression (Fig. 5). Furthermore, co-

expression increases secretory stress marginally further.

These results show that the majority of secretory stress in co-

expressing cells is due to D1.3 expression, and co-expression

elevates this stress only slightlymore. Increased activation of

the UPR suggests that more protein folding resources are

necessary to fold D1.3-Aga2p than are normally available in

the ER. The beneficial effects of BiP and PDI overexpression

support this point.

Increasing UPR Induction NegativelyImpacts Both D1.3 and BPTI Expressionin Co-Expressing Cells

Because elevated UPR levels are correlated with increased

levels of BPTI expression and decreased levels of D1.3

expression in co-expressing cells, the cause and effect

relationship between UPR and expression of each protein

was examined. A gene for the active spliced variant of Hac1

(Cox and Walter, 1996) was placed under the control of a

doxycycline repressible promoter (Gari et al., 1997). In this

way, titration of the UPR in co-expressing cells can be

performed by varying doxycycline concentration in the

media. An analysis of the UPR induced by this system,

measured by the UPR-GFP reporter gene, for doxycycline

concentrations between 0 and 10 mg/mL ofmedia is shown in

Figure 6A. Thismethod of gene induction results in a fivefold

span of UPR induction. The highest concentration of

doxycycline shows a secretory stress response similar to that

seen for the empty pRS316 control vector. Cytometric

analysis of D1.3 surface expression shows a strong negative

correlation between UPR induction and D1.3 surface display

(Fig. 6B). This negative correlation is also apparent for BPTI

surface expression (Fig. 6C). Furthermore, the surface

expression levels of both D1.3 and BPTI under the most

repressed conditions did not reach the expression levels in the

pRS316 control. This result would indicate that even small

levels of unregulated UPR are detrimental to expression, in

particular for D1.3 expression. Collectively these results

show that elevated UPR levels do not contribute to the

increase in BPTI expression uponD1.3 co-expression, but do

negatively impact D1.3 surface display.

Figure 4. High-level expression of the folding chaperones BiP and PDI in co-expressing cells improves D1.3 display (A) and has a small negative effect on

BPTI display (B) compared to wild-type (pRS316). BiP is expressed under galactose and GPD-driven promoters.


DOI 10.1002/bit

DISCUSSION

Although an attractive candidate for heterologous protein

production because of its eukaryotic secretory protein

folding machinery, S. cerevisiae typically secretes hetero-

logous proteins relatively poorly. Here, two proteins with

different secretory processing efficiencies are examined in

yeast simultaneously expressing both proteins. This con-

comitant expression allows for direct comparisons of the

secretory capacity for each protein, and demonstrates

directly that secretory pathway capacity is not static.

It is clear that yeast process BPTI more efficiently than

D1.3 both in their secreted and surface-displayed forms.

However, it is curious that co-expression has opposite effects

on the surface display of the two proteins. It is not clear why

co-expression of D1.3 has a stimulatory effect on BPTI

surface display. A first hypothesis would be that the increase

in secretory stress brought about by D1.3 expression is

beneficial to BPTI processing. However, the negative

correlation between BPTI surface expression and high levels

of UPR seems to rule out this possibility. It has been shown

that low copy expression of BPTI underutilizes the secretory

capacity of the cell, and protein production can bemaximized

by optimizing gene copy number (Parekh andWittrup, 1997).

Figure 6. Constitutive activation of the UPR as indicated by a UPR-GFP reporter gene (A) has a negative effect on expression of surface-displayed D1.3

(B) and BPTI (C). UPR activation was titrated by varying expression of the active Hac1ip spliced variant via a doxycycline repressible promoter.

Concentrations of doxycycline in the induction media are indicated. Awild-type control (pRS316) without constitutive UPR induction is also shown.

Figure 5. D1.3 expression imparts substantiallymore secretory stress than

BPTI expression as indicated by a UPR-activated GFP reporter gene.

Moreover, co-expressing BPTI and D1.3 results in an additional, small

elevation of secretory stress.



From this result, it can be inferred that there is a balance

between what is produced, what is secreted, and what is

degraded, and the flux of protein to any particular fate may be

influenced by the secretory stress of the cell. It may be that

D1.3 stimulates the cell to allow more BPTI to be directed

toward surface expression rather than ERAD or vacuolar

degradation.

Whereas expression of surface-displayed BPTI in JARDB

is below the potential of the cell to produce BPTI, it appears

that the expression of D1.3 in JARDB approaches the limit

that the cell will tolerate. This idea is evidenced by the

presence of D1.3-Aga2p in the vacuole and supernatant of

single protein expressing populations. What is even more

telling is that vacuolar D1.3 is not present inside every cell

although every cell has about the same level of surface

fluorescence. For reasons due to stochastic variation in

folding chaperones or epigenetic differences in the cellular

stress response, some of these singly expressing cells were

able to deal with surface-displayed D1.3 without routing it to

the vacuole. Furthermore, upon simultaneous expression of

BPTI, all of the cells show reduced surface-display levels,

elevated D1.3 retention in the vacuole, and increased D1.3 in

the supernatant. These results suggest that the additional

secretory load imparted by BPTI expression hinders the

processing of D1.3 causing it to be abnormally trafficked.

The decrease in D1.3 surface expression possibly has its

source in the ER. Perhaps BPTI, being a more stably folded

protein, is less sensitive to the UPR than D1.3. The relatively

small increase in UPR brought about by co-expression does

little to affect BPTI surface display but is enough to

negatively impact D1.3 surface display. It was determined

that co-expression of the ER folding chaperones BiP or PDI

reverses the surface display depression. The chaperones

could act by facilitating the Aga2-Aga1p linkage necessary

for surface display. The failure of this interaction could also

explain why more properly folded D1.3 is found in the

supernatant upon co-expression as the lack of Aga1p would

prevent cell wall anchorage. It has been shown that Aga2p

fusions can be secreted in the absence of Aga1p (Huang and

Shusta, 2005). Perhaps D1.3-Aga2p, lacking an Aga1p

fusion partner, is normally sorted to the vacuole; BPTI co-

expression saturates that sorting pathway causing D1.3-

Aga2p to overflow into the supernatant. Elevated levels of

vacuolar D1.3 in co-expressing cells certainly indicate

increased D1.3 flux toward the vacuole.

These studies have shown that the secretory stress

imparted on the cell as measured by the UPR is significantly

different for BPTI and D1.3. Clearly, the consequences of

folding D1.3 are felt in the ER. However, the lack of

chaperones does not explain why there is such a significant

net increase inD1.3 found in thevacuole and supernatant. It is

clear that the flux of D1.3 moving through post-Golgi

compartments has been greatly amplified. This increase is too

much to be accounted for by surface display depression

alone. Furthermore, this increase has cell-wide effects

including cellular swelling (due to an enlarged vacuole)

and reduced growth rate—a symptom not rescued by

chaperone co-expression. Assuming translational levels

remain the same, an increase in the flux of D1.3 in the

vacuole or surface directed pathways would indicate the

failure of another pathway normally utilized by protein

traffic. This blocked pathwaymay be postulated to be ERAD;

however, the relatively similar levels ofUPR in single and co-

expressing cells would indicate that ER stress is not

significantly different between the single expressing and

co-expressing populations. It is likely, however, that some

degradative pathway has been negatively impacted by co-

expression. Since BPTI is a serine protease inhibitor,

perhaps it directly inhibits some proteolytic activities in the

Golgi.

From these studies it appears that BPTI expression may

affect D1.3 surface-display by interfering with folding

processes. This interference causes drastic changes in

D1.3-Aga2 secretion and vacuolar trafficking as well as

decreased surface expression. Overexpression of BiP and

PDI relieves the surface expression problem suggesting that

ER chaperones may become limiting under co-expression

conditions. The origin of the post-Golgi flux increase is still

unclear. It is likely due to the saturation of a competing

degradative pathway. Screens of mutant libraries for reduced

vacuolar aggregation could help solve the mystery. The

characterization of this phenomenon could be fruitful as it

suggests a method for increasing the flux of heterologous

protein through the late secretory pathway as well as

providing a method for re-routing vacuole-targeted hetero-

logous proteins to the surface.

Special thanks to the Anne Robinson lab (University of Delaware) for

the UPR-induced GFP reporter construct and to the Peter Walter lab

(UCSF) for the constitutively activated Hac1 gene.

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Contrasting secretory processing of simultaneously expressed heterologous proteins in Saccharomyces cerevisiae