* Department of Yeast Genetics, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark; and Department of Biochemistry and Molecular Biology, Penn State University, University Park, Pennsylvania 16802
Aspects of protein disulfide isomerase (PDI)
function have been studied in yeast in vivo. PDI contains two thioredoxin-like domains, a and a, each of
which contains an active-site CXXC motif. The relative
importance of the two domains was analyzed by rendering each one inactive by mutation to SGAS. Such
mutations had no significant effect on growth. The domains however, were not equivalent since the rate of
folding of carboxypeptidase Y (CPY) in vivo was reduced by inactivation of the a domain but not the a
domain. To investigate the relevance of PDI redox potential, the G and H positions of each CGHC active site
were randomly mutagenized. The resulting mutant
PDIs were ranked by their growth phenotype on medium containing increasing concentrations of DTT. The
rate of CPY folding in the mutants showed the same
ranking as the DTT sensitivity, suggesting that the oxidative power of PDI is an important factor in folding in vivo. Mutants with a PDI that cannot perform oxidation reactions on its own (CGHS) had a strongly reduced growth rate. The growth rates, however, did not
correlate with CPY folding, suggesting that the protein(s) required for optimal growth are dependent on
PDI for oxidation. pdi1-deleted strains overexpressing
the yeast PDI homologue EUG1 are viable. Exchanging the wild-type Eug1p C(L/I)HS active site sequences
for C(L/I)HC increased the growth rate significantly,
however, further highlighting the importance of the oxidizing function for optimal growth.
FORMATION of correct disulfide bonds is essential for
proper folding of the majority of secretory proteins
in eukaryotic cells. Refolding of proteins containing more than one disulfide bond in vitro is stimulated by
the enzyme protein disulfide isomerase (PDI)1 (Givol et
al., 1965
Members of the thioredoxin family contain two catalytically active cysteine residues found in a CXXC motif. The
identity of the two X residues varies among the family
members, but catalysis is always mediated by reduction
and oxidation of an internal disulfide bond between the
cysteine residues of the active site (Martin, 1995 In the eukaryotic cell, PDI is localized to the ER. Glutathione and glutathione disulfide (GSSG) are believed to
function as the redox buffer since they are found at approximately equal millimolar concentrations in the ER,
providing a much more oxidizing environment than the cytoplasm (Hwang et al., 1992 In yeast, the structural gene for PDI, PDI1, is essential
for viability (Farquhar et al., 1991 Strains
Saccharomyces cerevisiae strains W303-1B Escherichia coli strains.
DH5 Media and Materials
Yeast cells were grown in standard YPD and SC media (Sherman, 1991 Plasmid Construction and Mutagenesis
Subcloning and transformation of E. coli and yeast were carried out using
standard procedures (Sambrook et al., 1989 Table I.
Plasmids Used
). PDI of mammalian origin has a characteristic a-b-b
-a
-c domain structure (Edman et al., 1985
). The a
and a
domains have sequence and structural homology to
thioredoxin (Kemmink et al., 1996
; Fig. 1 A). The b and b
domains are characterized by mutual homology. The
COOH-terminal c region is rich in acidic residues, and is
thought to be involved in binding of calcium ions (Lebeche et al., 1994
; Nigel Darby, EMBL, Heidelberg, personal communication).
Fig. 1.
(A) Sequences of PDI and Eug1p from yeast and
thioredoxin from E. coli surrounding the active site CXXC motifs
(boxed). A section of the a and a domains of PDI and Eug1p is
compared, and the location of the predicted secondary structure features is indicated by
2 (second
-sheet structure) and
2 (second
-helix structure). Residues that are found in a majority of
the sequences are underlined. (B) Reaction cycle for oxidation of
nascent polypeptide (ProteinSH/SH) catalyzed by PDI. The putative involvement of GSSG in the oxidation cycle is indicated. The
upper SH group of PDI (PDISH) indicates the NH2-proximal cysteine residue of the CXXC motif. This is the more reactive residue and will engage in the formation of mixed disulfides with the
substrate, here shown only for the protein substrate although a
similar reaction pathway exists for GSSG reduction. PDISH indicates the more COOH-proximal cysteine residue, which is involved in the formation of the internally oxidized form of PDI. In
mutant forms of PDI lacking the COOH-proximal cysteine, oxidation of substrate protein can occur, albeit inefficiently, by reduction of a mixed disulfide between glutathione and the PDI
NH2-proximal cysteine residue. (C) Isomerization reaction in
which the NH2-proximal cysteine residue of the CXXC motif attacks a wrongly formed disulfide of a substrate protein. The formation of mixed intermediate I allows a free SH group of the
substrate to form an alternative SS bond. Importantly, the more
COOH-proximal cysteine residue does not participate in this reaction cycle.
[View Larger Version of this Image (29K GIF file)]
). As the
name would indicate, isomerization of disulfide bonds is
an important feature of PDI. What is less widely acknowledged is that PDI may also play an important role in the
oxidation of newly synthesized proteins. Detailed biochemical analysis has shown that the a and a
domains of
PDI are capable of catalyzing two kinds of disulfide reactions (Creighton et al., 1980
; Darby and Creighton, 1995a
):
(a) oxidation reactions in which the intramolecular disulfide bond of the CGHC motif is transferred to a pair of
sulfhydryls in a substrate polypeptide (Fig. 1 B); and (b)
isomerization reactions in which disulfides are rearranged through the formation of a mixed disulfide between the
first cysteine residue of the CGHC motif and the substrate
(Fig. 1 C).
).
; LaMantia et al., 1991
;
Günther et al., 1991
; Scherens et al., 1991
). When PDI is
depleted from the cells, a secretory pathway marker protein accumulates in the ER, suggesting an impairment in
folding (Günther et al., 1991
; Tachibana and Stevens,
1992
). Despite the potentially central role that PDI plays
in the folding of newly synthesized secretory pathway proteins, little has been done to dissect the functional aspects
of its role in folding in vivo. In the present work, we have
analyzed the effects of various mutations in PDI1 on the folding of a yeast vacuolar protein, carboxypeptidase Y
(CPY). CPY has a number of virtues that make it a very
useful model for the study of in vivo folding and secretory
transport. Changes in molecular mass that accompany its
transport through different compartments of the secretory
pathway can be followed in pulse-labeling experiments
(Stevens et al., 1982
). Upon translocation into the ER, the
polypeptide receives four core N-glycosyl residues, resulting in the p1 form of proCPY, which has a molecular mass
of 67 kD. The core glycosyl structures are further modified
in the Golgi compartment to give the 69-kD p2 form of
proCPY. This form is sorted to the vacuole, where it is
processed to its mature form of 63 kD. The individual
transport steps are easily followed in 35S pulse-labeling experiments, and the total sequence of events has a half time
of ~6 min. As in mammalian systems, proteins that do not
fold into their correct three-dimensional structure are retained in the ER. Thus, ER retention is an indication of
misfolding. In a more direct assay, the in vivo folding rate
was monitored using a mutant form of CPY (CPY-3T)
that contains a new glycosyl acceptor site that is inaccessible in the folded protein. Treatment of whole cells with
DTT may block the folding of many disulfide bond-containing proteins by preventing oxidation of the cysteine residues of the newly synthesized proteins (Braakman et
al., 1991
). In yeast cells, DTT treatment results in accumulation of p1-proCPY (Simons et al., 1995
) and, in the case
of proCPY-3T, modification of the new glycosylation site
(Holst et al., 1996
). In strains expressing some mutant alleles
of PDI1, we found this modification was present on CPY-3T
at a level approaching that of DTT-treated cells. These observations are consistent with the view that not only is formation of the five disulfide bonds of the protein essential for correct folding, but also that mutations in PDI can affect the rate of folding in vivo even when no growth phenotype is obvious.
Materials and Methods
pdi1 (MAT
ade2-1 can1-100 ura3-1 leu2-3,112 trp1-1 his3-11,15
pdi1::HIS3) and M4143 (MAT
ade2-1 can1-100 ura3-1 leu2-3,112 trp1-1 his3-11,15
pdi1::HIS3
prc1-17)
containing either pCT37, pCT40 (Kurjan, 1985
; Tachibana and Stevens,
1992
), or pBH1464 was used for plasmid shuffling and characterization of
the different pdi1 mutants. M4143 was constructed by gene replacement
(Scherer and Davis, 1979
) of PRC1 using plasmid pWI-17 (Holst et al.,
1996
).
(Sambrook et al., 1989
) was used for
plasmid propagation, BMH71-18mutS (Kramer et al., 1984
; Zell and Fritz,
1987
), CJ236 (Kunkel et al., 1987
), and JM109 (Yanisch-Perron et al.,
1985
) were used for site-directed mutagenesis. Yeast strain JWY33-2B (MAT
ura3-52 leu2-3,112 trp1-1 sec18-1
pdi1::HIS3
prc1-17) was constructed by crossing M4143 with XCR101-12 (MATa ura3-52 sec18-1 his3
leu2-3,112) using standard genetic procedures (Sherman, 1991
).
).
E. coli was grown in LB, SOC, and 2× YT medium (Sambrook et al.,
1989
). Restriction enzymes, T4 DNA polymerase, T4 polynucleotide kinase, T4 DNA ligase, and Klenow polymerase were from Promega (Madison, WI). [35S]methionine was from DuPont NEN (Boston, MA). Zymolase 100-T was from Seikagaku Kogyo (Tokyo, Japan). Fixed Staphylococcus aureus cells were IgGsorb from The Enzyme Center (Malden, MA). Oligonucleotides were from DNA Technology (Aarhus, Denmark). Sequencing was performed using a Taq Dye DeoxyTM terminator cycle sequencing kit on an API 373A DNA Sequencer, both from Applied
Biosystems (Foster City, CA).
; Ito et al., 1983
). The plasmids
used in this study, the resulting PDI1 alleles, and the encoded enzymes are
listed in Table I. pBH1464 was constructed by subcloning of a KpnI-SacI
fragment from pCT38 (Tachibana and Stevens, 1992
) into the same sites
in pRS314 (Sikorski and Hieter, 1989
). pBH1514 was constructed by subcloning a KpnI-XbaI fragment from pCT38 into the same sites in pSELECT (Lewis and Thompson, 1990
). Site-directed mutagenesis was performed as described previously (Lewis and Thompson, 1990
) with the
modification described by Olesen and Kielland-Brandt (1993)
. For cloning purposes, a BamHI site was introduced by site-directed mutagenesis
into the PDI1 promoter region, generating pBH1857 after reconstitution
in an otherwise wild-type context. Pulse-labeling and growth experiments
showed no change in phenotype, as compared to the wild-type PDI1 (data
not shown). pBH1852, pBH1630, and pBH1692, containing serine residues instead of cysteine residues at the a site, the a
site, and both the a and the a
active sites, respectively, were generated by site-directed mutagenesis (Table I). pBH1680 was constructed by recloning of a KpnI-XbaI fragment from pBH1692 into pSELECT in the same sites. pBH1680
was used for site-directed mutagenesis to randomize the two middle
amino acids in either of the two active sites (CGHC). Table I lists the mutants reconstituted into an otherwise wild-type PDI1 context, except for
the already introduced alterations. Likewise, mutants with serine residues
instead of cysteine residues in the most COOH-proximal cysteine in either of the two active sites or in both active sites, were generated by site-directed mutagenesis in pBH1680 followed by a similar reconstitution
(Table I). EUG1 was mutated using conventional techniques (Kunkel et
al., 1987
; Herlitze and Koenen, 1990
) and subcloned into pBH1865 to
place the gene under PDI1 promoter control.
Characterization of pdi1 Mutants
Pulse labeling and immunoprecipitation were performed essentially as described by Winther et al. (1991). Yeast cells were grown in SC-trp-ura and
starved for sulfur in SC-trp-ura without (NH4)2SO4. [35S]methionine was
used for labeling instead of [35S]H2SO4. When indicated, DTT was added
to give final concentrations of 5 mM. For pulse labeling, PRC1 alleles encoding wild-type CPY or CPY with an extra N-glycosylation site (CPY-3T; Holst et al., 1996
) were reintroduced via plasmids pJW1433 (Ramos et
al., 1994
) and pBH994, respectively. The randomly mutagenized pdi1 mutants were screened for their ability to grow in the presence of different concentrations of DTT. The final concentration of DTT varied between 0 and 5 mM. Overnight cultures grown in the absence of DTT were diluted
104 times in water, aliquots of 10 µl were placed on freshly made SC plates
buffered to pH 5 with 50 mM NaH2PO4, and the plates were subsequently
incubated in a CO2 atmosphere to prevent oxidation of the DTT by O2.
After 48 and 72 h at 30°C, the mutants were ranked according to the maximal DTT concentration at which they were able to grow. Several independent experiments showed variation in ranking of no more than two positions.
Analysis of the Relative Importance of a and
a Domains
To evaluate the relative importance of the two thioredoxin-like domains of PDI, mutant forms of the protein
were constructed containing either of the two CGHC active site motifs converted to SGHS. Such mutations impair
the ability of the mutated active site to engage in disulfide
chemistry. Since PDI1 is an essential gene, we used a plasmid-shuffle procedure (Sikorski and Boeke, 1991) to introduce the mutant genes. Mutants were introduced on a
plasmid containing a TRP1 selectable marker into a
pdi1
strain that carried PDI1 on a plasmid with a URA3
marker. After transformation, the cells were forced to lose
the plasmid-containing wild-type PDI1 by plating on medium containing 5-fluoro orotic acid, which selects cells
that have lost URA3. Cells will be able to grow only if the
newly introduced PDI mutant form can complement the chromosomal pdi1 deletion. Consistent with the theory
that the essential activity of PDI is dependent on its ability
to engage in disulfide chemistry, we found that a SGHS-SGHS2 mutant is not able to complement
pdi1.
Cells producing the CGHC-SGHS or SGHS-CGHC
mutant enzymes as their sole PDI forms showed no
growth defect under normal conditions. To investigate
protein folding in vivo in these strains, we monitored the
rate of intracellular transport of CPY. Cells were pulse-labeled with 35S for 15 min, and were chased with nonradioactive sulfur for 0, 5, and 15 min before lysis and immunoprecipitation of CPY antigen. The precipitates were
subjected to SDS-PAGE, and the gels were exposed to
x-ray film or PhosphorImager screens. As described in the
introduction, CPY undergoes a number of modifications
in the secretory pathway that allow estimation of the rate
of transport between the ER, Golgi, and vacuole. Fig. 2 A
shows that while the rate of CPY maturation is essentially
the same in wild-type and CGHC-SGHS mutants, the half
time of CPY maturation is 15-20 min in the SGHS-CGHC
mutant. Since only folded proteins are allowed to exit the
ER, (Gething et al., 1986) this shows that the a domain
plays a more important part in folding of CPY than the a
domain.
Treatment of intact yeast or mammalian cells with DTT
results in ER accumulation of newly synthesized, disulfide-containing secretory proteins in a reduced state
(Braakman et al., 1991; Jämsä et al., 1994
). To test the
ability of the PDI mutants to oxidize proCPY after DTT
treatment, we carried out the following experiment. Cells
were treated with 5 mM DTT during labeling with 35S, after which they were washed and chased with nonlabeled,
DTT-free medium. As seen in Fig. 2 B (lanes 1, 4, and 7),
all strains accumulate p1-proCPY in the ER during a DTT
pulse. We find that the rate of maturation is comparable in
the wild-type cells and in those expressing the CGHC-SGHS form of PDI. In cells producing the SGHS-CGHC mutant, no mature CPY is found, even 60 min after DTT
washout. The simplest explanation for this effect on CPY
folding is that reoxidation of ER components is slower in
this mutant than in the CGHC-SGHS mutant or the wild
type.
Random Mutagenesis of the CGHC Sequence of the Thioredoxin Domains
The redox potential of thioredoxin and DsbA (an E. coli
analogue of PDI; Bardwell et al., 1991) is strongly dependent on the identity of the two central amino acid residues
of the CGHC motif (Grauschopf et al., 1995
; Chivers et al.,
1996
). To investigate the importance of the redox potential for both growth and ability to fold CPY in vivo, a library of C
C mutants (
denotes a random amino acid
residue) was constructed. The mutagenesis was carried out
on a plasmid encoding the SGHS-SGHS mutant. Two independent libraries of mutants were made so that each library converted only one of the two active sites from
SGHS to C
C. Each pool of mutant plasmids was introduced into a
pdi1 yeast strain and tested for complementation. Survival required the introduction of both the necessary cysteine residues as well as the mutagenesis of the
intervening GH sequence. From the mutant pools, a collection of 10 and 12 clones, respectively, were isolated and
sequenced (Table II). We have not analyzed the identity
or frequency of noncomplementing mutants. All complementing mutants, along with wild-type PDI, the CGHC-SGHS, and the SGHS-CGHC mutants, were further characterized by sensitivity to DTT. This test was chosen since
DTT is known to penetrate into the ER and interfere with the formation of disulfide bonds in nascent disulfide-containing proteins. We adjusted the pH in the plates to 5 and
incubated them in a CO2 atmosphere to reduce the spontaneous oxidation of the DTT. The wild-type and the
CGHC-SGHS mutant grow on 5 mM DTT. The SGHS-CGHC mutant, which showed increased sensitivity to
DTT in the pulse-labeling experiment, also shows a weaker
growth on plates containing DTT (Fig. 3). In Table II, the
mutants are sorted according their DTT sensitivity. Since
the DTT sensitivity is, in most cases, not an all-or-nothing
relationship, many of the mutants are classified as having
the same sensitivity threshold. Nevertheless, using media
containing a spectrum of DTT concentrations, a clear order of the ranking of growth rates was observed within each class. No significant growth phenotype was found under aerobic or anaerobic conditions in the absence of DTT
(Fig. 3). In the C
C mutants of DsbA, an increased DTT
sensitivity of the bacteria correlates with a more reducing
redox potential (Grauschopf et al., 1995
). By analogy, it is
likely that a similar ranking of the yeast PDI mutants reflects a ranking of redox potential.
Table II.
DTT Sensitivity of PDI C |
All mutants were analyzed further by pulse-chase labeling and immunoprecipitation of CPY, as described above,
and a correlation was found between rate of ER exit and
in vivo sensitivity towards DTT. Labeled immunoprecipitates of CPY from representative mutants are shown in
Fig. 4. The labeling shows that the rate of transport is reduced considerably for the mutants, as compared to the
wild type. The CGSC-SGHS mutant, which is sensitive to
1 mM DTT (Table II), has a half time of maturation only
slightly longer than the wild type (8-10 min). The CSGC-SGHS mutant has a half-time of maturation of ~15 min
and a DTT sensitivity threshold of 0.2 mM. In the most
DTT-sensitive mutant (CRRC-SGHS), proCPY is matured with a half time of ~30 min, as compared to 6 min
for cells containing wild-type PDI.
Oxidation-deficient Mutants
The ability of enzymes in the thioredoxin family to cycle
efficiently between an oxidized and reduced state depends
on the presence of an intramolecular disulfide bond in the
CXXC motif (Fig. 1 B). However, only the more NH2-proximal of the two cysteine residues plays an essential
role in the isomerization reaction. This is because only this
residue engages in the formation of mixed disulfides with
the substrate polypeptide (Fig. 1 C; Wunderlich et al.,
1995; Walker et al., 1996
).
After considering the effect of random changes to the
redox potential of PDI, we wished to analyze the most oxidation-deficient mutants that we could conceivably construct without the loss of isomerase activity. Efficient oxidation is possible only when both active-site cysteines are
present (Fig. 1, B and C). Thus, mutants of the CGHS-type should not be able to perform the internal redox
chemistry, but should still be able to function as isomerases. It has been shown previously that the CGHA mutant
forms of PDI will complement a pdi1 deletion (LaMantia
and Lennarz, 1993). Similarly, overexpressed EUG1, a
PDI homologue with a CXXS motif in both thioredoxin
domains, can support growth of a
pdi1 strain (Tachibana
and Stevens, 1992
). We constructed the CGHS-CGHS,
CGHS-SGHS, and SGHS-CGHS mutants and found, in
accordance with previous results, that they did support
growth. However, the three CGHS mutants all show reduced rates of growth. Furthermore, they are extremely sensitive to DTT on plates. While the slowest growth rate
is seen for the CGHS-SGHS mutant (Fig. 5 A), pulse-chase experiments showed that the CPY folding was most
severely affected in the SGHS-CGHS mutant (Fig. 5 B).
This shows that there is no direct correlation between rate
of growth and rate of intracellular CPY folding. This conclusion is supported further by the observation that the rate of CPY maturation in the CRRC-SGHS mutant is as
slow as that of the CGHS-SGHS mutant, while the growth
rate of the former is essentially the same as wild type. This
also suggests the existence of specific PDI substrates that
are essential for optimal cell growth and more dependent
than CPY on PDI catalysis.
Reconstruction of a Thioredoxin-like Active Site in Eug1p
The EUG1 gene was originally isolated by its homology to
PDI1 (Tachibana and Stevens, 1992). The Eug1p protein
is uniquely different from PDI since it has CLHS-CIHS in
place of the of CGHC-CGHC active site sequences. The
expression of EUG1 under normal growth conditions is
~5-10% of that of PDI1 (Tachibana and Stevens, 1992
). We found that EUG1 complemented the growth defect of
a pdi1 deletion when expressed under control of the PDI1
promoter (Fig. 6). It did so, however, only to an extent
similar to that of the CGHS-CGHS mutant of PDI, i.e.,
the growth rate was considerably reduced compared to
wild type. This suggested that the main reason that EUG1
does not fully complement
pdi1 is the absence of the
COOH-proximal active-site cysteines. To investigate this
hypothesis, we attempted to convert Eug1p to a more
PDI-like enzyme by changing the CLHS-CIHS sequences
to genuine thioredoxin-type active sites CLHC-CIHC. Indeed, under control of the PDI1 promoter, this EUG1 mutant allowed a
pdi1 strain to grow at an essentially wild-type rate (Fig. 6). The obvious interpretation of this result
is that the mutant Eug1p gained the ability to form internal cystine bridges in the two active sites, allowing oxidative activity as well as disulfide isomerase activity.
Assaying Folding In Vivo Using Glycosyl Modification of Buried Sites
To test the rate of folding in the PDI mutants more directly, we used a mutant form of CPY in which a new site
for asparagine-linked glycosylation has been introduced at
a buried position in the protein structure. The mutant protein, CPY-3T (Holst et al., 1996), is useful because the
level of glycosylation at the new site is dependent on the
folding rate of the protein. Thus, no glycosylation of this
site takes place under normal growth conditions, while in
the presence of DTT the mutant shows ~40% modification of the buried acceptor site. Overglycosylation results
in a shift in SDS-PAGE mobility approximately equal to
the p2 modification, making precise evaluation of the level
of overglycosylation difficult when the protein is not substantially retained in the ER. We have therefore expressed
the mutants in a sec18 strain that is blocked in the ER exit
at the nonpermissive temperature (Stevens et al., 1982
).
The only band immunoprecipitated from the PDI1 wild-type control strain corresponds to p1-proCPY (Fig. 7, lane
2), indicating that no modification is taking place at the
buried site. The strains expressing various mutant PDIs
show varying degrees of overglycosylation of CPY-3T.
The level of overglycosylation is highest in the pdi1 mutants with the slowest rate of ER exit found for wild-type
CPY. This confirms that the rate of CPY folding is reduced in these mutants.
In this work, we have addressed some basic questions concerning the function of PDI in vivo. There is ample biochemical evidence that PDI activity is exerted through the
action of two thioredoxin-like domains, a and a, which
have a very high level of mutual similarity. Using rat PDI
expressed in yeast, Laboissiere et al. (1995)
showed that
the simultaneous mutation of the CGHC active-site sequences of these domains to SGHS generates an enzyme that is unable to support yeast growth. Our mutagenesis of
yeast PDI does not support an earlier proposal that yeast
PDI possesses an essential "chaperone" function (LaMantia and Lennarz, 1993
). The combined evidence indicate
that the ability of PDI to engage in disulfide chemistry is
essential for the viability of yeast.
Considering the mutual sequence similarity and the in
vitro evidence that both thioredoxin domains are able to
engage in disulfide chemistry, we wished to investigate the
relative importance of the two domains for folding of
CPY. CPY is an exceedingly well-characterized model
substrate in terms of biosynthesis and folding both in vivo and in vitro (Stevens et al., 1982; Winther et al., 1991
; Ramos et al., 1994
; Holst et al., 1996
), and requires the presence of at least some of its five disulfide bonds for folding
(Jämsä et al., 1994
). We have used two methods for monitoring the folding rate in vivo. One relies on the presence
of a quality control system in the ER (Ramos et al., 1994
;
Gething et al., 1986
). This mechanism ensures that only
correctly folded proteins are allowed to exit the ER and
proceed through the secretory pathway. Our other assay
for in vivo folding takes advantage of a mutant form of
CPY that contains an N-linked glycosylation site at a buried position in the protein structure. This site becomes
partially glycosylated if folding is compromised (Holst et
al., 1996
). In both assays for folding, we find that a mutant
enzyme that contains only an active a site (the CGHC-SGHS mutant) is as efficient in folding CPY as the wild-type enzyme. The SGHS-CGHC mutant, on the other
hand, shows a significantly slower CPY folding. This is the
first in vivo evidence that the two thioredoxin domains are
functionally different. This might reflect differences in substrate specificity between the domains, but as we shall
see below, could also be explained by differences in redox
potential.
The intramolecular disulfide bond between the two cysteine residues of thioredoxin-like proteins can be characterized with respect to its redox potential. Thioredoxins
from various sources have fairly reducing redox potentials,
while PDIs are generally characterized by a very oxidizing
active-site cystine bond (Bardwell and Beckwith, 1993).
This is true in spite of the observation that the three-dimensional structures of the various thioredoxin-like domains are virtually superimposable (Martin, 1995
; Kemmink et al., 1996
). In DsbA, an enzyme from E. coli with a
function similar to that of PDI, the relevance of the two
central residues in the CGHC motif has been investigated
extensively (Grauschopf et al., 1995
). Changing these residues had marked effects on the redox potential of the enzyme. DsbA mutants showed increased sensitivity to DTT
for folding of a chimeric malF-
-galactosidase fusion protein in vivo. Importantly, the sensitivity was inversely related to the oxidative power, which was determined on the
purified mutant enzymes in vitro. Thus, it appears that the
wild-type E. coli version of PDI is able to overcome the reducing power of DTT in an in vivo folding reaction. In
yeast and higher eukaryotes, DTT is able to penetrate into
the cells and inhibit the folding of disulfide bonded proteins (Braakman et al., 1991
). In particular, this has been
shown for CPY in yeast (Jämsä et al., 1995; Simons et al.,
1995
). We find that wild-type cells are seriously inhibited for growth on plates containing 10 mM DTT at pH 5 under
anaerobic conditions; however, cells will grow at 5 mM
DTT. The ability of cells to grow on DTT-containing medium is strongly dependent on PDI function. We have
shown that the CGHC-SGHS and SGHS-CGHC mutants
have different growth rates on DTT plates. While the
CGHC-SGHS mutant grew as well as the wild type on 5 mM DTT, the SGHS-CGHC mutant showed increased
sensitivity and grew like the wild type only on plates containing 3 mM DTT or less. This is in accordance with the
observation on human PDI that the a domain is more oxidizing than the a
domain (Darby and Creighton, 1995b
).
It is also interesting to compare these data with results from Walker et al. (1996)
, following the oxidative refolding of RNase using similar mutants of human PDI in vitro.
In these experiments, the oxidative folding of RNase was
faster in the presence of a PDI with a mutated a
site than
in the presence of a PDI with a mutated a site. Taken together, these results indicate that the initial oxidative formation of disulfide bonds may be a rather important aspect of PDI function. The difference in the rate of CPY
folding by PDI mutants disrupted in the a and a
domains
might reflect differences in their ability to oxidize substrate. Mutagenesis of the cysteines engaged in disulfide bonds in CPY has shown that several of these are important for folding in vivo (Jakobsen, A., and J.R. Winther,
unpublished observations). However, the actual rates of
ER exit probably cannot be directly correlated to in vitro
observations since characterization of CPY propeptide
mutants has shown that rates of folding in vivo and in vitro
can only be related qualitatively (Lunde, C., and J.R. Winther, unpublished observations).
To investigate the importance of the redox potential of
PDI, we randomly mutagenized the two central amino
acid residues in the CGHC motif in both the a and a
domains. The mutants were initially selected for complementation of a chromosomal pdi1 deletion. Sequencing a
number of randomly chosen mutants showed a clear overrepresentation of the helix-breaking residues Gly and Pro
(Table II). This bias is not unreasonable considering the
presence of the CGHC sequence at the beginning of helix
2 of the thioredoxin fold (Fig. 1 A). Apart from this trend,
we do not find any obvious characteristics or similarities
among these sequences. The mutants showed little or no
growth phenotype under normally oxidizing conditions.
The screening procedure, however, would probably implicitly select against mutant forms giving very slow growth
phenotypes. Many of the mutants proved to be extremely
DTT sensitive (Table II). A priori, it might not be obvious
that DTT should inhibit growth of the cells because of its
effect on disulfide bond formation. There could, in principle, be several reasons for the toxic effects of DTT. These
results, however, indicate a direct link between PDI structure/function and growth rate on various concentrations of
DTT, and they imply that toxicity is evoked specifically
through inhibition of the folding of disulfide-bonded
secretory proteins. In accordance with this hypothesis, invertase, a secretory protein without disulfide bonds, is secreted normally in the presence of DTT at concentrations that completely blocked proCPY ER exit (data not shown;
Jämsä et al., 1994
). This suggests that the secretory pathway as such is not perturbed by DTT treatment, but that
the effect is specific to proteins containing disulfide bonds.
By analogy to DsbA, we propose that our DTT-sensitive
PDI mutants are altered in their redox potential (Fig. 4).
The mutants are affected in their rate of CPY folding in
the ER, as demonstrated by the reduced rate of exit from
the ER of CPY in the PDI mutant backgrounds. The reduced folding rate is also seen in a more direct way by the
observation that CPY-3T (Holst et al., 1996) is glycosylated at a buried N-glycosylation site in the PDI mutants
but not in the wild type. Thus, the overglycosylation of the
CPY-3T mutant would strongly suggest that the PDI mutants are affected in early folding events associated with
the introduction of disulfide bonds. The importance of the
redox potential for "PDI function" has also been highlighted in studies by Chivers et al. (1996)
. Here, a signal
peptide was introduced in front of E. coli thioredoxin to
target it to the ER. In such a construct, thioredoxin is not
able to complement a PDI deletion. However, C
C mutants were selected by their ability to complement a pdi1 deletion, and it was found that only mutants with a more
oxidizing redox potential could complement the yeast
PDI1 deletion. This supports the notion that oxidation is
an important, albeit not essential, function of PDI in terms
of supporting yeast growth.
To examine extreme effects on deficiency in oxidative
power, we constructed CGHS mutants that are unable to
form the normal active site disulfide bonds. It is likely that
such mutations would not abolish all PDI activity, since
they are not lethal for yeast and CHGS mutants are still
able to catalyze oxidation reactions by formation of a
mixed disulfide with glutathione (Wunderlich et al., 1995;
Walker et al., 1996
). CPY folding in cells expressing the
CGHS-SGHS and SGHS-CGHS mutants was not significantly slower than in the most DTT-sensitive C
C mutant (e.g., CRRC-SGHS). Nascent proCPY might be oxidized even in the absence of the oxidative power of PDI
by the endogenous GSH-GSSG redox buffer in the ER (Hwang at al., 1992) or by another other PDI homologue.
MPD1, for example, is able to suppress the lethality of a
pdi1 deletion when expressed from a high copy plasmid
(Tachikawa et al., 1995
) or under the control of the PDI1
promoter (Holst, B., and J.R. Winther, unpublished observations). Although this enzyme is not normally produced
at levels that would sustain growth in the absence of PDI,
it may provide a background of oxidizing power seen in the CGHS mutants.
Despite the similarity between the most extreme CC
mutants and the CGHS mutants in the rate of CPY folding, there is a marked difference in the way they affect the
growth rate. This suggests that other substrates, which are
required for optimal growth rates, have a stronger requirement for the oxidative power of PDI. It is somewhat surprising that the CGHS-SGHS mutant grows more slowly than the SGHS-CGHS mutant, since it seems to contradict
the conclusion that the a site is more important for CPY
folding than the a
site. However this may be because the
a
domain is more important for the growth-essential substrate(s) than the a domain.
The yeast PDI-like protein Eug1p does not contain the second cysteine residue of the CXXC motif, yet it is able to sustain viability when overexpressed. EUG1, under the control of the PDI1 promoter, complements a pdi1 deletion, and the growth rate of such cells is comparable to that of a CGHS-CGHS PDI mutant. In view of the current interest in the relevance of PDI activity for yeast survival, we investigated the effect of mutating the CLHS-CIHS active-site sequences of Eug1p "back" to thioredoxin-like structures: CLHC-CIHC. Presumably, wild-type Eug1p can isomerize disulfide bonds, but it can oxidize only by first forming a mixed disulfide with GSH. The altered Eug1p should be able to form an internal active-site disulfide bond, which would greatly facilitate its ability to act as an oxidant. The observation that these mutants, when expressed from the PDI1 promoter, complemented the pdi1 deletion to an almost wild-type growth rate indicates that genuine PDI activity can be achieved by the altered Eug1p. It also underscores the importance of the oxidative function of PDI. The implication that Eug1p is able to adapt a thioredoxin-type mechanism is interesting from both an enzymatic and an evolutionary point of view. Sequencing of the yeast genome has revealed an ancient duplication that is reflected the presence of two homologous copies of most genes. It is likely that EUG1 and PDI1 constitute such a pair since they belong to the same duplication "block" (http://acer.gen.tcd.ie/~hwolfe/yeast/topmenu.html). Also, the apparent absence of Eug1p homologues of nonyeast origin suggests that Eug1p has evolved from PDI fairly recently. This may also explain why the enzyme mechanism can be changed by a single amino acid substitution. The fact that CPY folding occurs as slowly in the Eug1p mutant (not shown) as in a CRRC-SGHS PDI mutant is not surprising since it is probably not very oxidizing. In vitro characterization is in progress to compare biochemical properties of both wild-type and mutant forms of PDI and Eug1p.
Received for publication 18 February 1997 and in revised form 23 June 1997.
Address all correspondence to Dr. Jakob R. Winther, Department of Yeast Genetics, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark. Tel: +45 3327 5282. Fax: +45 3327 4765. E-mail: jrw{at}crc.dkWe wish to thank Anette W. Bruun for excellent technical assistance, as well as Morten C. Kielland-Brandt, Mathilde Lerche, and Vibeke Westphal for critical reading of the manuscript. Nigel Darby is thanked for stimulating discussions and for providing data before publication.
CPY, carboxypeptidase Y; GSSG, glutathione disulfide; PDI, protein disulfide isomerase.
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