(Received for publication, August 31, 1995; and in revised form, September 27, 1995 )
From the
Protein-disulfide isomerase (PDI) is an abundant protein of the
endoplasmic reticulum that catalyzes dithiol oxidation and disulfide
bond reduction and isomerization using the active site CGHC. Haploid pdi1 Saccharomyces cerevisiae are inviable, but can be
complemented with either a wild-type rat PDI gene or a mutant
gene coding for CGHS PDI (shufflease). In contrast, pdi1
yeast cannot be complemented with a gene coding for SGHC PDI. In vitro, shufflease is an efficient catalyst for the
isomerization of existing disulfide bonds but not for dithiol oxidation
or disulfide bond reduction. SGHC PDI catalyzes none of these
processes. These results indicate that in vivo protein folding
pathways contain intermediates with non-native disulfide bonds, and
that the essential role of PDI is to unscramble these intermediates.
Protein-disulfide isomerase (PDI; ()EC 5.3.4.1)
constitutes approximately 2% of the protein in the endoplasmic
reticulum (ER). PDI has been shown to catalyze the in vitro oxidation of protein sulfhydryl groups and reduction and
isomerization of protein disulfide bonds (1, 2; Fig. 1). The
products of catalysis by PDI depend on the dithiol/disulfide reduction
potential of the substrate and the solution. The enzyme itself can
exist in either a reduced or oxidized state or as a mixed disulfide
with a substrate. Although PDI is the most efficient known catalyst of
oxidative protein folding(3, 4) , it also participates
in cellular processes that do not exploit its enzymatic
activity(5, 6) .
Figure 1: Reactions catalyzed by PDI in vitro. PDI catalyzes the oxidation of dithiols and reduction of disulfide bonds (top) and the isomerization of disulfide bonds (bottom). In this work, the substrate for dithiol oxidation was reduced RNase A, that for disulfide reduction was insulin, and that for disulfide bond isomerization was scrambled RNase A.
Mature PDI from rat contains two active sites with the sequence WCGHCK (7) . The C terminus of rat PDI ends with the sequence KDEL, the signal for retention of proteins in the mammalian ER(8) . PDI also has at least one site that can bind to peptides(9, 10) . The amino acid sequence of Saccharomyces cerevisiae PDI is approximately 30% identical with that of rat PDI, and the regions containing the active sites are conserved completely(11, 12, 13, 14) . Mature PDI from S. cerevisiae contains five putative N-glycosylation sites and a C terminus ending with HDEL, the S. cerevisiae equivalent of KDEL(13, 15) .
The role of PDI in vivo is unclear. In S.
cerevisiae, pdi1 mutants are
inviable(11, 12, 13, 14) . In E.
coli, the PDI analog dsbC is necessary for the
formation of native disulfide bonds in many periplasmic
proteins(16) . Studies based on the complementation of pdi1
S. cerevisiae have provided some clues as to the
role of PDI. Tachibana and Stevens (17) showed that the
overexpression of EUG1, which codes for an ER protein with
active-site sequences WCLHSQ and WCIHSK, allows pdi1
cells to grow. Also, LaMantia and Lennarz (18) found that pdi1
cells can be rescued by a mutant PDI that cannot
catalyze dithiol oxidation.
To determine why the PDI gene
is essential for the growth of S. cerevisiae, we have mutated
a cDNA that codes for PDI and have tested the ability of the resulting
mutant proteins to support the growth of pdi1 S.
cerevisiae. These results, coupled with in vitro analyses
of catalysis, demonstrate that the essential role of PDI is not related
to the net formation of protein disulfide bonds. Rather, the role of
PDI is to act as a ``shufflease,'' a catalyst of the
isomerization of existing disulfide bonds (Fig. 1, bottom).
Yeast cells were grown at 30 °C in rich or defined medium containing glucose (1% w/v) prepared as described(20) . 5-Fluoroorotic acid (5-FOA) addition was as described(21) . Visible absorbance measurements were made on a Cary 3 spectrophotometer thermostatted with a Cary temperature controller. Samples were diluted to read A = 0.1-0.5 at 600 nm. Yeast were transformed as described(22) .
The NheI/XmaIII fragment of pMAL3.1 was inserted into pMAL10 that had been digested with NheI and XmaIII, to yield plasmid pMAL12. The codons for cysteine residues in the second active site of the PDI cDNA in pMAL12 were changed to those for a serine residue using oligonucleotide ML13: ATGCTCCCTGG(G/T)CCGGACACTGCAAGCAGCTAGCGCTAGAAAGCTT or ML14: TCCTGGTGTGGGCAT(G/T)CGAAGCAGCTAGCGCTAGAAAGCTT. The regions in the resulting plasmids that code for PDI were amplified by using the polymerase chain reaction. The amplified fragments were digested with NheI and XmaIII and ligated to the NheI/XmaIII fragment of pMAL112 and pMAL122 generating plasmids pMAL312 (C35S/C379S PDI) and pMAL322 (C38S/C382S PDI).
To discern whether the C-terminal HDEL sequence of S. cerevisiae PDI is important for its function in vivo, we replaced the C-terminal KDEL sequence of C35S/C379S and C38S/C382S PDI with HDEL. The NcoI/NheI fragments of pMAL312 and pMAL322 were inserted into pMAL5.1 (19) that had been digested with NcoI and XmaIII, to yield plasmids pMAL512 (C35S/C379S/K486H) and pMAL522 (C38S/C382S/K486H).
Catalysis of disulfide reduction was assayed by monitoring the reduction of porcine insulin (Sigma) in the presence of GSH, as described(27) .
Catalysis of disulfide isomerization was assayed by monitoring the regain in activity of scrambled RNase A (Sigma), which has non-native disulfide bonds, as described(19) .
Figure 2:
Complementation of pdi1 S.
cerevisiae. Haploid cells carrying a URA3 plasmid that
directs the production of S. cerevisiae PDI were transformed
with a TRP1 plasmid that directs the production of a test PDI.
Transformants (5 for each plasmid, 1 is shown and the other 4 gave the
same result) were cultured, and then grown for 4 days on solid
tryptophan dropout medium (top) or solid medium containing
5-FOA (bottom), which selects for cells that have lost the URA3 plasmid. Row 1, SGHC PDI; row 2, CGHS
PDI; row 3, SGHC PDI (HDEL); row 4, CGHS PDI (HDEL); row 5, S. cerevisiae PDI.
Immunoblots of membrane fractions extracted before plasmid shuffling
showed that rat PDI was present in all transformants (Fig. 3A). Immunoblots of membrane fractions from cells
that complemented the pdi1 deficiency showed that rat PDI
was also present in these cells (Fig. 3B). Immunoblots
probed with antibodies to S. cerevisiae PDI show that this
protein is absent from the rat PDI complemented cells (data not shown).
Figure 3:
Membrane-bound protein in complemented pdi1 S. cerevisiae. Immunoblots were probed with
antibodies to bovine PDI. A, before plasmid shuffling. Lane 1, SGHC PDI; lane 2, CGHS PDI; lane 3,
SGHC PDI (HDEL); lane 4, CGHS PDI (HDEL); lane 5, S. cerevisiae PDI. B, after plasmid shuffling. Lane 1, S. cerevisiae PDI; lane 2, empty; lane 3, CGHS PDI; lane 4, CGHS PDI
(HDEL).
The abilities of wild-type and mutant PDIs to support the growth of pdi1 S. cerevisiae is shown in Table 1. Cells
complemented with PDI containing a C-terminal KDEL or HDEL sequence had
indistinguishable doubling times (data not shown). The data described
hereafter were obtained from the KDEL constructs.
Here, we have expanded the assessment of PDI mutants to include all three enzymatic assays on wild-type PDI and the two relevant mutant enzymes. Our results are listed in Table 1, and the results of PDI assays are summarized in Table 2. Briefly, we found that wild-type rat PDI had dithiol oxidation activity (measured by an increase in activity of reduced RNase A) comparable to that of PDI isolated from bovine liver. SGHC and CGHS PDI had negligible dithiol oxidation activity. Wild-type rat PDI had disulfide reduction activity (measured by the cleavage of porcine insulin) comparable to that of PDI isolated from bovine liver. SGHC PDI and CGHS PDI had negligible disulfide reduction activity. Wild-type and CHGS rat PDI had isomerization activity (measured by an increase in activity of scrambled RNase A) comparable to PDI from bovine liver. SGHC PDI had negligible isomerization activity.
Early work on PDI suggested that its cysteine residues were
essential for its enzymatic activity. Carboxymethylation or
carbamoylmethylation caused irreversible
inactivation(29, 30) . In addition, PDI was shown to
be inhibited by arsenite or Cd, behavior diagnostic
of enzymes with active site dithiol groups(31, 32) .
Thus, a cysteine residue was suspected to be responsible for the
enzymatic activity of PDI.
In 1991, pdi1 S. cerevisiae cells were shown to be inviable(13) . Still, the question
remained: What cellular process is impaired by the absence of PDI? PDI
catalyzes dithiol oxidation and disulfide bond reduction and
isomerization(1) . On the other hand, PDI can bind to peptides (9, 10) and is part of cellular complexes in which the
role of its enzymatic activity has not been
explored(5, 6) . These data have lead many to suggest
that the essential role of PDI is unrelated to its enzymatic
activities(18, 33, 34, 35, 36, 37) .
To illuminate the cellular process that is impaired by the absence
of PDI, we have mutated each cysteine residue in its active site and
studied the resultant proteins in vitro and in vivo.
Since each PDI monomer has two CGHC active sites, our two mutant
proteins are actually double mutants in which either the first or
second cysteine residue in each active site is replaced by a serine. We
find that CGHS PDI and wild-type PDI are able to complement a pdi1 strain of S. cerevisiae (Table 1). In
contrast, SGHC PDI is unable to compensate for this deficiency.
Replacing two sulfur atoms with oxygen atoms is unlikely to have a significant impact on the ability of PDI to bind to peptides or to otherwise act in nonenzymic roles. The mutations do, however, have a significant effect on catalysis by PDI. The results of in vitro PDI assays show that CGHS PDI catalyzes the shuffling of disulfide bonds with efficiencies comparable to that of the wild-type enzyme (Table 1). But, unlike the wild-type enzyme, CGHS PDI does not catalyze the oxidation of dithiols or the reduction of disulfide bonds. Apparently, the ability of PDI to form an intramolecular disulfide bond is necessary for it to catalyze the oxidation or reduction of a substrate efficiently. SGHC PDI catalyzes none of these three processes. Thus, the essential function of PDI is enzymic, but does not relate to the net formation of disulfide protein bonds. Rather, the role of PDI in vivo is to act as a shufflease (Fig. 1, bottom).
Our results and those of others expose the
critical functional group in the CXXC motif of PDI. The S.
cerevisiae EUG1 gene complements pdi1 S. cerevisiae.
Each active site of Eug1p (WCLHSQ and WCIHSK) contains only a single
cysteine residue (17) . Wild-type Eug1p is therefore analogous
to the shufflease mutant of PDI. Thioredoxin (Trx) catalyzes disulfide
bond reduction in the cytosol of eukaryotes and prokaryotes in the
active site: WCGPCK(38) . Although the three-dimensional
structure of PDI is unknown, that of E. coli Trx has been
determined by both x-ray diffraction analysis and NMR
spectroscopy(39, 40) . In the Trx structure, the most
pronounced deviation from an almost spherical surface is a protrusion
formed by residues 29-37, which includes the active site. The
sulfhydryl group of Cys-32, which has a low pK
, is
exposed to the solvent while that of Cys-35 is recessed. The results of
chemical modification studies and pK
determinations on PDI are parallel to those on Trx(41) ,
suggesting that the reactivity of the active sites is similar. In
addition, PDI is a substrate for thioredoxin reductase, which suggests
that the three-dimensional structures of the active sites are similar.
Recently, we demonstrated that CGPS Trx but not SGPC Trx can complement pdi1
S. cerevisiae.
Thus, the essential
functional group in the CXXC motif is the sulfhydryl group of
the N-terminal cysteine residue.
If a CXXS sequence can replace the CXXC motif, why does PDI have a CXXC motif? A CXXC motif could be less susceptible to inactivation by adventitious oxidation to a hindered mixed disulfide or a sulfenic acid (S-OH) because it can escape by forming an intramolecular disulfide bond. In addition, having CXXC and CXXS motifs from endogenous PDI and Eug1p, respectively, could provide cells with a selective advantage.
Catalysis of dithiol oxidation or disulfide bond reduction depends on the redox environment(42, 43) . In contrast, during catalysis of disulfide bond isomerization, the substrate does not undergo a net change in oxidation state (Fig. 1). The simplest mechanism for catalysis of an isomerization reaction begins with the attack of a thiolate ion on a protein disulfide, forming a mixed disulfide(44) . Then, the protein thiolate produced can attack another protein disulfide bond. Finally, the resulting thiolate can attack the mixed disulfide to release the catalyst, unaltered. Such an isomerization reaction would be driven by the search for the most stable conformation of the substrate protein.
Much evidence suggests that dithiol oxidation is random during the early stages of protein folding(45, 46) . Classic studies on the oxidative folding of reduced bovine protease trypsin inhibitor suggest that non-native intermediates accumulate during the folding process (47) . In contrast, recent work argues that the well-populated intermediates contain only native disulfide bonds(48) . Still, to reach the final conformation, these intermediates must rearrange by forming species with non-native disulfide bonds. PDI has been shown to catalyze this process by rescuing kinetically trapped intermediates(49) . Thus, PDI activity may be required either in a normal protein folding pathway or for rescuing proteins that have become misfolded or aggregated. Our results link the disulfide bond isomerization activity of PDI with cell viability. Thus, as proposed by Anfinsen (50) more than 30 years ago, the essential function of PDI is to isomerize non-native disulfide bonds, to be a shufflease.