From the
The microsomal triglyceride transfer protein (MTP) is a
heterodimer composed of the multifunctional enzyme, protein
disulfide-isomerase, and a unique large, 97 kDa, subunit. It is found
as a soluble protein within the lumen of the endoplasmic reticulum of
liver and intestine and is required for the assembly of very low
density lipoproteins and chylomicrons. Mutations in MTP which result in
an absence of MTP function have been shown to cause
abetalipoproteinemia. Here, the gene encoding the MTP 97-kDa subunit of
an abetalipoproteinemic subject, which we have previously demonstrated
lacks MTP activity and protein (Wetterau, J. R., Aggerbeck, L. P.,
Bouma, M.-E., Eisenberg, C., Munck, A., Hermier, M., Schmitz, J., Gay,
G., Rader, D. J., and Gregg, R. E.(1992) Science 258,
999-1001), was isolated and sequenced. A nonsense mutation, which
predicts the truncation of the protein by 30 amino acids, was
identified. To investigate if this apparently subtle change in MTP
could explain the observed absence of MTP, protein disulfide-isomerase
was co-expressed with either the normal or mutant MTP 97-kDa subunit in
Sf9 insect cells using a baculovirus expression system. Although there
were high levels of expression of both the normal and mutant forms of
the MTP 97-kDA subunit, only the normal subunit was able to form a
stable, soluble complex with protein disulfide-isomerase. These results
indicate that the carboxyl-terminal 30 amino acids of the MTP 97-kDa
subunit plays an important role in its interaction with protein
disulfide-isomerase.
The microsomal triglyceride transfer protein (MTP)
Lipid binding studies and kinetic analysis of MTP-catalyzed
transport of lipid between membranes indicated that MTP binds and
shuttles lipid molecules between membranes(10) . The amino acid
sequence of both components of the MTP complex have been deduced from
cDNA sequences(3, 11) . However, the structural features
of the two components of MTP which are important for the formation of
the protein complex, MTP-membrane interaction, and the lipid molecule
binding properties of MTP, are not known.
Here, the identification
of a nonsense mutation in the 97-kDa subunit of MTP from a subject with
abetalipoproteinemia is reported. It has previously been shown that
intestinal biopsies from this subject lack MTP activity and
protein(4) . The predicted translation product of the gene is
truncated by 30 amino acids. Characterization of this mutant expressed
in Sf9 insect cells demonstrate that the mutant protein is not able to
form a stable, soluble complex with PDI, indicating that the
carboxyl-terminal 30 amino acids of the MTP 97-kDa subunit are required
for the formation of a normal MTP complex. In addition to the
implications regarding PDI-97-kDa subunit interactions, these findings
also have important implications for future studies of
structure-activity relationships in the microsomal triglyceride
transfer protein.
The mutant 97-kDa subunit cDNA construct,
pVL-MTP864, containing a G to T nucleotide change at bp 2593 of the
cDNA, was prepared by PCR-directed mutagenesis (15) of the
normal cDNA and subcloned into pVL1393 as described above for
pVL-MTP894.
The baculovirus transfer vector, pVL-PDI, was
constructed from a cDNA for PDI obtained by a reverse trancriptase
reaction with mRNA from HepG2 cells, followed by PCR amplification of
the reaction product utilizing primers designed from cDNA sequence
published previously (11). SmaI restriction sites were
incorporated into the primers used to amplify the 5` and 3` ends.
Overlapping PCR products were ligated to produce the full-length cDNA.
The product, containing 17 bp of the 5`-untranslated sequence, the
whole coding region, and 29 bp of the 3`-untranslated sequence was
subcloned into pUC18 by blunt end ligation into the SmaI site
of the vector. The cDNA sequence was verified by direct sequencing of
the PCR product and then excised from pUC18 with BamHI and EcoRI. The fragment was then ligated into the BamHI-EcoRI sites of pVL1393.
Following centrifugation, the
supernatant containing the soluble MTP was dialyzed against 0.015 M Tris, pH 7.4, 0.04 M NaCl, 0.001 M EDTA, 0.02%
NaN
DNA from an abetalipoproteinemic subject was isolated and
used to construct a genomic library. We had previously demonstrated
that intestinal biopsies from this subject had no detectable MTP
activity or protein. Genomic clones containing inserts encoding exons
1-3 and 5-18 of the gene for the 97-kDa subunit of MTP were
isolated and characterized. Exon 4 cDNA was obtained by PCR
amplification of the isolated DNA. The entire coding region, the
intron-exon boundaries, and 250 bp upstream of the transcription start
site were sequenced. Studies characterizing the MTP gene promoter have
indicated that the first 250 bp upstream of the transcription start
site direct the correct cell type specific expression of reporter
genes(16) . The entire sequence agreed with that of the normal
gene(13) , with the exception of a G to T mutation at a base
corresponding to base 2593 downstream from the translational start site
of the cDNA. This mutation changes the Gly
The mutation at bp 2593 creates a new NlaIII restriction site in the DNA sequence. To further
characterize the defect in this subject, a NlaIII digest was
performed on a 343-bp PCR-amplified fragment of genomic DNA surrounding
the mutation. As illustrated in Fig. 1, control DNA produced a
single band indicating the absence of a NlaIII site in exon
18, while the DNA from the abetalipoproteinemic subject was digested
completely into two fragments of 192 and 151 bp. This suggests that
both copies of the MTP 97-kDa subunit gene of the abetalipoproteinemic
subject contain the G to T mutation. However, the possibility of a
deletion in exon 18 of one of the copies of the gene has not been
formally excluded.
Following a 48-h co-infection in 60-mm dishes, the cells were
harvested, and aliquots of the media and total cells removed. The
remaining cells were pelleted, washed, homogenized, treated with
deoxycholate to ensure release of soluble MTP protein from the
microsomal fraction, and centrifuged at 100,000
As a
control, immunoblot analysis of soluble protein present in the media
was performed to exclude the possibility that a portion of the mutant
97-kDa subunit, which had not formed a complex with PDI, was secreted
from the cells. The 97-kDa subunit was not present at detectable levels
in the media from cells infected with viruses expressing either MTP
subunit alone or from cells co-infected with viruses expressing the
mutant large subunit and PDI. A minor amount of soluble 97-kDa protein
was present in the media from cells co-infected with viruses expressing
the normal 97-kDa subunit and PDI (data not shown), suggesting that
normal retention mechanisms of the endoplasmic reticulum were not able
to retain the high levels of expressed protein.
Analysis of naturally occurring mutations has been a
productive approach for identifying important structure-function
relationships in proteins. MTP mutations associated with
abetalipoproteinemia reported to date (3, 5, 6) all predict severe abnormalities in
the 97-kDa subunit of MTP and thus do little to elucidate the
structure-function relationships in MTP. Here we report the
identification of a nonsense mutation in the 97-kDa subunit of an
abetalipoproteinemic subject which predicts an MTP large subunit
truncated by only 30 amino acids. Previously, it has been demonstrated
that this subject lacks MTP activity and that the 97-kDa subunit is
undetectable in either a soluble or total cell protein fraction from an
intestinal biopsy obtained from this subject(4) . Whether this
apparently subtle change in MTP could explain the absence of MTP
protein in this subject is not obvious.
To investigate if the
nonsense mutation could result in a complete absence of MTP in
intestinal biopsies from the abetalipoproteinemic subject, PDI was
co-expressed with either the normal MTP 97-kDa subunit or the mutant
subunit in Sf9 insect cells using a baculovirus expression system. Sf9
cells have endogenous PDI activity(17) , however the protein
that expresses this activity did not form an active transfer protein
complex when the cells were infected with the 97-kDa subunit only.
Although the anti-bovine PDI antibody used to detect PDI by immunoblot
analysis in this study cross-reacts with mouse and human PDI, there was
no detectable signal from Sf9 cells not infected with heterologous PDI (Fig. 4, lane1). This suggests that there is
not a high degree of homology between mammalian and the insect cell
proteins that express PDI activity. Thus MTP expression in Sf9 cells is
dependent on co-expression of the MTP large subunit and PDI.
High
levels of PDI and the MTP 97-kDa subunit (mutant or normal) were
expressed following co-infections. When the activity and MTP protein in
a soluble protein extract from the Sf9 cells were compared, MTP
activity and 97-kDa subunit were detected in the soluble fraction
obtained from cells expressing the normal protein, but neither was
detected in the soluble fraction from the cells expressing the mutant,
although clearly both MTP subunits were available to form a complex.
These results suggest that the mutant protein was unable to form a
normal complex with PDI.
The absence of detectable MTP 97-kDa
subunit in intestinal biopsies from the abetalipoproteinemic subject
most likely is a result of degradation of the mutant MTP 97-kDa
subunit, which is unable to form a complex with PDI. Proteins that are
folded incorrectly, or subunits of an oligomeric protein that are not
properly assembled into a protein complex, are diverted to a
degradation pathway(18) . Malfolded or unassembled proteins
often form transient soluble complexes with a chaperone protein prior
to degradation(19) . Using the baculovirus expression system,
the high levels of expression may have exceeded the capacity of the Sf9
cells to either degrade the MTP 97-kDa subunit or to form a soluble
complex between it and a chaperone protein. Previous biochemical
analysis of MTP has indicated that the 97-kDa subunit of MTP forms an
insoluble aggregate unless it is associated with PDI(20) . Thus,
finding the mutant subunit in an insoluble form in the total cell
extract, but not in a soluble protein extract, is expected when the
mutant protein is unable to form a complex with PDI.
The possibility
that the mutant subunit forms a complex with PDI which is subsequently
rapidly degraded and thus not detected, cannot be fully excluded.
However, the similar levels of normal and mutant large subunits
following co-expression with PDI, the absence of detectable mutant
97-kDa subunit degradation products by immunoblot analysis, the
apparent recovery of all of the mutant large subunit protein in the
100,000
These findings
predict that mutations in the PDI subunit of MTP which affect its
ability to form a complex with the 97-kDa subunit, would also cause
abetalipoproteinemia. Although 11 different defective 97-kDa alleles
have been reported for abetalipoproteinemic
subjects(3, 5, 6) , a mutation in PDI that
causes abetalipoproteinemia has not been described to date. This
indicates that the region of PDI that interacts with the 97-kDa subunit
of MTP may have a critical function independent of its role in MTP.
The results of this study have important implications regarding
future structure-function analysis of MTP. Carboxyl-terminal deletion
analysis is a common approach to investigate the functional role of
various regions of a protein. However, this study demonstrates that
deletions as short as 30 amino acids at the carboxyl terminus of the
large subunit interrupt its interaction with PDI and that a fully
translated 97-kDa subunit may be required for complex formation.
Alternative strategies, which utilize assembled protein, will be
required to elucidate the functional roles of the carboxyl-terminal
portions of the MTP large subunit.
We thank the University of Cincinnati for providing
the antibodies used in this study.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)is a heterodimer composed of protein
disulfide-isomerase (PDI) and a unique large, 97 kDa, subunit that
confers lipid transfer activity to the
complex(1, 2, 3) . Studies of MTP in subjects
with abetalipoproteinemia, a rare autosomal recessive disease
characterized as a defect in the assembly of plasma lipoproteins that
contain apolipoprotein B (apoB), indicated that an absence of MTP
function is a cause of
abetalipoproteinemia(3, 4, 5, 6) . These
findings demonstrated that MTP is required for the assembly of very low
density lipoproteins in the liver and chylomicrons in the intestine.
Co-expression of MTP and truncated forms of apoB in cell lines which
normally do not secrete apoB-containing lipoprotein particles (HeLa and
COS cells), further demonstrated that MTP expression is sufficient to
allow nonhepatic, nonintestinal cell lines to efficiently synthesize
and secrete apoB particles with densities similar to those secreted
from hepatic-derived cell lines(7, 8, 9) .
Isolation and Characterization of Genomic
Clones
Genomic DNA was isolated from the blood of a 52-year-old
male abetalipoproteinemic patient. The clinical profile of this
individual has been described previously(12) , and we recently
demonstrated that MTP activity and protein are not detectable in
intestinal biopsies obtained from this subject(4) . The DNA was
used to construct a DASH genomic library by Stratagene. Two
million recombinant phage plaques from the library were screened for
genomic sequences homologous to the 97-kDa subunit of MTP with
radiolabeled bovine and human MTP cDNA as described
previously(13) . Twelve phage inserts were isolated and
characterized by hybridization to cDNA fragments encoding exons
1-18. Overlapping phage inserts containing all exons, except exon
4, were identified and sequenced directly as described
previously(13) . The sequence for exon 4 was obtained following
a polymerase chain reaction (PCR) amplification of genomic DNA
corresponding to exon 4 and subcloning of the PCR product into pUC18.
Analytical Digest Analysis of Mutant DNA
Genomic
DNA was prepared from blood from a control subject and from the
abetalipoproteinemic subject. A 343-bp genomic fragment surrounding bp
2593 of the cDNA (corresponding to bp 111 5` of the intron-exon
junction for exon 18 through bp 2745 of the cDNA) was amplified by PCR
from the genomic DNA of the abetalipoproteinemic and normal control
subject. Forward (5`-CTCAGATTGCTTTGGAAC-3`) and reverse
(5`-TCATGCCACATTGTGTCC-3`) primers were designed based on sequence
obtained from human genomic clones(13) . Equal aliquots of the
PCR reaction products were removed from both samples, and half of each
aliquot was digested with NlaIII. Digested and undigested DNA
from each subject was loaded into separate wells, electrophoresed
through a 1.4% agarose gel, and visualized by ethidium bromide
staining.
Construction of the Baculovirus Transfer Vectors,
pVL-MTP894, pVL-MTP864, and pVL-PDI
The baculovirus transfer
vector pVL-MTP894, encoding the 97-kDa large subunit, was generated by
PCR amplification of overlapping fragments of a human 97-kDa subunit
cDNA described previously(3) . The 5` most primer contained a BamHI site followed by sequence -3 to +18 of the
cDNA, and the 3`-most primer contained sequence +2667 to
+2686 of the cDNA followed by an XhoI site. The
overlapping PCR fragments were ligated, and the ligated product was
subcloned into the BamHI-XhoI sites of the
pBluescript vector. The cDNA sequence was verified by sequencing of
this pBluescript subclone. The pBluescript subclone insert was cut at
the 3` end with XhoI. A fill-in reaction with T4 DNA
polymerase (14) was performed in the presence of 0.02 M nucleotides to create a blunt end at the XhoI site for
ligation into the SmaI site of the baculovirus transfer
vector. The subclone was then digested with BamHI to remove
the full-length cDNA from pBluescript. The cDNA was subcloned into the BamHI-SmaI sites of the baculovirus transfer vector
pVL1393 (Invitrogen Corp.) to produce a final cDNA containing 3 bp of
the 5`-untranslated sequence, the entire coding region, and 7 bp of the
3`-untranslated sequence.
Transfection and Isolation of Recombinant
Viruses
Recombinant baculovirus transfer vectors were
co-transfected into Sf9 cells with wild-type Autographa californica nuclear polyhedrosis virus DNA by cationic liposome-mediated
transfection. Methods for transfection; screening for recombinant
viruses VL-MTP894, VL-MTP864, and VL-PDI; isolation of plaque purified
viruses; and the production of large scale, high-titer stocks are
described in the manufacturer's instructions (Invitrogen Corp.)
supplied with the MaxBac kit, product K822-03.
Expression of Recombinant Proteins in Sf9
Cells
Sf9 cells were cultured at 27 °C in ExCell 401
serum-free media (JRH Biosciences) in suspension in Erlenmeyer or
spinner flasks. Alternatively, they were cultured as monolayers in
60-mm dishes in 1 TNM-FH media supplemented with 10% fetal
bovine serum (Life Technologies, Inc.). To produce recombinant
proteins, Sf9 cells were seeded at a density of 2.0
10
cells/ml in suspension culture or 2.5
10
cells/60-mm dish. For expression studies, the cells were infected
at a multiplicity of infection of 5 with recombinant viruses VL-MTP894
and VL-MTP864, or 50 for VL-PDI. The same multiplicity of infections
were used for co-infections. Preliminary experiments were performed in
suspension culture to optimize the expression with respect to time
course, multiplicity of infection, and the ratio of viruses expressing
PDI and MTP 97-kDa subunit.
Analysis of Recombinant Proteins in Sf9 Cells
The
cells were harvested 48 h after infection in 60-mm dishes, and aliquots
of the soluble media and the total cell protein were taken for analysis
of MTP protein as described below. The remaining cells were washed
twice with phosphate-buffered saline and homogenized with a Polytron in
0.05 M Tris, pH 7.4, 0.05 M KCl, 0.005 M EDTA, 0.002 M phenylmethylsulfonyl fluoride, 0.2 mM leupeptin. One-half volume of 0.54% deoxycholate, pH 7.5, was
added to the homogenate to release MTP from the microsomes. The
suspension was incubated on ice for 30 min and centrifuged for 1 h at
100,000 g.
and assayed for MTP activity by measuring the rate of
radiolabeled triglyceride (TG) transfer from donor to acceptor egg
phosphatidylcholine small unilamellar vesicles as a function of cell
protein as described previously(4) . Aliquots of total cell
protein, the pellets from the 100,000
g centrifugation, and the supernatant from the 100,000
g centrifugation were solubilized in 2% sodium dodecyl sulfate,
heated at 95 °C for 5 min and fractionated by SDS, 8%
polyacrylamide gel electrophoresis. Samples were immunoblotted with
rabbit anti-bovine 97 kDa or anti-bovine PDI antibodies as described
previously(4) . Horseradish peroxidase-conjugated secondary
antibody and a colorimetric reaction were used to detect immunoreactive
proteins.
codon in exon
18 of the gene to a stop codon. The predicted translation product is
truncated by 30 amino acids. This mutation was found on three
independent DNA inserts.
Figure 1:
Analytical digest analysis of the
nonsense mutation. A 343-bp fragment of exon 18 surrounding bp 2593 of
the cDNA was amplified by PCR from the genomic DNA of a control subject
and the abetalipoproteinemic subject. Half of each sample was digested
with NlaIII. The digested and remaining undigested DNA from
the control and abetalipoproteinemic subject was electrophoresed in a
1.4% agarose gel. The relative positions of molecular weight standards
(in kilobases) are shown in the leftmargin. Lanes1 and 2 are the 343-bp PCR fragment
from the control subject, undigested and NlaIII-digested,
respectively. Lanes3 and 4 are the
corresponding fragment from the abetalipoproteinemic subject,
undigested and NlaIII-digested, respectively. The presence of
the G to T nucleotide base change in both copies of the MTP gene of the
abetalipoproteinemic subject is indicated by complete digestion of the
fragment with NlaIII into two smaller fragments of 192 and 151
bp.
To determine whether this nonsense mutation,
which predicts a protein truncated by 30 amino acids, could explain the
absence of MTP activity and protein in intestinal biopsies, PDI was
co-expressed with either the normal or mutant 97-kDa subunit using a
baculovirus expression system. Preliminary experiments in suspension
culture were used to optimize the expression conditions. In control
experiments, MTP activity was not measurable in those cells infected
with viruses expressing the normal 97-kDa subunit or PDI alone (data
not shown), demonstrating the requirement of both subunits for MTP
activity. Recombinant viruses were then used to co-infect Sf9 cells in
monolayer culture with either the normal 97-kDa subunit and PDI
constructs or the mutant 97-kDa subunit and PDI constructs.
g for
1 h. Following dialysis, MTP activity in the 100,000
g supernatant was assayed by measuring the rate of
protein-stimulated transport of radiolabeled triglyceride from donor to
acceptor small unilamellar vesicles. As shown in Fig. 2,
MTP-catalyzed TG transfer was readily detected in the supernatant from
cells co-infected with viruses expressing the normal 97-kDa subunit
(VL-MTP894) and PDI (VL-PDI). No activity was measurable in supernatant
from cells co-infected with viruses expressing the mutant 97-kDa
subunit (VL-MTP864) and PDI, even at protein concentrations that
yielded almost 30% transfer in the co-infection with the normal 97-kDa
subunit.
Figure 2:
Baculovirus expression of the normal
97-kDa subunit-PDI complex and the mutant 97-kDa subunit-PDI complex.
Sf9 cells were co-infected with either VL-MTP894 and VL-PDI or
VL-MTP864 and VL-PDI as described under ``Materials and
Methods.'' Protein from the 100,000 g supernatant
fraction following deoxycholate treatment of the cells was assayed for
TG transfer activity. Activity is expressed as the percent of
C-radiolabeled triglyceride transferred from donor
vesicles to acceptor vesicles in a 1-h assay as a function of
protein.
To investigate why MTP activity was not detected in Sf9
cells co-infected with viruses expressing the mutant 97-kDa subunit and
PDI, aliquots representing 2.5% of either the soluble media protein,
total cell protein, 100,000 g supernatant, or 100,000
g cell pellet protein were fractionated by
SDS-polyacrylamide gel electrophoresis and immunoblotted with anti-97
kDa or anti-PDI antibodies ( Fig. 3and 4). Analysis of total cell
protein revealed similar levels of expression of the large subunit from
cells co-infected with viruses expressing either the normal or mutant
97-kDa subunits and PDI (Fig. 3A, lanes2 and 4, respectively). Higher levels of expression of the
97-kDa subunit were observed in cells infected with viruses expressing
the normal subunit alone (Fig. 3A, lane1). This was likely a result of increased efficiency by
the cells for production of a single heterologous protein. As expected,
the mutant 97-kDa subunit ran slightly below that of the normal,
indicating expression of a truncated protein.
Figure 3:
Immunoblot analysis of MTP expressed in
Sf9 cells. Aliquots of the total cell protein, the 100,000 g pellet, and 100,000
g supernatant (which
contains soluble MTP released from the microsomal fraction after
treatment of homogenized Sf9 cells with deoxycholate) were subjected to
SDS-polyacrylamide gel electrophoresis and immunoblotted with rabbit
antibodies to the 97-kDa subunit of MTP. In all panels, lane1 contains protein from Sf9 cells infected with the
VL-MTP894 construct alone, lane2 contains protein
from Sf9 cells co-infected with VL-MTP894+VL-PDI, lane3 contains protein from Sf9 cells infected with the
VL-PDI construct alone, and lane4 contains protein
from Sf9 cells co-infected with the mutant MTP construct,
VL-MTP864+VL-PDI. A, 2.5% of the total cell
protein; B, 2.5% of the total 100,000
g supernatant protein; C, 2.5% of the total 100,000
g pellet protein. The mobility of the 97-kDa subunit of the
MTP complex is indicated to the left of the figure. The
relative positions of molecular mass standards (in kDa) are shown to
the right of the figure.
In the 100,000 g supernatant, normal soluble 97-kDa subunit protein was
present at high levels from cells co-infected with this construct and
PDI (Fig. 3B, lane2). In contrast,
the mutant subunit was not detected in the soluble fraction from cells
co-infected with viruses expressing the mutant construct and PDI (Fig. 3B, lane4), nor was the normal
subunit detected in cells infected with viruses expressing the normal
construct alone (Fig. 3B, lane1). In
the 100,000
g cell pellet, representing protein from
the nonsoluble cellular fraction, the 97-kDa subunit was present in
both the cells infected with viruses expressing the normal 97-kDa
subunit only and in the cells co-infected with viruses expressing the
mutant 97-kDa subunit and PDI (Fig. 3C, lanes1 and 4, respectively). However, the 97-kDa
subunit was negligible in the pellet from cells co-infected with
viruses expressing the normal 97-kDa subunit and PDI (Fig. 3C, lane2). The nonsoluble
97-kDa subunit found in the 100,000
g pellet from
cells infected with normal 97 kDa or co-infected with PDI and mutant 97
kDa (Fig. 3C, lanes1 and 4,
respectively) appears to represent all of the 97 kDa produced by the
cells (see Fig. 3A, lanes1 and 4). Immunoblots with anti-PDI antibody demonstrated similar
levels of expression of PDI in both the total cell protein (data not
shown) and the 100,000
g supernatant from both normal
and mutant co-infections (Fig. 4, lanes2 and 4, respectively).
Figure 4:
Immunoblot analysis of PDI expressed in
Sf9 cells. Aliquots of 100,000 g supernatant protein
were subjected to SDS-polyacrylamide gel electrophoresis and
immunoblotted with rabbit antibodies to PDI. Lanes1-4 contain protein from Sf9 cells infected as
described for Fig. 3. The relative positions of molecular mass
standards (in kDa) are shown to the right of the
figure.
The complete absence of the mutant 97-kDa
subunit in the supernatant from cells co-infected with viruses for this
construct and PDI and its presence in the 100,000 g pellet suggests that despite the availability of both subunits for
complex formation, the expressed mutant MTP large subunit does not form
a complex with PDI. As a result, it aggregates within the endoplasmic
reticulum and cannot be released in a soluble form utilizing the
deoxycholate method effective for the normal protein. This result is
identical to those observed in cells expressing the normal large
subunit only, where PDI is unavailable for complex formation.
g cell pellet, and the known insoluble nature
of the MTP 97-kDa subunit (20) provides strong support that the
defect is in the formation of the MTP complex. These findings indicate
that the carboxyl terminus of the 97-kDa subunit plays an important
role in its interaction with PDI and that a fully translated 97-kDa
subunit may be required for normal complex formation.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.