©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A 30-Amino Acid Truncation of the Microsomal Triglyceride Transfer Protein Large Subunit Disrupts Its Interaction with Protein Disulfide-isomerase and Causes Abetalipoproteinemia (*)

Beverly Ricci (1), Daru Sharp (1), Edward O'Rourke (2), Bernadette Kienzle (1), Laura Blinderman (1), David Gordon (1), Connie Smith-Monroy (1), Gordon Robinson (1), Richard E. Gregg (1), Daniel J. Rader (3), John R. Wetterau (1)(§)

From the (1)Department of Metabolic Diseases and the (2)Department of Molecular Biology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000 and the (3)Department of Medicine, Division of Medical Genetics, University of Pennsylvania, Philadelphia, Pennsylvania 19104-4283

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

The microsomal triglyceride transfer protein (MTP)()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) .

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.


MATERIALS AND METHODS

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.

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.

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.

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 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.


RESULTS

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 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.

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.


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.

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 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.

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.


DISCUSSION

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 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.

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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Bristol-Myers Squibb, P. O. Box 4000, Princeton, NJ 08543. Tel.: 609-252-4254; Fax: 609-252-6964.

The abbreviations used are: MTP, microsomal triglyceride transfer protein; PDI, protein disulfide-isomerase; apoB, apolipoprotein B; PCR, polymerase chain reaction; bp, base pair(s); TG, triglyceride.


ACKNOWLEDGEMENTS

We thank the University of Cincinnati for providing the antibodies used in this study.


REFERENCES
  1. Wetterau, J. R., Combs, K. A., Spinner, S. N., and Joiner, B. J. (1990) J. Biol. Chem.265, 9800-9807
  2. Wetterau, J. R., Aggerbeck, L. P., Laplaud, P. M., and McLean, L. R. (1991) Biochemistry30, 4406-4412 [Medline] [Order article via Infotrieve]
  3. Sharp, D., Blinderman, L., Combs, K. A., Kienzle, B., Ricci, B., Wager-Smith, K., Gil, C. M., Turck, C. W., Bouma, M.-E., Rader, D. J., Aggerbeck, L. P., Gregg, R. E., Gordon, D. A., and Wetterau, J. R. (1993) Nature365, 65-69 [Medline] [Order article via Infotrieve]
  4. 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) Science258, 999-1001 [Medline] [Order article via Infotrieve]
  5. Shoulders, C. C., Brett, D. J., Bayliss, J. D., Narcisi, T. M. E., Jarmuz, A., Grantham, T. T., Leoni, P. R. D., Bhattacharya, S., Pease, R. J., Cullen, P. M., Levi, S., Byfield, P. G. H., Purkiss, P., and Scott, J.(1993) Hum. Mol. Genet.2, 2109-2116 [Abstract]
  6. Shoulders, C. C., Brett, D. J., Narcisi, T. M. E., Leiper, J. M., Chester, S. A., Bayliss, J. D., Reid, J., and Scott, J.(1994) Circulation90, I-186 [Abstract]
  7. Gordon, D. A., Jamil, H., Sharp, D., Mullaney, D., Yao, Z., Gregg, R. E., and Wetterau, J.(1994) Proc. Natl. Acad. Sci. U. S. A.91, 7628-7632 [Abstract]
  8. Leiper, J. M., Bayliss, J. D., Pease, R. J., Brett, D. J., Scott, J., and Shoulders, C. C.(1994) J. Biol. Chem.269, 21951-21954 [Abstract/Free Full Text]
  9. Patel, S. B., and Grundy, S. M.(1994) Circulation90, I-186 [Abstract]
  10. Atzel, A., and Wetterau, J. R.(1993) Biochemistry32, 10444-10450 [Medline] [Order article via Infotrieve]
  11. Pihlajaniemi, T., Helaakoski, T., Tasanen, K., Myllylä, R., Huhtala, M.-L., Koivu, J., and Kivirikko, K. I.(1987) EMBO J.6, 643-649 [Abstract]
  12. Lackner, K. J., Monge, J. C., Gregg, R. E., Hoeg, J. M., Triche, T. J., Law, S. W., and Brewer, H. B., Jr.(1986) J. Clin. Invest.78, 1707-1712 [Medline] [Order article via Infotrieve]
  13. Sharp, D., Ricci, B., Kienzle, B., Lin, M. C. M., and Wetterau, J. R. (1994) Biochemistry33, 9057-9061 [Medline] [Order article via Infotrieve]
  14. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K.(1989) Current Protocols in Molecular Biology, Vol. 1, p. 3.5.11, John Wiley and Sons, Inc., New York
  15. Higuchi, R., Krummel, B., and Saiki, R.(1988) Nucleic Acids Res.16, 7351-7367 [Abstract]
  16. Hagan, D. L., Kienzle, B., Jamil, H., and Hariharan, N.(1994) J. Biol. Chem.269, 28737-28744 [Abstract/Free Full Text]
  17. Vuori, K., Pihlajaniemi, T., Myllylä, R., and Kivirikko, K. I. (1992) EMBO J.11, 4213-4217 [Abstract]
  18. Klausner, R. D., and Sitia, R.(1990) Cell62, 611-614 [Medline] [Order article via Infotrieve]
  19. Gething, M.-J., and Sambrook, J.(1992) Nature355, 33-45 [Medline] [Order article via Infotrieve]
  20. Wetterau, J. R., Combs, K. A., McLean, L. R., Spinner, S. N., and Aggerbeck, L. P.(1991) Biochemistry30, 9728-9735 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.