Protein Disulfide Isomerase Is a Component of the Microsomal Triglyceride Transfer Protein Complex*

A bovine liver protein which catalyzes the transfer of triglyceride between membranes has previously been isolated from the lumen of the microsomal fraction. When further purified about 100-fold, two polypeptides of molecular mass 58,000 and 88,000 were identified (Wetterau, J. R., and Zilversmit, D. B. (1985) Chem. Phys. Lipids 38, 205-222). We demonstrate here that the two polypeptides (referred to as 58-kDa and 88-kDa, respectively) are associated in a protein-protein complex, and that the triglyceride transfer activity is associated with this complex. Antibodies specific for either polypeptide immunoprecipitated both the 58-kDa and 88-kDa polypeptides as well as the lipid transfer activity. The 58-kDa subunit of the microsomal transfer protein complex was identified as protein disulfide-isomerase (PDI) (EC 5.3.4.1) by 1) a comparison of the amino-terminal sequence of PDI and the 58-kDa subunit of the transfer protein, 2) a comparison of the reverse phase high performance liquid chromatography peptide maps of CNBr digests of PDI and the lipid transfer protein, 3) immunoprecipitation competition experiments in which PDI was found to compete with the lipid transfer protein for immunoprecipitation by the anti-58-kDa polyclonal antibodies, 4) immunological cross-reactivity of the microsomal triglyceride transfer protein complex with polyclonal antibodies raised against PDI, and 5) the appearance of protein disulfide isomerase activity following the dissociation of purified microsomal transfer protein complex with guanidine HCl. In conclusion, the microsomal triglyceride transfer protein has a multi-subunit structure which is unique compared to other intracellular lipid transfer proteins which have been described to be single polypeptides. The unexpected finding that PDI is a component of the microsomal triglyceride transfer protein complex suggests a new previously undescribed role for protein disulfide isomerase.

A bovine liver protein which catalyzes the transfer of triglyceride between membranes has previously been isolated from the lumen of the microsomal fraction. When further purified about loo-fold, two polypeptides of molecular mass 58,000 and 88,000 were identified (Wetterau, J. R., and Zilversmit, D. B. (1985) Chem. Phys. Lipids 38,[205][206][207][208][209][210][211][212][213][214][215][216][217][218][219][220][221][222] (TG),' cholesteryl ester, and, to a lesser extent, phosphatidylcholine (PC) between membranes has been isolated from bovine (1) and rat liver (2). It has also been detected in the intestinal mucosa of rats; however, it was not detected in appreciable quantities in the brain, heart, kidneys, or plasma. When rat liver homogenates were fractionated, most of the TG transfer activity was found in the microsomal fraction. The protein appears to reside within the lumen of the microsomes (2) and thus has been designated the microsomal lipid transfer protein (MTP). This neutral lipid transfer protein appears unique in that it prefers to transfer TG relative to cholesteryl ester.
The lipid transport properties as well as the tissue and cellular location of MTP has led to speculation that it may play a role in plasma lipoprotein biogenesis (2-4). Recent evidence suggests that plasma very low density lipoproteins are assembled by the sequential addition of lipid to preexisting nascent particles in the lumen of the endoplasmic reticulum and Golgi apparatus (4-7). MTP may mediate the transport of newly synthesized triglyceride into nascent very low density lipoproteins in the liver or chylomicrons in the intestine.
MTP has been purified from bovine liver by a series of five column chromatography steps (8). It has an apparent molecular weight of about 220,000, as determined by the elution position of lipid transfer activity on a calibrated Sephadex G-200 gel permeation column. Transfer activity could be recovered from nondenaturing polyacrylamide electrophoresis gels at a position which coincided with that of the single stained protein band. When the protein was electrophoresed in the presence of SDS (in the presence or absence of /3mercaptoethanol), two bands of molecular masses 58,000 and 88,000 were observed. This data suggested that a 58-kDa-88-kDa protein complex may be the lipid transfer protein. Alternatively, the MTP preparations may contain two independent proteins which have similar properties and co-purify. The purpose of this work was to distinguish between these possibilities and to begin to characterize the components of MTP.
Polyclonal antibodies specific for the 58-kDa and 88-kDa polypeptides were generated in rabbits. Immunoprecipitation with either anti-58-kDa or anti-88-kDa removed both the 58-kDa and 88-kDa polypeptides from solution, as well as the triglyceride transfer activity. The 58-kDa subunit was iso- TFA, trifluoroacetic acid; Ts, 3,5,3'-triiodo-L-thyronine. lated, and the sequence of the amino terminal 25 amino acids was determined.
The sequence was found to have perfect homology with the bovine microsomal protein, protein disulfide isomerase (PDI). The identity of the 58-kDa polypeptide as PDI was further supported by biochemical and immunochemical characterization of MTP. We propose that the microsomal protein which catalyzes the transfer of triglyceride between membranes is a complex of two proteins. The smaller, 5%kDa component is protein disulfide isomerase.

RESULTS
The Microsomal Triglyceride Transfer Protein Is a Protein Complex-A highly purified preparation of the microsomal triglyceride transfer protein contains two polypeptides of molecular mass 58,000 and 88,000. To obtain a tool to investigate the role of these two proteins in the lipid transport process, polyclonal antibodies against the isolated 58-kDa and 88-kDa polypeptides were generated. The specificity of each antiserum was examined by immunoblot analysis of bovine liver homogenate and purified MTP, following their fractionation by SDS-PAGE (see Fig. 1). Anti-58-kDa reacted with only a 58,000 molecule mass protein in the whole liver homogenate or purified MTP. Anti-88-kDa reacted with an 88,000 molecular mass protein in the whole liver homogenate and purified MTP. Anti-88-kDa in whole liver homogenate also reacted with a protein with a mobility intermediate between the 58-kDa and 88-kDa proteins. The relationship between this and the 88-kDa protein is not known. Because this cross-reactivity does not influence experiments performed with purified MTP, its origin was not pursued at this time.
To determine if the 58-kDa and 88-kDa polypeptides actually formed a 58-kDa-88-kDa protein complex, purified MTP was iodinated to a specific activity of 1000 cpm/ng of protein and then immunoprecipitated with antibodies specific for each polypeptide. Using Bolton-Hunter reagent for iodination, approximately 10 times more lz51 was incorporated into the 88-kDa polypeptide than the 58-kDa polypeptide. Fig. 2, left panel, illustrates the immunoprecipitation performed with the anti-58-kDa antiserum. Preimmune IgG immunoprecipitated neither the 58-kDa polypeptide nor the 88-kDa polypeptide. However, postimmune anti-58-kDa IgG was equally effective in immunoprecipitating the 58-kDa and 88-kDa polypeptides. Since anti-58-kDa did not recognize the 88-kDa polypeptide (Fig. l), these data suggest that the immunoprecipitation of the 88-kDa polypeptide occurs because it forms a stable complex with the 58-kDa polypeptide.
A similar experiment was performed with the anti-88-kDa antiserum (Fig. 2, right panel).
The preimmune IgG did not immunoprecipitate either polypeptide, whereas the postimmune antiserum immunoprecipitated both '251-labeled polypeptides. These results confirm that the two polypeptides form a 58-kDa-88-kDa protein complex.
To determine if the triglyceride transfer activity was associated with the 58-kDa-88-kDa protein complex, both antisera were used to immunoprecipitate TG transfer activity. Neither preimmune antiserum was able to deplete transfer activity from solution in an immunoprecipitation reaction. In contrast, both the anti-58-kDa and anti-88-kDa antisera were able to deplete the solution of transfer activity (Fig. 3). These results, in conjunction with the lz51-MTP immunoprecipitation results, demonstrate that the triglyceride transfer protein is a complex of two proteins of molecular weights 58,000 and 88,000. per l-h incubation) was 15.5 rt 0.6 (+ S. D.) and 21.9 -C 2.5, respectively. phase Cl6 column and eluted with a linear gradient of increasing 0.1% TFA, acetonitrile in 0.1% TFA, water. The 58-kDa polypeptide eluted at 52% acetonitrile. The recovery of the 58-kDa polypeptide was about 10% of what was applied to the column. The 88-kDa polypeptide could not be recovered with this procedure.  amino acid sequence analysis of the HPLC 58-kDa polypeptide. Two amino acids were identified in some cycles. The most probable sequence for the protein (based upon recovery of amino acids at each cycle with consideration for our typical recovery of each amino acid from standard proteins) matched exactly the sequence of bovine protein disulfide isomerase (17). Portions of a second, minor sequence were apparent. This second sequence also was identical with that of PDI; however, it appeared to be two amino acids shorter than the native form of PDI. For example, what appeared in cycle 4 for the most probable sequence, appeared in cycle 2 of the minor sequence.

Identification of Protein Disulfide Isomerase in the Purified
Protein disulfide isomerase was isolated from bovine liver by a modification of the procedure of Hillson et al. (14). PDI and the 58-kDa subunit of MTP co-migrated on SDS-PAGE (Fig. 4). To confirm the identity of the MTP 58-kDa subunit, MTP and conventionally purified PDI were digested with cyanogen bromide, and reverse phase HPLC peptide maps were generated (Fig. 5). Peptides from the PDI digest (middle panel) can be clearly identified in the MTP digest (see arrows, top panel), providing further evidence that the 58-kDa subunit of MTP is protein disulfide isomerase. Particularly evident are the cluster of peptides at around 40% solvent B and the two major peaks at 46 and 48% B. Few peptides which appear unique to MTP (and therefore thought to represent digested 88-kDa polypeptide) could be identified. The poor recovery of the 88-kDa peptides is consistent with our inability to recover the intact 88-kDa polypeptide from HPLC. The 88-kDa polypeptide apparently has properties which are not amenable to reverse phase HPLC purification. were tested for their ability to compete with lz51-MTP or triglyceride transfer activity in an immunoprecipitation reaction with the anti-58-kDa antibody. Following the immunoprecipitation of ""I-MTP with 50 ~1 of anti-58-kDa, only 15% of the radiolabeled 58-kDa and 88-kDa polypeptides remained in solution (Fig. 6, 0 pg of competing PDI). When increasing concentrations of HPLC-isolated 58-kDa polypeptide were added to the immunoprecipitation reactions, increasing amounts of radiolabeled protein remained in the supernatant.
This demonstrated that the HPLC-isolated 58-kDa protein competed with the '*"I-58-kDa-88-kDa protein complex in the immunoprecipitation.
Similar competition was observed with PDI isolated by a modification of the method of Hillson et al. (14). Fig. 7 illustrates that the 58-kDa protein isolated by HPLC or PDI isolated by a modification of the method of Hillson et al. (14) compete for the immunoprecipitation of TG transfer activity by anti-58-kDa.
Collectively, these results indicate that the 58-kDa polypeptide of the lipid transfer protein complex is protein disulfide isomerase.
Polyclonal Antibodies Raised Against Protein Disulfide Isomerase Cross-react with the Microsomal Triglyceride Transfer

Protein
Complex-Polyclonal antibodies against PDI were raised in rabbits and tested for their ability to cross-react with MTP. Fig. 8 is an immunoblot analysis of a bovine liver homogenate and MTP. Anti-PDI cross-reacts with the 58,000 molecular weight component of MTP.
Anti-PDI was tested for its ability to immunoprecipitate MTP. As is illustrated in Fig. 9, these antibodies which were raised against a protein which has no TG transfer activity PDI and MTP were digested with CNBr as described under "Materials and Methods." A control, blank digest, was performed in which protein was omitted. An aliquot of digested MTP (top panel) or PDI (middle panel) or control digest (bottom panel) was loaded on a CIR reverse phase column and eluted with a linear gradient of 0.1% TFA, acetonitrile (solvent B) in 0.1% TFA, water (solvent A). Protein was detected by optical density at 220 nm. Arrows are included in the top panel to indicate peaks in the MTP digest which correspond to peaks in the PDI digest.
(see Table II) readily deplete TG transfer activity from solution.
Treatment of MTP with a Denaturant, Guanidine HCl, Results in the Loss of TG Transfer Activity and Enhanced Expression of Protein Disulfide Isomerase Activity-Protein disulfide isomerase catalyzes the rearrangement of protein disulfide bonds. It can reactivate denatured proteins by correctly pairing their cysteine residues into disulfide bonds, thus allowing the protein to fold to its enzymatically active state (for review, see Ref. 18). We tested the ability of MTP to express PDI activity by measuring its ability to reactivate previously reduced and denatured ribonuclease. Table II is a comparison of the TG transfer activity and protein disulfide isomerase activity expressed by MTP and PDI. Native PDI expresses no TG transfer activity, even at protein concentrations over 1000 times higher (up to 350 pg tested) than that normally used to measure MTP transfer activity. MTP has some disulfide isomerase activity, but in this example, only about 5% that of an equal mass of PDI. PDI and MTP were isolated independently from three differ- of MTP results in a complete loss of TG transfer activity; however, a 6-7-fold increase in disulfide isomerase activity was observed. PDI, which was treated in a similar manner as a control, lost some disulfide isomerase activity. In summary, the native lipid transfer protein complex expresses little disulfide isomerase activity. Once the complex is disrupted by guanidineHC1, the lipid transfer activity is lost and the disulfide isomerase activity expressed increases to a level comparable to native PDI. (The specific activity of MTP is expected to be lower than PDI due to the mass of the 88-kDa polypeptide).

DISCUSSION
The data reported here indicate that the microsomal triglyceride transfer protein is a complex of two proteins of molecular mass 58,000 and 88,000. Polyclonal antibodies specific for either the 58-kDa or 88-kDa polypeptide immunoprecipitated both the 58-kDa and 88-kDa polypeptides.
In addition, both antisera immunoprecipitated triglyceride transfer activity. The 58-kDa subunit of MTP was identified as protein "Disulfide isomerase activity was measured as the increase in activity of reduced and denatured ribonuclease following a 20-min incubation at 37 "C in the presence of PDI or MTP. The spontaneous reactivation of ribonuclease was subtracted from the total reactivation to determine the protein-stimulated reactivation. Ribonuclease activity was a measure of the RNA digested in a 7-min assay at 37 "C. Digested RNA was ouantitated bv oatical densitv at 260 nm followine a perchloric acid, &any1 acetate precipitation-of undigested RNA: The disultide isomerase activity is expressed as the change in the ribonuclease activity (AA& per mg of MTP or PDI. The activity was measured four times, and the results are expressed as the average * S.D. See "Materials and Methods" for additional details of the assay. disulfide isomerase by 1) comparison of the amino-terminal sequence of the two proteins, 2) peptide mapping experiments, 3) immunoprecipitation competition experiments with PDI, 4) immunological cross-reactivity of MTP with antibodies raised against PDI, and 5) the expression of protein disulfide isomerase activity by MTP. The association of PDI with the 88-kDa polypeptide in MTP inhibits the expression of disulfide isomerase activity.
The two-subunit structure of MTP is curiously unique compared to other lipid transfer proteins. Previously characterized intracellular lipid transfer proteins contain a single peptide with a molecular weight in the range of 8,000 to 35,000 (see Refs. 19 and 20 for reviews). The plasma neutral lipid transfer protein is a glycoprotein with an apparent molecular mass of around 74,000 (21,22). The protein sequence, derived from the cDNA sequence, indicates this protein is translated as a 53,000 molecular mass polypeptide (23). PDI catalyzes the rearrangement of disulfide bonds in proteins (18). fn uivo, PDI is believed to play a role in the formation of disulfide bonds in newly synthesized proteins within the lumen of the endoplasmic reticulum (ER). This appears to promote the proper folding of disulfide-bonded proteins (24). A similar process has been replicated in vitro with a variety of proteins including ribonuclease (25), egg white lysozyme (26), soybean trypsin inhibitor (27), and mouse IgM (28). Although PDI mRNA has been identified in a variety of tissues, it is found in particularly high concentrations in cells which actively synthesize and secrete disulfidebonded proteins. Its concentration appears to be regulated in concert with the level of disulfide-bonded protein synthesis and secretion which is occurring in the cell (28)(29)(30).
Interestingly, PDI also functions as the /3 subunit of prolyl 4-hydroxylase (31). Pihlajaniemi et al. (32) determined that both proteins (PDI and the fl subunit of prolyl4-hydroxylase) are the products of a single gene. Prolyl4-hydroxylase has an apparent molecular mass of around 240,000 daltons with two LY subunits of 64,000 daltons and two /3 subunits of around 60,000 daltons (see Ref. 33 for review). It catalyzes the formation of 4-hydroxyproline, an important residue in the stability of the collagen triple helical structure. The (Y subunit must be in a tetrameric protein complex with PDI to express this activity. PDI only expresses about one-half of its disulfide isomerase activity when complexed in prolyl 4-hydroxylase. In contrast, the association of PDI with the 88-kDa subunit in MTP appears to drastically decrease the expression of disulfide isomerase activity. In addition to its role in protein assembly and prolyl 4hydroxylase, bovine liver PDI has been reported to be a thyroid hormone binding protein. Ta binds to PDI with a dissociation constant of 57 nM (17). Recently, a protein which appears highly similar if not identical with PDI was identified as the glycosylation site binding protein, a component of oligosaccharyl transferase (34). These results, in conjunction with those we have presented here, suggest that PDI is a multifunctional protein that participates in a variety of cellular processes.
The association between the 88-kDa subunit and PDI may play a role in the intracellular targeting of the lipid transfer activity. Proteins which contain the carboxyl-terminal sequence, Lys-Asp-Glu-Leu, or are associated with a protein which contains this sequence, appear to be selectively retained in the ER (35). For example, PDI, BiP (the immunoglobin heavy chain binding protein), and grp94 all contain this sequence and all are retained in the ER. In addition, improperly folded proteins or /3-glucuronidase which do not contain the retention sequence are thought to be retained in the ER because they form a complex with proteins which are retained, BiP (36) and egasyn (37), respectively.
Perhaps PDI, which contains the four-amino acid carboxyl-terminal retention sequence, is playing a similar role in the retention of the 88-kDa polypeptide within the ER. The role of the subunits in the lipid transfer protein complex is not known. One of the two subunits may be the TG transfer protein and the second subunit may have another, yet to be defined role. Alternatively, both subunits may be required to express lipid transfer activity. If one subunit is the transfer protein, one would predict it is 88-kDa as 58-kDa, PDI, in a purified form does not express TG transfer activity (see Table II). To date, we have been unable to isolate catalytically active 88-kDa. Treatment of MTP with detergents, denaturants, or chaotropic agents at concentrations necessary to disrupt the MTP complex appear to inactivate MTP.* The characterization of 88-kDa has been further hampered by its tendency to degrade following its dissociation from PDT and our difficulty isolating it by HPLC. Our working model of the microsomal triglyceride transfer protein complex is that the uncharacterized 88-kDa component is the catalytic subunit or that this subunit when complexed with PDI confers transfer activity to the protein complex. Based upon our inability to isolate catalytically active 88-kDa, we hypothesize that both subunits are required for an active lipid transfer protein. The role of PDI in the TG transfer protein complex is intriguing.
The interfacial interaction between the two subunits may be necessary to create the active site or to stabilize the transfer protein in an active conformation.
The role of PDI in the transfer protein complex may have similarities to its role in prolyl4-hydroxylase. The a subunit of this tetrameric protein has no catalytic activity unless it is complexed with PDI (33). One could speculate that the ability of PDI to catalyze the rearrangement of disulfide bonds within a protein may play a role in the lipid transfer process. Perhaps this modulates conformational changes during the transfer reactions. A role for cysteine residues in neutral lipid transport may be suggested by the sensitivity of the plasma neutral lipid transfer protein to sulfhydryl reagents (38).