Lipoprotein lipase (LPL) ()is the major enzyme responsible for the hydrolysis of triglyceride-rich lipoproteins in plasma(1) . Genetic deficiency of LPL causes type I hyperlipoproteinemia syndrome, which is characterized by a significant increase of chylomicron levels in plasma and a marked increase in plasma triglyceride levels(2, 3) .

Functional LPL is a homodimeric glycoprotein with a subunit of 448 amino acids(4) . LPL is synthesized in parenchymal cells of tissues such as adipose tissue, heart, skeletal muscle, brain, and ovary(5, 6) . After synthesis the enzyme is secreted and bound to heparan sulfate proteoglycans on the luminal surface of the capillary endothelium(7) . At this site, LPL is rate-limiting for the hydrolysis and removal of triglycerides associated with chylomicrons and very low density lipoproteins(8) . The monoglycerides and fatty acids liberated by the LPL reaction are further processed for tissue storage or oxidation. An important part of LPL regulation is a tissue-specific event that is associated to post-translational modifications of the enzyme(9, 10, 11) . This modifications might be essential for the expression of LPL catalytic activity. Among these, asparagine-linked glycosylation (12) and dimerization of the protein (13) have been suggested as interrelated processes that confer catalytic activity to LPL(14) .

LPL secretion might also be a crucial regulatory point in the physiological action of the enzyme. Most secretory proteins such as LPL share a common biosynthetic origin in the rough endoplasmic reticulum (rER), from which they are transported to the Golgi complex. In the trans-Golgi network proteins destined to the regulated secretory pathway are sorted from those to be constitutively secreted or sent to lysosomes for intracellular degradation. Secretory pathways involve vesicular transfer to the plasma membrane followed by the secretory event itself and exocytic discharge of vesicle contents(15) . Genetic analysis of LPL deficiency, site-directed mutagenesis, and cellular expression of altered LPL cDNAs in heterologous systems have revealed that some missense changes lead to impaired or altered LPL secretion(16) , suggesting that both LPL secretion and enzyme activity are very sensitive to single amino acid exchanges. However, to date, the only single residue studied in some detail with a clear role in LPL secretion is Asn. Replacement of Asn by Ala completely abolishes LPL enzyme activity, leading to the production of an inactive LPL, which accumulates inside the rER(12, 17) .

By searching for mutations affecting the LPL gene in Type I hypertriglyceridemic patients, Ameis et al.(18) found that the substitution of a G for an A at nucleotide position 680 of human LPL cDNA, which produced a replacement of glycine 142 by a glutamic acid in the mature LPL protein, led to an inactive enzyme that was not efficiently secreted. The aim of the present study was to elucidate the intracellular destination of the nonsecreted LPL protein to gain further insight into the the mechanism of LPL secretion. We produced, by site-directed mutagenesis, a mutant hLPL carrying the substitution of a glutamic acid for glycine 142 (G142E hLPL), and we transfected COS1 cells with this construction and the wild type one (WT hLPL). The results obtained demonstrated that G142E hLPL presented reduced secretion compared with the WT hLPL, that heparin had no evident effect on the secretion of this mutant protein, and that after transport through the Golgi complex, lysosomes were the final degradation site of this altered, nonfunctional LPL. The findings reported suggest that the missorting of the mutant hLPL to lysosomes could act as a quality control mechanism of the secretory process of LPL.

MATERIALS AND METHODS

LPL Mutagenesis

Cell Culture and Transfection

Heparin Treatment and Cell Lysis

Western Blotting Assays

Heparin-Sepharose Chromatography, ELISA, and Activity Assays

Metabolic Labeling, Radioimmunoprecipitation, and Fluorography

After protein determination cell extracts were precleared with a rabbit preimmune serum conjugated with protein G-Sepharose (Sigma) for radioimmunoprecipitation. The protein G-Sepharose had been previously blocked with nontransfected cell extracts during an incubation of 1 h on a rotating device at 4 °C. The precleared cell extract expressing the protein was then incubated for at least 3 hours on a rotating device at 4 °C with a rabbit anti-hLPL (obtained from a rabbit immunized with recombinant human LPL expressed in a bacterial system) conjugated to blocked protein G-Sepharose. Immunoprecipitates were washed (six times) in a buffer containing 1% (w/v) Triton X-100, 2 mM EDTA in PBS. For endoglycosidase H digestion we used the procedure described by Hobman et al.(28) . The original immunoprecipitates as well as the tubes containing the endoglycosidase H digestion mix were eluted at 100 °C in SDS-gel sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 2% 2-mercaptoethanol, 10% glycerol, 0.1% bromphenol blue) and separated on an SDS-10% polyacrylamide gel at 200 V. Gels were fixed in methanol/acetic acid/water for 30 min, washed in water for 1 h, and soaked in amplifying buffer (sodium-salicilate) for 30 min before drying and exposure to Kodak X-Omat-S film at -80 °C. Protein bands were quantitated using the Ambis system.

Immunofluorescent Labeling and Confocal Microscopy

For confocal microscopy studies we used a Leica TCS 4D (Leica Lasertechnick GmbH, Heidelberg, Germany) confocal scanning laser microscope adapted to an inverted Leitz DMIRBE microscope. Colocalization analysis was made by the Multicolor software (version 2.0, Leica Lasertechnick). The confocal colocalization, defined as the topographical overlapping of fluorescent markers (fluorescein isothiocyanate-green and TRITC-red) for two cellular components (Lamp1 protein and LPL), was represented in a cytofluorogram in which the area where both markers overlap was indicated in yellow. By image treatment using the confocal system and to better illustrate the cellular sites where both proteins colocalized, we generated new images where colocalization is indicated in white (see Fig. 5).

Figure 5: Double immunofluorescent detection using confocal microscopy of LPL and Lamp1 protein on transfected COS1 cells. A shows the Lamp1 (A1) and LPL (A2) labeling of WT-expressing cells, A3 indicates the superposition of the anterior images, and A4 shows the colocalization image analysis (in white) (small arrow points to the rER and large arrow to the Golgi compartment). The same sequence of images for G142E hLPL-expressing cells is shown in B, where B1 and B2 reveal single labeling of Lamp1 and LPL, respectively, B3 the superposition and B4 the colocalization analysis of both proteins at the confocal microscope. A cytofluorogram indicating the colocalization area in both cases is represented in C1 for the WT hLPL-expressing cell and C2 for the G142E hLPL-expressing cell. Bars, 20 µm.

Immunoelectron Microscopy

RESULTS

Mutant G142E hLPL Is Deficiently Secreted by Transfected COS1 Cells

Western blotting assays of cell lysates from wild type (WT)- and mutant hLPL-transfected cells revealed, in both cases, a single band of 58 KDa similar to that of bovine LPL (55 KDa) as shown in Fig. 1A, indicating that the mutant protein did not present gross alterations in its electrophoretic mobility. Western blot assays of extracellular medium from cells transfected with the mutant LPL did not show any band, whereas the secreted WT hLPL was clearly detected (Fig. 1B). This difference was not due to poor expression of the mutant protein, since RNAs coding for the two proteins were equally expressed, as seen with RNA blots (results not shown). In addition, Western blot (Fig. 1A) and ELISA assays (result shown in Fig. 3C) revealed at least equivalent amounts of protein in cell extracts from WT hLPL and G142E transfectants (Fig. 1A). These results indicated that the secretion of G142 hLPL was defective.

Figure 1: A, WT hLPL and G142E hLPL in extracts from COS1 transfected cells. Equal amounts of cell lysates from WT hLPL- (lane 1) and G142E hLPL- (lane 2) transfected cells were subjected to Western blot analysis using the 5D2 antibody for detection on the nitrocellulose filter. In the first lane 0.5 µg of bovine LPL (bLPL), acting as a control, were loaded (arrow). B, media (20 µl) from nontransfected COS1 cells (NT), from WT hLPL- (lane 1) and G142E hLPL-transfected cells (lane 2), and 0.5 µg of bLPL (bovine LPL) were separated in a SDS-polyacrylamide gel, and LPL was detected by Western blot with the same monoclonal 5D2 antibody. In A and B arrows point to bLPL (small arrow, 55 KDa) and to LPL from transfected cells (large arrow, 58 KDa). Note that no band corresponding to LPL is detected in medium from G142E hLPL-transfected cells. The results are representative of four separate experiments.

Figure 3: Effect of heparin on the secretion of WT LPL and G142E hLPL from transfected COS1 cells. A, equal amounts of cell extracts from WT hLPL and G142E hLPL COS1 expressing cells treated without (-h) or with heparin (+h) (10 units/ml) for 24 h, were assayed by Western blotting. Lane 1 corresponds to WT hLPL- and lane 2 to G142E hLPL-expressing cells. The LPL detection was carried out with the 5D2 monoclonal antibody. The band on the last lane corresponds to the control of bovine LPL (bLPL, 55 KDa). Heparin affinity of G142E hLPL. Media (B) and cell extracts (C) from WT hLPL- and G142E hLPL-expressing cells were injected in a heparin-Sepharose affinity chromatography column to check the heparin affinity of both expressed proteins. The system used was the fast protein liquid chromatography system from Pharmacia, and 30 fractions of 1 ml were collected after elution in an NaCl gradient and were quantified for LPL immunoreactivity using ELISA assays. The ELISAs were carried out using polyclonal rabbit antibodies for coating and the monoclonal 5D2 antibody for detection.

This was further confirmed using ELISA assays of media which allowed us to evaluate the mass of extracellular LPL (expressed in absorbance units at 492 nm). The mass of secreted hLPL from mutant-expressing cells was reduced by approximately 75% compared with that of the WT-transfected cells (Fig. 2) as previously reported by Ameis et al.(18) . Untransfected COS1 cells expressed no LPL since almost no absorbance at 492 nm was detected in the culture medium. LPL activity of medium and cell extracts from WT- and G142E hLPL-expressing cells was next examined, revealing no lipolytic activity in the case of the mutant protein (results not shown).

Figure 2: ELISAs of media from untransfected COS1 cells, WT hLPL-transfected cells, and G142E hLPL-transfected cells were performed using polyclonal rabbit antibodies for coating and the monoclonal 5D2 antibody for detection. Absorbance at 492 nm, indicating LPL mass levels in media, was measured. The results are representative of three separate experiments.

Secretion of G142E hLPL Is Not Sensitive to Heparin

To determine the heparin affinity of both the WT and the mutant proteins, we tested culture cell media and cell extracts on heparin-Sepharose affinity chromatography columns, as described under ``Materials and Methods.'' Although the amount of mutant LPL in medium was 75% of that of the WT hLPL (as described earlier) both the WT-LPL and the mutant eluted maximally at a molarity of 0.6 M for the monomeric LPL and 1 M for the dimeric LPL in the NaCl gradient (Fig. 3B). It is worth remarking that most of the LPL in medium was found in its monomeric form (eluting at 0.6 M in the NaCl gradient) since transfected cells were incubated at 37 °C for 48 h and LPL monomerizes at high temperature (4) . In cell lysates the G142E hLPL was present at higher levels than the WT hLPL, and both proteins also eluted at the same (1 M) NaCl molarity (Fig. 3C). These results suggest that the affinity for heparin was the same for both the mutant and the WT proteins (Fig. 3, B and C).

Intracellular Levels of G142E hLPL Increase upon Leupeptin Treatment

Figure 4: Pulse-chase assays of newly synthesized WT hLPL and G142E hLPL expressed in COS1 cells. A, WT hLPL- (solid line) and G142E hLPL-transfected (broken line) cell monolayers were pulse-labeled with [S]methionine, and different chases at 0, 1, 2.5, 5, 6.5 h were performed as described under ``Materials and Methods.'' Cell extracts were obtained following the current protocol, and next they were immunoprecipitated using polyclonal rabbit antibodies. Immunoprecipitates were loaded in an SDS-PAGE gel for fluorography (arrow points to the LPL band). Dried gels were analyzed with the Ambis scanning system and software. Percentage of the time zero total counts are represented on the y axis. Chase times are represented on the x axis. B, pulse-chase assay of newly synthesized WT and G142E hLPL expressed in COS1 cells. Transfected monolayers were treated with 5 µM leupeptin just after the [S]methionine pulse. Cell extracts were obtained at different chase times and immunoprecipitated and assayed as described in panel A. The Ambis analysis allowed us to represent the total counts of LPL bands. The results are representative of three separate experiments.

In order to test this hypothesis, we incubated the cells with leupeptin (a lysosomal protease inhibitor) (27) during the pulse-chase experiments. If the disappearance of LPL seen in the previous experiment had been due to lysosomal degradation, inhibition of lysosomal proteases would have restored the cellular level of mutant LPL. In fact, as shown in Fig. 4B, we found that leupeptin treatment increased the intracellular level of the G142E hLPL, indicating that the inhibition of lysosomal degradation had a marked effect on the fate of the mutant protein.

Mutant G142E hLPL Is Diverted to Lysosomes

To study the specific intracellular localization of the G142E hLPL at higher resolution we performed immunoelectron microscopy assays. We found out that, as described in our previous study(17) , cells expressing the WT hLPL presented most of the LPL labeling located in the rER, inside intracellular vesicles and at the plasma membrane. Immunogold labeling detecting LPL in cells expressing the mutant protein was also found in the rER and Golgi stacks (Fig. 6). Thus it is reasonable to assume that the mutant protein entered the Golgi complex. This result was confirmed by double immunolocalization experiments using the monoclonal 5D2 antibody for LPL and the polyclonal antibody against -galactosyltransferase (30) of the trans-Golgi. The confocal analysis of this immunodetection confirmed a high colocalization index of both markers (not shown). Furthermore, endoglycosidase H digestion assays showed that the WT and mutant LPL had the same behavior toward this glycosidase, thus indicating the Golgi processing of the G142E hLPL (results not shown).

Figure 6: G142E hLPL is found in the Golgi compartment. Ultrathin cryosections of G142E hLPL-transfected cells were incubated with the 66 anti-LPL antibody and detected with 15-nm gold particles as described under ``Materials and Methods.'' A, the mutant LPL (arrows) can be found in Golgi stacks, which are better seen at higher magnification (B). Arrows point to gold particles detecting mutant LPL. g, Golgi. Bar for A, 800 nm. Bar for B, 500 nm.

Single immunogold detections revealed that mutant G142E hLPL was also present inside autophagic vesicles resembling lysosomes (Fig. 7A). To confirm the lysosomal nature of these vesicles we carried out double immunoelectron microscopy studies in which LPL and the Lamp1 lysosomal protein were labeled with 10- and 15-nm gold particle respectively (Fig. 7B). The results obtained indicated that most of the mutant LPL was found in Lamp1-positive vesicles, meaning that the lysosomal compartment might likely be the intracellular localization site of the altered enzyme.

Figure 7: Immunogold labeling of G142E hLPL-transfected COS1 cells for LPL and Lamp1 protein A. Ultrathin cryosections of COS1 cells expressing the G142E hLPL were incubated sequentially with the 66 polyclonal antibody anti-LPL, rabbit anti-chicken antibody, and protein-A coupled to 15-nm gold particles. B, cryosections were incubated sequentially with the 66 anti-LPL antibody visualized with 10-nm gold particles (small arrows) and with the anti-Lamp1 rabbit polyclonal antibody known to recognize the Lamp1 protein of the lysosomal membrane. The Lamp1 labeling was detected with protein A coupled to 15-nm gold particles (large arrows). L, lysosomes. Bar, 300 nm.

DISCUSSION

Our results suggest that secretion of the G142E hLPL is defective and heparin-insensitive and that the protein is missorted and diverted to lysosomes for degradation.

Several studies have indicated that most LPL regulation occurs post-translationally. N-linked glycosylation at Asn of hLPL is essential in the development of the enzyme activity(12, 32) , and the absence of N-glycosylation at this residue leads to impaired LPL secretion and rER accumulation of the mutant protein(12, 17) . However, the model that emerges from the present results (obtained by cellular expression of mutant G142E hLPL) is clearly different, although both mutant proteins are retained inside the cell. In contrast to N43A hLPL, the present results suggest that G142E hLPL leaves the endoplasmic reticulum, as has been shown by immunofluorescence and electron microscopy; reaches the Golgi complex, as indicated by immunogold labeling, confocal analysis of double immunolocalizations and endoglycosidase H sensitivity of the mutant enzyme; and, instead of being secreted to the extracellular medium, is diverted to lysosomes for degradation, as demonstrated using leupeptin treatment, immunofluorescence, and immunogold detection studies. Thus, although the mutation G142E in hLPL leads to impaired secretion, as happens with the N43A hLPL, the intracellular fate of these mutants is different. The molecular and cellular basis of the different intracellular behavior of this mutant G142E hLPL protein is not clear at present.

Glycoproteins processed by the trans-Golgi network can be sorted and sent to (i) lysosomes, (ii) constitutive secretory vesicles, (iii) regulated secretory vesicles and (iv) constitutive-like secretory vesicles(15) . Studies performed to date indicate that LPL secretion is a complex event (see (6) for a complete review). Pulse-chase experiments have revealed that in adipocytes approximately 80% of the newly synthesized LPL is degraded(33, 34) . The main intracellular site for LPL degradation seems to be lysosomes, since leupeptin subtantially reduces the rate of LPL degradation(34) . It has also been suggested that some LPL degradation may also occur in the rER(35) . This result appears to be controverted by other authors who report no rER LPL degradation in Brefeldin A-treated Chinese hamster ovary cells(36) . To explain the high degree of lysosomal LPL degradation observed, two different models have been proposed: Ailhaud (14) proposed that active LPL homodimer is sorted either directly from the trans-Golgi to the lysosomes or the constitutive secretory pathway or to the regulated pathway, where the exocytosis of LPL from intracellular secretory vesicles might be accelerated by heparin by an unknown mechanism. It has been suggested that the control of LPL efflux from the Golgi compartment represents the main post-translational regulation of LPL secretion(27, 37) . In contrast, Cisar et al. (11) proposed another model of LPL secretion in which newly synthesized enzyme would be transported to the cell surface, where it would bind to heparan sulfate proteoglycan receptors; LPL would then be either released to the extracellular medium or internalized via the receptor and either degraded in the lysosomes or recycled back to the cell surface. In our experimental system cells expressing the WT hLPL showed some degree of intracellular degradation (mostly in the lysosomes), since leupeptin treatment decreased the intracellular disappearance of the protein. Furthermore, secretion of WT hLPL to the extracellular medium was highly sensitive to heparin, indicating that although COS1 cells normally do not express LPL, they at least process the protein similarly to normal LPL-synthesizing cells. On the other hand, cells transfected with G142E hLPL and treated with leupeptin showed a 3-fold increase in LPL intracellular level. The mutant G142E hLPL had normal heparin binding capacity, but its secretion was not stimulated to the level of the WT hLPL upon heparin treatment, indicating that the intracellular compartment where G142E hLPL is retained might be slightly sensitive or insensitive to heparin modulation. Preliminar experiments performed by incubating transfected COS cells with antibodies against LPL indicated that at least part of the G142E hLPL reached the plasma membrane of the expressing cells. Contrary to what was observed in WT hLPL expressing cells, this membrane-bound mutant LPL was not sensitive to heparin release, suggesting that the membrane component that binds mutant LPL might not be a heparan sulfate proteoglycan. The combination of our data demonstrates that the mutant G142E hLPL follows a different intracellular pathway from that of the WT hLPL, and we suggest that both pathways diverge at the level of the trans-Golgi network. The observation that some of the G142E hLPL escapes intracellular degradation and is detected in culture medium cannot be explained at present.

Many active domains have been described in the present LPL molecule according to its homology with pancreatic lipase(38) . These domains include three clusters that confer the electrostatic potential to the molecule, the 5-loop, the lipid binding site, the C-terminal domain, the lid or surface loops covering the active site, and the active or catalytic site itself(39) . These domains play a crucial role in LPL function, and they are composed of several residues or clusters, which are clearly implied in the tertiary fold of the enzyme. So far, none of the domains described seems to include the Gly residue reported in this paper. However, the amino acid sequence surrounding glycine 142 is highly homologous in the several species whose LPL sequences are known. In their paper Ameis et al. (18) suggested that the reason for this high local sequence conservation might derive from its proximity to the catalytic center of the enzyme (serine 132). Thus, the mutation G142E, which substitutes a large, negatively charged glutamic acid for a small, neutral glycine residue, disrupts the enzymatic function of LPL ( (18) and this study). Since other missense mutations in the enzyme domains lead to the formation of inactive proteins that are normally secreted from the expressing cells, the results reported in this paper suggest that the Gly residue could have an additional role, ensuring the proper secretion of the molecule.

At this point, the question to be elucidated concerns the molecular events that direct the mutant G142E hLPL to lysosomes for degradation. One possible explanation would be that the replacement of a glycine with a glutamic acid at position 142 of LPL produced a gross effect on the tertiary fold of the molecule, preventing its efficient secretion. This hypothesis appears unlikely since aberrant proteins carrying major conformational changes are retained within the rER by chaperone proteins, such as BiP and calnexin(40) . This is the case of the N43A mutant hLPL(17) . Another hypothesis is that this single amino acid change could create a specific missorting sequence signal that could lead the protein to the lysosomal compartment. As far as we know this is not the case, since no homology has been found between the amino acid sequence created by the mutation and any defined specific lysosome-targeting domains. Finally we can consider the possibility that the mutant G142E hLPL had a different oligosaccharide processing as a consequence of the amino acid change, which could confer affinity for lysosomal sorting to the mutant LPL through the mannose 6-phosphate targeting. The presence of the mannose 6-phosphate signal of lysosomal enzymes is extensively reported (41). Since this mechanism appears saturable and mannose 6-phosphate receptors travel to the plasma membrane, this could constitute an explanation of why some of the G142E hLPL is detected at this site and is secreted. Obviously, further studies will be necessary to elucidate the precise molecular mechanisms that contribute in the missorting of G142E hLPL to lysosomes. The finding that the G142E hLPL is missorted to lysosomes and degraded suggests that this mechanism might represent an intracellular protein quality control system that would ensure the viability of the cell.

FOOTNOTES

*

This work was supported by the Comission Interministerial de Ciencia y Tecnologia (Grant SAF-92-0897), the Fondo de Investigaciones Sanitarias from the Ministerio de Sanidad (Grant 93/0423E), National Institutes of Health Grant HL47151, the International Life Science Institute (ILSI), and the European Community (BIOMED PL-921243). 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. Tel.: 34-3-4021550; Fax; 34-3-4112967; :senen{at}porthos.bio.ub.es.

()

The abbreviations used are: LPL, lipoprotein lipase; hLPL, human LPL; PBS, phosphate-buffered saline; rER, rough endoplasmic reticulum; WT, wild type; ELISA, enzyme-linked immunosorbent assay; TRITC, tetrarhodamine isothiocyanate.

ACKNOWLEDGEMENTS

REFERENCES

1. Olivecrona, T., and Bengtsson-Olivecrona, G. (1987) Lipoprotein Lipase (Borensztajn, J., ed) pp. 15-77, Evener Publishers, Inc., Chicago
2. Brunzell, J. D. (1983) Metabolic Basis of Inherited Disease (Scriver, C. K., Beaudet, A. L., Sly, W. S., and Valle, D., eds) pp. 1165-1180, McGraw-Hill Book Co., New York
3. Eckel, R. H. (1989) N. Engl. J. Med. 320, 1060-1068 [Abstract]
4. Iverius, P., and Östlund-Lindqvist, A. M. (1976) J. Biol. Chem. 251, 7791-7795 [Abstract/Free Full Text]
5. Camps, L., Reina, M., Llobera, M., Vilaró, S., and Olivecrona, T. (1990) Am. J. Physiol. 258, 673-681
6. Braun, J. E. A., and Severson, D. L. (1992) Biochem. J. 284, 337-347
7. Jackson, R. L., Baker, H. N., Gillian, E. B., and Gotto, A. M. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 1942-1945 [Abstract/Free Full Text]
8. Olivecrona, T., and Bengtsson-Olivecrona, G. (1993) Curr. Opin. Lipidol. 4, 187-196
9. Semenkovich, C. F., Wims, M., Noe, L., Etienne, J., and Chan, L. (1989) J. Biol. Chem. 264, 9030-9038 [Abstract/Free Full Text]
10. Doolittle, M. H., Ben-Zeev, O., Elovson, J., Martin, D., and Kirchgessner, T. G. (1990) J. Biol. Chem. 265, 4570-4577 [Abstract/Free Full Text]
11. Cisar, L. A., Hoogewerf, A. J., Cupp, M., Rapport, C. A., and Bensadoun, A. (1989) J. Biol. Chem. 264, 1767-1774 [Abstract/Free Full Text]
12. Semenkovich, C. F., Luo, C. C., Nakanishi, M. K., Chen, S-H., Smith, L. C., and Chan, L. (1990) J. Biol. Chem. 265, 5429-5433 [Abstract/Free Full Text]
13. Osborne, J. C., Bengtsson-Olivecrona, G., and Lee, N. S. (1985) Biochemistry. 24, 5606-5611 [CrossRef][Medline] [Order article via Infotrieve]
14. Ailhaud, G. (1990) Clin. Biochem. 23, 343-347 [CrossRef][Medline] [Order article via Infotrieve]
15. Halban, P. A., and Irminger, C. M. Biochem. J. 299, 1-18
16. Santamarina-Fojo, S. (1992) Curr. Opin. Lipidol. 3, 86 [CrossRef]
17. Buscà, R., Pujana, M. A., Pognonec, P., Auwerx, J., Deeb, S., Reina, M., and Vilaró, S., (1995) J. Lipid. Res. 36, 939-951 [Abstract]
18. Ameis, D., Kobayashi, J., Davis, R. C., Ben-zeev, O., Malloy, M. J., Kane, J. P., Lee, G., Wong, H., Havel, R. J., and Schotz, M. C. (1991) J. Clin. Invest. 87, 1165-1170
19. Auwerx, J. H., Deeb, S., Brunzell, J. D., Wolfbauer, G., and Chait, A. (1989) Biochemistry 28, 4563-4567 [CrossRef][Medline] [Order article via Infotrieve]
20. Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) Methods Enzymol. 154, 367-382 [Medline] [Order article via Infotrieve]
21. Miyazaki, J. I., Takaki, K., Tashiro, F., Tominega, A., Takatsu, K., and Yamamura, K. (1989) Gene (Amst.) 79, 269-277
22. Niwa, H., Yamamura, K., and Miyazaki, J. (1991) Gene (Amst.) 108, 193-200
23. Kriegler, M. (1990) Gene Transfer and Expresion: A Laboratory Manual , pp. 99-100, M. Stockton Press
24. Östlund-Lindqvist, A. M., and Boberg, J. (1977) FEBS Lett. 83, 231-236 [CrossRef][Medline] [Order article via Infotrieve]
25. Vilella, E., Joven, J., Fernández, M., Vilaró, S., Brunzell, J. D., Olivecrona, T., and Bengtsson-Olivecrona, G. (1993) J. Lipid Res. 34, 1555-1564 [Abstract]
26. Ramirez, I., Kryski, A. J., Ben-zeev, O., Schotz, M. C., and Severson, D. L. (1985) Biochem. J. 232, 229-236 [Medline] [Order article via Infotrieve]
27. Vannier, C., Deslex, S., Pradines-Figuères, A., and Ailhaud, G. (1989) J. Biol. Chem. 264, 13199-13205 [Abstract/Free Full Text]
28. Hobman, T. C., Woodward, L., and Farquhar, M. G. (1992) J. Cell Biol. 118, 795-811 [Abstract/Free Full Text]
29. Carlsson, S. R., Roth, J., Piller, F., and Fukuda, M. G. (1988) J. Biol. Chem. 263, 18911-18919 [Abstract/Free Full Text]
30. Nilsson, T., Lucocq, M. C., Mackay, D. C., and Warren, G. (1991) EMBO J. 10, 3567-3575 [Medline] [Order article via Infotrieve]
31. Slot, J. W., Geuze, H. J., Gigengack, S., Lienhard, G. E., and James, D. E. (1991) J. Cell Biol. 113, 123-135 [Abstract/Free Full Text]
32. Masuno, H., Schultz, C. J., Park, J-W., Blanchette-Mackie, E. J., Mateo, C., and Scow, R. O. (1991) Biochem. J. 277, 801-809
33. Cupp, M., Bensadoun, A., and Melford, K. (1987) J. Biol. Chem. 262, 6383-6388 [Abstract/Free Full Text]
34. Vannier, C., and Ailhaud, G. (1989) J. Biol. Chem. 264, 13206-13216 [Abstract/Free Full Text]
35. Ben-Zeev, O., Doolittle, M. H., Davis, R. C., Elovson, J., and Schotz, M. C. (1992) J. Biol. Chem. 267, 6219-6227 [Abstract/Free Full Text]
36. Pradines-Figuères, A., Vannier, C., and Ailhaud, G. (1990) J. Lipid Res. 31, 1467-1476 [Abstract]
37. Winkler, F. K., D'Arcy, A., and Hunziker, W. (1990) Nature 343, 771-774 [CrossRef][Medline] [Order article via Infotrieve]
38. van Tilbeurg, H., Roussel, A., Lalouel, J-M., and Cambillau, C. (1994) J. Biol. Chem. 269, 4626-4633 [Abstract/Free Full Text]
39. Shamu, C. E., Cox, J., and Walter, P. (1994) Trends Cell Biol. 4, 56-60
40. Damhs, N., Lobel, P., and Kornfeld, S. (1989) J. Biol. Chem. 264, 12115-12118 [Free Full Text]

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