©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Thrombospondin 3 Is a Pentameric Molecule Held Together by Interchain Disulfide Linkage Involving Two Cysteine Residues (*)

Aziz Qabar (1), Laura Derick (3), Jack Lawler (4), Vishva Dixit (1) (2)(§)

From the (1) Department of Pathology and (2) Department of Cellular and Molecular Biology, University of Michigan School of Medicine, Ann Arbor, Michigan 48109, the (3) Department of Biomedical Research, St. Elizabeth's Hospital of Boston, Boston, Massachusetts 02135, and the (4) Department of Pathology, Brigham and Women's Hospital, Harvard University School of Medicine, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The thrombospondins (TSPs) are a family of 5 distinct gene products designated TSP1, -2, -3, -4, and COMP, for cartilage oligomeric matrix protein. TSP1, the prototypical member, is a trimeric extracellular matrix molecule implicated in cell migration and development. TSP1 trimer formation is mediated by interchain disulfide linkage involving two NH-terminal cysteines. TSP3, a recent addition to the family, is a developmentally regulated heparin binding protein that is similar in sequence to the COOH terminus of TSP1 but has a distinct NH terminus. This has raised the question of the oligomeric nature of TSP3 and identification of the cysteine residues involved in oligomer formation. We demonstrate, using a combination of deletional and site-directed mutagenesis and rotary shadowing electron microscopy, that TSP3, like TSP4 and COMP, is a pentameric molecule. TSP3 is held together by interchain disulfide linkage involving just two cysteine residues, Cys-245 and Cys-248.


INTRODUCTION

Thrombospondin 1 (TSP1),() the first described and best studied member of the TSP family, is a large trimeric heparin binding protein that is synthesized by a variety of cell lines and incorporated into the extracellular matrix where it differentially modulates cell adhesion in a cell type-specific manner (see Refs. 1-3 for reviews). The function of the other TSPs (TSP2, -3, -4, and COMP, for cartilage oligomeric matrix protein) at present is unclear, although it is evident that they are all highly expressed in vertebrate development (4, 5, 6, 7, 8) . TSP3, for example, is predominantly expressed in the developing lung (6) , whereas COMP expression is limited to cartilage following the initial phase of chondrogenesis (8) . The TSPs are modular proteins and both TSP1 and TSP2 possess NH-terminal heparin binding domains (3) . Two cysteine residues (Cys-252 and Cys-256 in TSP1 and TSP2) that are involved in interchain disulfide linkage and trimer assembly are just distal to the heparin binding domain (9) . Conservation of these cysteines in TSP1 and TSP2, coupled to their high degree of overall homology, allows for the expression of heterotrimers composed of both polypeptides (10) . The heparin binding domain is followed by a short stretch of sequence with homology to procollagen, and by three distinct repeating motifs designated Types 1, 2, and 3. There are 3 copies of the Type 1 repeat in TSP1 and TSP2. The putative melanoma cell adhesive sequence, VTCG (11), and a novel heparin binding sequence, WSXW (12) , that mediates binding of certain cells to TSP are included in the Type 1 repeats. The Type 2 repeats have homology to epidermal growth factor and 3 copies are present in TSP1 and TSP2 (3) . The Type 3 repeats are enriched in aspartic acid and constitute the calcium binding domain that is capable of mediating significant conformational alterations in the molecule, some of which, like the unmasking of a reactive thiol in TSP1, may have functional consequence (13) . In stark contrast to TSP1 and TSP2, the other TSPs (TSP3, -4, and COMP) have a distinct structure. Notably, they possess a distinct NH terminus, no procollagen or Type 1 repeats, and contain an extra Type 2 repeat (3) . Previous studies have shown TSP4 (14) and COMP to be pentamers (15) , and the two cysteines involved in COMP interchain disulfide linkage have been identified as Cys-68 and Cys-71 (16) . These cysteines are in an equivalent position to Cys-252 and Cys-256 of TSP1 since COMP lacks the approximately 200 amino acid NH-terminal domains that are found in the other thrombospondins (17) . TSP3 was shown to be a disulfide-linked higher order oligomer that was larger than a trimer. However, its precise oligomeric nature was undefined. Using deletional and site-directed mutagenesis and rotary shadowing electron microscopy, we show that TSP3, like TSP4 and COMP, is a pentameric molecule. In addition, the two cysteine residues involved in disulfide linkage are unambiguously identified.


MATERIALS AND METHODS

Construction of Expression Plasmids

A full-length coding HindIII/NotI TSP3 cDNA fragment (6) was directionally subcloned into the Epstein-Barr virus based episomal expression vector pCEP4 that also contains a hygromycin resistance cassette (InVitrogen, San Diego, CA). TSP1 and TSP2 cDNAs lacking both the 5`- and 3`-untranslated regions were obtained by PCR employing previously cloned mouse TSP1 and TSP2 cDNAs as template (10) , Vent DNA polymerase (New England Biolabs, Beverly, MA), and upstream primers with an ATG start codon (bold) and downstream primers containing a stop codon (italics). For ease of cloning, the primers contained custom restriction sites (underlined). TSP1 (upstream primer), 5`-GCACAAAGCTTCCACCATGGAGCTCCTGCGGGGACTA 3`, HindIII; TSP1 (downstream primer), 5`-TGATTGGCAGCGGCCGCTTAGGAATCTCGACACTCGTATTT-3`, NotI; TSP2 (upstream primer), 5`-CACAGCTAGCGCCACCATGCTCTGGGCACTGGCCCTG-3`, NheI; TSP2 (downstream primer), 5`-AGCTGGAGCGCGGCCGCCTAGGCATCTCTGCACTCATACTT-3`, NotI.

The two PCR products were directionally subcloned into pCEP4 using the unique restriction sites NheI/NotI for TSP2 and HindIII/NotI for TSP1. The resulting constructs were fully sequenced to ensure fidelity of PCR amplification.

Transfections

293T cells, a human renal epithelial cell line, were stably transfected with plasmid expression constructs encoding mouse TSP1, TSP2, or TSP3 using the calcium phosphate coprecipitation method as described previously (6, 10) . Following hygromycin selection (100 µg/ml), individual colonies were subcloned and tested for expression of each TSP by metabolic labeling and immunoprecipitation.

Expression and Purification of Mouse TSP1, -2, and -3

293T cells expressing recombinant TSP were grown in complete Eagle's minimum essential medium (BioWhittaker, Walkersville, MD) supplemented with 10% bovine calf serum for 24 h, following which the medium was replaced with serum-free insulin, transferrin, and selenium-supplemented Eagle's minimum essential medium (Collaborative Biomedical Products, Bedford, MA) for 72 h. Conditioned medium was mixed with a 50% slurry of heparin-Sepharose and incubated for 8-12 h at 4 °C on a rotating shaker. The heparin-Sepharose slurry was poured into a 1-ml column and washed extensively with 0.15 M NaCl, 20 mM Tris-HCl (pH 8.0), and bound thrombospondin step-eluted by the application of 0.5 M NaCl in the same buffer. 500-µl aliquots were collected and analyzed by SDS-acrylamide gel electrophoresis and Coomassie Blue staining.

Generation of a Polyclonal TSP3 Peptide Antibody

A synthetic peptide, RLRGPSRPSPC, from the NH-terminal end of TSP3 corresponding to amino acid residues 107-116 was synthesized and covalently conjugated to keyhole limpet hemocyanin as a carrier protein via the free -SH group of the carboxyl-terminal cysteine residue (Multiple Peptide Systems, San Diego, CA). 2 mg of the conjugated peptide was emulsified with 500 µl of TiterMax (Vaxcel Inc., Norcros, GA) and injected into New Zealand White female rabbits subcutaneously. Subsequent injections were carried out using incomplete Freund's adjuvant.

Construction of TSP3 Truncations and Mutagenesis

Four TSP3 truncations were derived by PCR using a common 5`-oligonucleotide primer containing an ATG start codon (bold) and a custom HindIII restriction site (underlined), 5`-AGTGAAGCTTCTAAGCGGCATGGAGAAGCCG-3`, and four different downstream oligonucleotide primers (antisense strand) having an in-frame TAA stop codon (italics) and custom NotI restriction site (underlined). The truncations and downstream primers were designated according to the identity of the COOH-terminal amino acid (Glu-696, Asp-437, Cys-268, and Gly-249): Glu-696 primer: 5`-AAGTCTGTGCGGCCGCCTTCTGCACTTTACTCCGGGCA-3`, NotI; Asp-437 primer: 5`-TGGTCCGGGCGGCCGCCTATATCTTAGTCAGGCCCACA-3`; Cys-268 primer: 5`-TAGCCCGGGCGGCCGCACACTTCTTAGAAGTCCACGCC-3`; Gly-249 primer: 5`-CTGCAGTGGCGGCCGCGCTCGTGTTAACCGCACAC-3`.

The four truncated molecules were subcloned into the cytomegalovirus promoter-based mammalian expression vector pcDNA3 (InVitrogen, San Diego, CA) using the unique sites HindIII/NotI.

Finally, the two cysteine residues (at positions 245 and 248) were altered to serine residues in the native full-length TSP3 molecule by site-directed mutagenesis using the ``Altered Sites'' kit (Promega, Madison, WI) according to the manufacturer's instructions. Two point mutations were introduced at nucleotides 797 and 806 (TGT to TCT and TGC to TCC, respectively; indicated in bold), using the following antisense mutagenic primer: 5`-CTCGTGGAAACCGGACACCTGAGACTCCATGATGGT-3`.

Expression of Truncated Molecules

293T cells were transiently transfected with each truncation/mutant construct and expression detected by metabolic labeling and immunoprecipitation essentially as described in previous publications (6, 10) , using the TSP3 peptide antibody.

Electron Microscopy

Thrombospondin-3 in TBS containing 2 mM CaCl was diluted 1:5 or 1:10 with 70% glycerol, 0.15 M ammonium acetate, and 0.2 mM CaCl just prior to spraying. A parallel sample was adjusted to 5 mM in EDTA prior to mixing with 70% glycerol and 0.15 M ammonium acetate prepared without CaCl. The samples were sprayed and rotary shadowed with platinum from an angle of 6° to the horizontal by the method of Tyler and Branton (18) . A shadow thickness of approximately 12 Å was used. Replicas were coated with a 100-Å thick supporting film of carbon from an angle of 90°.


RESULTS AND DISCUSSION

Expression and Purification of Recombinant Mouse TSP1, TSP2, and TSP3

Recombinant TSP was purified from three day serum-free 293T cell-conditioned medium transfected with the appropriate TSP expression construct. 293T cells have previously been shown not to synthesize any endogenous TSP and are therefore ideal for the expression of recombinant TSPs (10) . On average, conditioned medium from ten 10-cm diameter dishes allowed for the purification of 5-10 µg of TSP3 and 1-5 µg each of TSP1 and TSP2. The purification process consisted of application of the serum-free medium to a heparin-Sepharose column which, after extensive washing, was step eluted in a high salt (0.5 M NaCl) buffer. The predominant protein in the resulting eluate was the recombinant TSP as evidenced by SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining (Fig. 1).


Figure 1: Purified recombinant TSPs. Recombinant mouse TSP1, -2, and -3 proteins were purified from serum-free conditioned medium of transfected 293T cells by heparin-Sepharose affinity chromatography. Purified TSP was resolved on a 7.5% SDS-polyacrylamidegel and stained with Coomassie Blue. The predominant protein in each sample is the recombinant TSP. Molecular mass markers in kilodaltons are shown to the left.



Characterization of TSP1, -2, and -3 Antibodies

To establish the specificity of each TSP antibody, an immunoblot analysis of the recombinant TSPs was carried out (Fig. 2). Production of rabbit polyclonal antisera against the divergent NH terminus of TSP1 and TSP2 using bacterially expressed recombinant fusion protein as immunogen has been described previously (10) . The TSP3 antibody was raised against a peptide corresponding to residues 107-116 that are unique to TSP3. Fig. 2clearly shows that the predominant protein recognized by each antibody is the corresponding TSP. Also of note (see Fig. 1and Fig. 2) are the relative molecular masses of the three TSPs in the presence of reductant, with TSP2 being the largest (190 kDa), followed by TSP1 (180 kDa), and TSP3 being the smallest (160 kDa). In contrast, in the absence of reductant (Fig. 3), native TSP3 migrates slower than either native TSP1 or TSP2 which are both trimeric. Since the apparent subunit molecular mass of TSP3 is smaller than that of TSP1 and TSP2, it follows that in the native nonreduced state TSP3 must be larger than a trimer.


Figure 2: Specificity of TSP1, TSP2, and TSP3 antibodies. Conditioned medium from 293T cells transfected with each TSP construct was resolved by SDS-polyacrylamide gel electrophoresis in the presence of reductant, transferred to nitrocellulose, and probed with TSP1, TSP2, or TSP3 antisera. The antibodies recognize the corresponding TSP (arrow).




Figure 3: Migration of recombinant TSP1, -2, and -3 in the absence of reductant. Transfected cells were metabolically labeled with [S]cysteine and methionine and labeled proteins in the medium immunoprecipitated with the corresponding antibody. Samples were resolved by SDS-polyacrylamide gel electrophoresis and subjected to autoradiography. TSP3 is the slowest migrating species, followed by TSP2, and then TSP1.



Expression of Truncated Forms of TSP3

The deduced amino acid sequence of native TSP3 contains 47 cysteine residues (6) , most of which are presumably involved in intrachain disulfide linkages. To identify the cysteine residues involved in interchain disulfide linkages and oligomerization, four COOH-terminal truncated molecules, each having a defined number of cysteine residues, were generated by PCR, subcloned into a mammalian expression vector (pcDNA3), and expressed by transient transfection in 293T cells. Metabolic labeling and immunoprecipitation using TSP3 NH-terminal polyclonal antiserum revealed that each of the four truncations (Glu-696, Asp-437, Cys-268, and Gly-249) was synthesized and present in both the cell layer and medium compartments (Fig. 4A). In the presence of reductant, the apparent molecular mass of each species corresponded to the number of amino acid residues expressed, i.e. Glu-696 was 75 kDa, Asp-437 was 47 kDa, Cys-268 was 29 kDa, and Gly-249 was 27 kDa. In the absence of reductant (Fig. 4B), all four species migrated substantially slower, consistent with the formation of oligomers. The two shortest truncations (Gly-249 and Cys-268) migrate with an apparent molecular mass of 150 kDa consistent with the formation of a pentameric species. The two larger truncations (Glu-696 and Asp-437) also migrate with appreciably reduced mobility, barely entering the gel system, presumably due to oligomer formation.


Figure 4: Expression of TSP3 truncations in the presence and absence of reductant. 293T cells transfected with the indicated constructs were metabolically labeled with [S]methionine and cysteine and subjected to immunoprecipitation with the TSP3 peptide antibody. Medium and cell layer samples were resolved by SDS-polyacrylamide gel electrophoresis in the presence of reductant (A) or in the absence of reductant (medium alone, panel B). In the presence of reductant, TSP3 truncations of the predicted size are found in both the cell layer and medium. In the absence of reductant, there is a clear shift of all the truncations to a higher molecular mass oligomeric form. Also shown (panel C) is a schematic representation of a monomeric chain of TSP3 with cysteine residues numbered and truncations indicated by the identity of the COOH-terminal residue. The four cysteine residues present in the Gly-249 truncation are circled.



Mutagenesis of Cys-245 and Cys-248 in Native TSP3

Since the shortest truncation (Gly-249) contained only four cysteine residues (Cys-124, Cys-183, Cys-245, and Cys-248) and was capable of oligomerization to a pentameric structure, it followed that, at the very least, a pair of these cysteines had to be involved in pentamer assembly. Given that COMP does not possess the equivalent first pair of cysteines (17) and that the second pair of NH-terminal cysteine residues are involved in TSP1 trimer assembly (9) , it appeared likely that this second analogous pair of cysteines in TSP3 (Cys-245 and Cys-248) was responsible for interchain disulfide linkage and pentamer assembly.

To confirm that Cys-245 and Cys-248 were involved in oligomerization of the intact native TSP3 molecule, these two residues were altered to serine by site-directed mutagenesis of a full-length TSP3 cDNA. Following subcloning into the pcDNA3 expression vector and transient transfection of 293T cells, the expression of native and mutant TSP3 was monitored by metabolic labeling and immunoprecipitation in the absence and presence of reductant (Fig. 5).


Figure 5: Cys-245 and Cys-248 are critical for oligomerization of native TSP3. Native TSP3 (Wt TSP3) or TSP3 with cysteine 245 and cysteine 248 altered to serine (Mut TSP3) were expressed in 293T cells, metabolically labeled, and immunoprecipitated with a TSP3 peptide antibody, resolved by SDS-polyacrylamide gel electrophoresis either in the absence (-) or presence (+) of reductant and visualized by autoradiography. In the absence of reductant, there is a complete lack of oligomerization of the mutant TSP3, implicating Cys-245 and Cys-248 in oligomer assembly. A cartoon interpretation of the results is shown.



Native TSP3, as expected, migrated as an oligomer in the absence of reductant and a monomeric species in the presence of reductant. Mutant TSP3, with both Cys-245 and Cys-248 altered to serine, also migrated as a monomer in the presence of reductant, but in the absence of reductant, there was a complete absence of oligomerization. In fact, the only discernible species had an electrophoretic mobility greater than the reduced monomeric form, presumably due to intrachain disulfide linkage producing a more compact, faster migrating form of the molecule.

These results confirmed the critical role played by Cys-245 and Cys-248 in oligomerization of native TSP3 and also suggested that the remaining 45 cysteines were not involved in interchain disulfide linkage but rather intrachain linkage or free sulfhydryls.

Electron Microscopy of TSP3

Electron microscopy of TSP3 revealed well defined globular structures that were connected by thin-flexible regions of polypeptide (Fig. 6). In those molecules where the globular structures are not overlapping and are clearly defined, six globular domains are present. Five of these appear at the ends of the thin-flexible regions and the sixth is near the site where the thin connecting regions are connected to each other. Comparison with the published images of TSP1, TSP4, and COMP indicates that the 5 globular domains at the ends of the thin-flexible regions are the COOH-terminal and the single globular domain near the site where the polypeptides are connected is formed by the NH-terminal domains of all of the subunits in close proximity (15, 19) . Like TSP1 and TSP4, the globular domains at the ends of the thin-flexible regions undergo a conformational change when calcium is removed from the molecule (Fig. 6), consistent with the presence of the calcium binding Type 3 repeats at the COOH terminus of the molecule. In the presence of calcium the globular domains are 10.8 ± 1.3 nm in diameter (all dimensions are given as mean ± S.D. and are not corrected for thickness of replicating metal). When calcium is removed from the sample prior to electron microscopy, the diameter of these globular domains decreases to 8.9 ± 1.1 nm and the length of the thin-flexible region increases. These data are consistent with a pentameric model of TSP3 in which each subunit is composed of a single small globular domain at the NH-terminal, a site where the subunits are connected, a thin-flexible region of extended polypeptide, and five globular domains containing the calcium sensitive Type 3 repeats at the COOH-terminal.


Figure 6: Electron microscopy of purified recombinant TSP3. Samples were prepared for rotary shadowing in the presence (A) or absence (B) of 200 µM CaCl. Bar = 50 nm.



It has been proposed that the cysteine residues that form the interchain disulfides of TSP1 and COMP are included in -helical segments of polypeptide (9, 16) . These -helices reportedly assemble to form three- or five-stranded bundles that drive trimer or pentamer formation of TSP1 or COMP, respectively. Deletion of polypeptide that includes this region results in defective trimer formation of TSP1, even though the cysteine residues are still present (20, 21) . The data presented here and the similarity of the COMP and TSP3 sequences around the corresponding cysteine residues indicates that a similar mechanism may result in pentamer formation in TSP3. In the TSP1, TSP2, and TSP3 genes these regions are included in a single exon (22, 23, 24) .

Whereas the similarities in the sequences around the interchain disulfides may provide the common basis for multimerization, the differences may result in TSP1 and TSP2 forming trimers and TSP3, TSP4, and COMP forming pentamers. In the former group, there are three amino acids between the cyteines that form the interchain disulfides and in the latter group there are two amino acids. If these regions are helical, then both cysteines are on the same side of the helix with either spacing. However, the additional residue between the interchain disulfides of TSP1 and TSP2 will change the relative positions of the cysteine residues. In addition, the cysteines that form the interchain disulfides are near the NH-terminal of the predicted -helix in TSP1 and TSP2, and they are near the COOH-terminal of the predicted -helix in TSP3, TSP4, and COMP. We hypothesize that these differences result in the -helices packing together to form bundles of either three or five strands.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant HL45351 (to J. L.) and CA58182 (to V. M. D.). 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.

§
Established Investigator of the American Heart Association. To whom correspondence should be addressed: The University of Michigan Medical School, Dept. of Pathology, 1301 Catherine St., Ann Arbor, MI 48109-0602. Tel.: 313-747-0264; Fax: 313-764-4308; E-mail: vishva.dixit@med.umich.edu.

The abbreviations used are: TSP, thrombospondin; COMP, cartilage oligomeric matrix protein; PCR, polymerase chain reaction.


ACKNOWLEDGEMENTS

We gratefully acknowledge Karen O'Rourke for invaluable advice and technical assistance.


REFERENCES
  1. Frazier, W. A.(1991) Curr. Opin. Cell Biol. 3, 792-799 [Medline] [Order article via Infotrieve]
  2. Bornstein, P.(1992) FASEB J. 6, 3290-3299 [Abstract/Free Full Text]
  3. Adams, J., and Lawler, J.(1993) Curr. Biol. 3, 188-190
  4. Iruela-Arispe, M. L., Liska, D. J., Sage, E. H., and Bornstein, P. (1993) Dev. Dyn. 197, 40-56 [Medline] [Order article via Infotrieve]
  5. Corless, C. L., Mendoza, A., Collins, T., and Lawler, J.(1992) Dev. Dyn. 193, 346-358 [Medline] [Order article via Infotrieve]
  6. Qabar, A. N., Lin, Z., Wolf, F. W., O'Shea, K. S., Lawler, J., and Dixit, V. M.(1994) J. Biol. Chem. 269, 1262-1269 [Abstract/Free Full Text]
  7. Lawler, J., Duquette, M., Whittaker, C. A., Adams, J. C., McHenry, K., and DeSimone, D. W.(1993) J. Cell Biol. 120, 1059-1067 [Abstract]
  8. Hedbom, E., Antonsson, P., Hjerpe, A., Aeschlimann, D., Paulsson, M., Rosa-Pimentel, E., Sommarin, Y., Wendel, M., Oldberg, A., and Heinegard, D.(1992) J. Biol. Chem. 267, 6132-6136 [Abstract/Free Full Text]
  9. Sottile, J., Selegue, J., and Mosher, D. F.(1991) Biochemistry 30, 6556-6562 [Medline] [Order article via Infotrieve]
  10. O'Rourke, K. M., Laherty, C. D., and Dixit, V. M.(1992) J. Biol. Chem. 267, 24921-24924 [Abstract/Free Full Text]
  11. Tolsma, S. S., Volpert, O. V., Good, D. J., Frazier, W. A., Polverini, P. J., and Bouck, N.(1993) J. Cell Biol. 122, 497-511 [Abstract]
  12. Vogel, T., Guo, N. H., Krutzsch, H. C., Blake, D. A., Hartman, J., Mendelovitz, S., Panet, A., and Roberts, D. D.(1993) J. Cell. Biochem. 53, 74-84 [Medline] [Order article via Infotrieve]
  13. Sun, X., Skorstengaard, K., and Mosher, D. F.(1992) J. Cell Biol. 118, 693-701 [Abstract]
  14. Lawler, J., McHenry, K., Duquette, M., and Derick, L.(1995) J. Biol. Chem. 270, 2809-2814 [Abstract/Free Full Text]
  15. Morgelin, M., Heinegard, D., Engel, J., and Paulsson, M.(1992) J. Biol. Chem. 267, 6137-6141 [Abstract/Free Full Text]
  16. Efimov, V. P., Lustig, A., and Engel, J.(1994) FEBS Lett. 341, 54-58 [CrossRef][Medline] [Order article via Infotrieve]
  17. Oldberg, A., Antonsson, P., Lindblom, K., and Heinegard, D.(1992) J. Biol. Chem. 267, 22346-22350 [Abstract/Free Full Text]
  18. Tyler, J. M., and Branton, D.(1980) J. Ultrastruct. Res. 71, 95-102 [Medline] [Order article via Infotrieve]
  19. Lawler, J., Derick, L. H., Connolly, J. E., Chen, J. H., and Chao, F. C.(1985) J. Biol. Chem. 260, 3762-3772 [Abstract]
  20. Prochownik, E. V., O'Rourke, K., and Dixit, V. M.(1989) J. Cell Biol. 109, 843-852 [Abstract]
  21. Lawler, J., Ferro, P., and Duquette, M.(1992) Biochemistry 31, 1173-1180 [Medline] [Order article via Infotrieve]
  22. Wolf, F. W., Eddy, R. L., Shows, T. B., and Dixit, V. M.(1990) Genomics. 6, 685-691 [Medline] [Order article via Infotrieve]
  23. Shingu, T., and Bornstein, P.(1993) Genomics. 16, 78-84 [CrossRef][Medline] [Order article via Infotrieve]
  24. Bornstein, P., Devarayalu, S., Edelhoff, S., and Disteche, C. M. (1993) Genomics 15, 607-613 [CrossRef][Medline] [Order article via Infotrieve]

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