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Molecular Cloning and Expression Analysis of the Human Rab7 GTP-ase Complementary Deoxyribonucleic Acid
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
229, 887–890 (1996)
1897
Molecular Cloning and Expression Analysis of the Human Rab7 GTP-ase Complementary Deoxyribonucleic Acid1 R. Vitelli, M. Chiariello, D. Lattero, C. B. Bruni,2 and C. Bucci Dipartimento di Biologia e Patologia Cellulare e Molecolare ‘‘L. Califano’’ and Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Universita` degli Studi di Napoli ‘‘Federico II,’’ Via S. Pansini 5, 80131, Naples, Italy Received November 18, 1996 Rab7 is a small GTP-ase localized on late endosomes, which regulates late endocytic membrane traffic in mammalian cells. Moreover it has been shown that this protein has a fundamental role in the cellular vacuolation induced by the cytotoxin VacA of Helicobacter pylori. We report here for the first time the isolation of a cDNA encoding human Rab7 from a placenta cDNA library. The open reading frame for human Rab7 encodes a protein of 207 amino acids which exhibits high homology with the mouse, rat, and dog counterparts. Northern blot analysis of total RNAs isolated from different cell lines with a cDNA probe containing the entire open reading frame revealed two mRNA transcripts of 2.5 and 1.8 kilobases. The isolation of human Rab7 cDNA will allow further characterization of its function in normal and pathological states. q 1996 Academic Press
The Rab family of Ras-related GTP binding proteins regulates intracellular membrane traffic in eukaryotic cells. Rab proteins are localized to the cytoplasmic surface of specific intracellular membrane compartments and their involvement in the regulation of membrane traffic has been studied in yeast and higher eukaryotes (1, 2). These proteins are anchored to the membrane by a geranyl-geranyl group that is added to the C-terminal cysteines and that is important for their function (3). cDNAs for Rab7 have been previously isolated from rat, dog, mouse, yeast and plants (410). Functional data on yeast and plant Rab7 suggested an important role of this protein in the late endocytic pathway (7, 8). Recently the fundamental role of Rab7 in the late endocytic pathway has been demonstrated also in mammalian cells, using mutants that interfered with the ability of the protein to bind or hydrolyze GTP (11-13). The late endosomal compartment, where Rab7 resides, appears to be involved in the site of some human and mammal disease such as the Chediak-Higashi syndrome (14) or the vacuolar degeneration of epithelial cells induced by the VacA toxin of Helicobacter pylori (15). In both cases the formation of giant late endocytic organelles that bear Rab7 on them was observed (14, 15). The involvement of Rab7 in the cellular vacuolation induced by the Helicobacter pylori VacA cytotoxin has recently been demonstrated (16). Dominant negative mutants of Rab7 prevented vacuolisation, while the wild type or costitutively active mutants slightly increased the extent of vacuolisation. These data suggest that the vacuoles formed by the VacA toxin are probably formed by fusion of late endocytic structures in a process that requires a functional Rab7 (16). In order to be able to study the function of rab7 in normal and pathological states and to understand whether this protein could be involved in the etiology and/or in the pathogenesis 1 2
GenBank Accession No. is X93499. Corresponding author. Fax:/39-81-7703285. 887 0006-291X/96 $18.00 Copyright q 1996 by Academic Press All rights of reproduction in any form reserved.
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Vol. 229, No. 3, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. Comparison of the amino acid sequences of human, rat, mouse, dog and yeast Rab7. Potential geranylgeranylation sites are indicated by asterisks. Conserved residues in all the species are shaded.
of diseases that involve the late endosomal compartment, we have cloned and sequenced the human Rab7 cDNA from a placenta cDNA library. MATERIALS AND METHODS cDNA cloning and sequencing. A lgt 11 human placenta cDNA library (17) was screened. In detail 51105 phage plaques were plated and lifted onto nitrocellulose filters. Duplicate filters were screened using the rat Rab7 coding sequence (Bucci, 88). Hybridization was carried out using 7% SDS and 0.5M NaPi pH7.2 at 507C (18). Filters were washed with 1% SDS and 40mM NaPi pH7.2 at 507C as described (18). Restriction maps and sequence analysis of seven positive clones showed that they were all derived from the same RNA and that the longest one contained the full length open reading frame. This cDNA clone was studied in more detail. The cDNA clone is 2264 bp long. The cDNA insert was restricted with EcoRI and three EcoRI fragments of approximately 1.1, 0.7 and 0.4 kb were subcloned in pGEM4z and sequenced by dideoxynucleotide chain termination method on both strands (19). To ensure that the three fragments were colinear we have amplified and sequenced by PCR (polymerase chain reaction) on the phage DNA the two regions between the three fragments excluding the presence of small additional EcoRI fragments. The human Rab7 cDNA and deduced amino acid sequence were analyzed and compared with those of mouse, rat, dog and yeast using the GeneWorks program. Northern blot analysis. Total RNAs from different cell lines were prepared and denatured as described (6). The RNAs were then electrophoresed in a denatured 1.2% agarose gel and tranferred to a nylon membrane. The membrane was then hybridized with a 32P-labeled 1150bp EcoRI fragment spanning the coding region of human Rab7 or with a 32P-labeled 1400bp BamHI fragment containing the coding region of the human GAPDH, in 7% SDS and 0.5M NaPi pH7.2 at 607C (18). After 16 hrs the filters were washed at 557C as described (18) and were autoradiographed using Kodak XAR5 film for 16 hrs at 0707C.RNA.
RESULTS AND DISCUSSION
In order to investigate the structure and function of human Rab7, we cloned and sequenced the human cDNA encoding this protein. A human cDNA clone encoding rab7 was isolated from a placenta cDNA library in lgt 11 (17) by screening with the rat Rab7 coding sequence. The cDNA clone is 2264 bp long and it shows an open reading frame (ORF) encoding a protein of 207 amino acids. The complete sequence of 2264 bp is available from the EMBL/ Gene Bank Data Libraries under the accession number X93499. The nucleotide sequence of 888
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6914$$$621
12-06-96 07:12:33
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Vol. 229, No. 3, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 2. Northern blot analysis of human Rab7 mRNAs. Total RNAs (10 mg) were extracted from a meningioma and from HepG2, A1251, Hela, Aro and Fro cells. The RNAs were then electrophoresed in a denatured 1.2% agarose gel and tranferred to a nylon membrane. The membrane was then hybridized with a 32P-labeled 1150bp EcoRI fragment spanning the coding region of human Rab7 (A) or with a 32P-labeled 1400bp BamHI fragment containing the coding region of the human GAPDH (B) as described (18). After 16hrs the filters were washed at 507C as described (18) and were autoradiographed using Kodak XAR5 film for 16 hrs at 0707C. The relative migration of the ribosomal RNAs (28S and 18S) is indicated on the left of A.
the coding region of the human cDNA clone shares 91%, 92%, 95% homology with mouse, rat and dog respectively (data not shown). Figure 1 shows the deduced amino acid sequences of human Rab7 compared with that of mouse, rat, dog and yeast peptides. At the amino acid level the identity is 99% between human and mouse, rat, and dog and 61% between human and yeast. The conserved GTP-binding motifs GXXXXGKT/S, GIT, DXXG and NKXD (20) are all present in the G-domain of human Rab7. The two C-terminal cysteine required for the prenylation and conserved in the human cDNA clones are indicated by an asterisk (Fig. 1) To analyze the expression of human rab7 we performed a Northern blot analysis on total RNA extracted from a meningioma and from different human cells lines: HepG2 (liver carcinoma), A1251 (kidney carcinoma) , Hela (uterous carcinoma), ARO (human anaplastic thyroid carcinoma) and FRO (human thyroid follicular carcinoma) (21-24) (Fig. 2A). The filter was hybridized first with the EcoRI fragment of 1150 bp containing the entire ORF of the human rab7 cDNA and then with a probe of human GAPDH (Fig. 2B). The analysis showed two messenger RNA for Rab7 of about 2.5 and 1.7 kb similarly to what was been previously found in mouse and rat (4, 6). The two mRNAs were present in all the cell lines examined. However there was a strong difference in the amount of mRNAs among the different cell lines. The two mRNAs for Rab7 were very abundant in A1251 cells while they were barely visible in HepG2. The amount of the GAPDH mRNA, instead was quite similar in the different cell lines. The fact that the expression of the Rab7 mRNAs is altered in A1251 and HepG2 cells 889
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BBRC 5857
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6914$$$621
12-06-96 07:12:33
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Vol. 229, No. 3, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
that come from two different kind of tumours, could suggest an important role of this protein in these pathologies. The high degree of conservation between the human and the yeast Rab7 protein (61%) indicates a critical structure-function relationship in the Rab7 protein conserved throughout the evolution similarly to what happens for other GTP-binding proteins of the same family (25). For instance the mammalian Rab1 protein non only shows a very high homology with the yeast counterpart YPT1 but is also able to complement its function (26). The isolation of the human Rab7 cDNA will allow us to study in detail the role of this protein in the endocytic pathway both in human cell lines and in normal or pathological states. This could be very significant especially considering that the late endosomal compartment appears to be involved in the pathogenesis of several important human diseases. ACKNOWLEDGMENTS Rosalba Vitelli is supported by a fellowship from the Associazione Italiana per la Ricerca sul Cancro (AIRC). This work was partially supported by a grant from the European Economic Community (to C.B.)
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.
Pfeffer, S. R. (1994) Curr. Opin. Cell Biol. 6, 522–526. Simons, K., and Zerial, M. (1993) Neuron 11, 789–799. Magee, T., and Newman, C. (1992) Trends in Cell Biol. 2, 318–323. Bucci, C., Frunzio, R., Chiariotti, L., Brown, A. L., Rechler, M. M., and Bruni, C. B. (1988) Nucleic Acids Res. 16, 9979–9993. Chavrier, P., Parton, R. G., Hauri, H. P., Simons, K., and Zerial, M. (1990) Cell 62, 317–329. Vitelli, R., Chiariello, M., Bruni, C. B., and Bucci, C. (1995) Biochim. Biophys. Acta 1264, 268–270. Wichmann, H., Hengst, L., and Gallwitz, D. (1992) Cell 71, 1131–1142. Cheon, C.-I., Lee, N.-G., Siddique, A.-B. M., Bal, A. K., and Verma, D. P. S. (1993) EMBO J. 12, 4125–4135. Drew, J. E., Brown, D., and Gatehouse, J. A. (1993) Plant Mol. Biol. 21, 1195–1199. Haizel, T., Merkle, T., Turk, F., and Nagy, F. (1995) Plant Physiol. 108, 59–67. Feng, Y., Press, B., and Wandinger-Ness, A. (1995) J. Cell Biol. 131, 1435–1452. Me´resse, S., Gorvel, J.-P., and Chavrier, P. (1995) J. Cell Sci. 108, 3349–3358. Vitelli, R., Santillo, M., Lattero, D., Chiariello, M., Bifulco, M., Bruni, C. B., and Bucci, C. (1996) J. Biol. Chem., in press. Burkhardt, J., Wiebel, F. A., Hester, S., and Argon, Y. (1993) J. Exp. Med 178, 1845–1856. Papini, E., de Bernard, M., Milia, E., Bugnoli, M., Zerial, M., Rappuoli, R., and Montecucco, C. (1994) Proc. Natl. Acad. Sci. USA 91, 9720–9724. Papini, E., Satin, B., Bucci, C., de Bernard, M., Telford, J., Rappuoli, R., Zerial, M., and Montecucco, C. (1996) EMBO J., in press. Young, R. A., and Davis, R. W. (1983) Proc. Natl. Acad. Sci. 80, 1194–1200. Church, G. M., and Gilbert, W. (1984) Proc. Natl. Acad. Sci. USA 81, 1991–1995. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463–5467. Bourne, H., Sanders, D., and McCormick, F. (1991) Nature 349, 117–127. Fagin, J. A., Matsuo, K., Karmakar, A., Chen, D. L., Tang, S. H., and Koeffler, H. P. (1987) J. Clin. Invest. 91, 179–184. Knowles, B. B., Howen, C. C., and Aden, D. P. (1980) Science 209, 497. Martin, G. G., and Shapiro, D. J. (1985) Biochim. Biophys. Acta 837, 163–172. Rosen, S. W., Weintraub, B. D., and Aaronson, S. A. (1980) J. Clin. Endocrinol. Metab. 50, 834–841. Valencia, A., Chardin, P., Wittinghofer, A., and Sander, C. (1991) Biochemistry 30, 4637–4648. Haubruck, H., Prange, R., Vorgias, C., and Gallwitz, D. (1989) EMBO J. 8, 1427–1432.
890
AID
BBRC 5857
/
6914$$$621
12-06-96 07:12:33
bbrca
229, 887–890 (1996)
1897
Molecular Cloning and Expression Analysis of the Human Rab7 GTP-ase Complementary Deoxyribonucleic Acid1 R. Vitelli, M. Chiariello, D. Lattero, C. B. Bruni,2 and C. Bucci Dipartimento di Biologia e Patologia Cellulare e Molecolare ‘‘L. Califano’’ and Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Universita` degli Studi di Napoli ‘‘Federico II,’’ Via S. Pansini 5, 80131, Naples, Italy Received November 18, 1996 Rab7 is a small GTP-ase localized on late endosomes, which regulates late endocytic membrane traffic in mammalian cells. Moreover it has been shown that this protein has a fundamental role in the cellular vacuolation induced by the cytotoxin VacA of Helicobacter pylori. We report here for the first time the isolation of a cDNA encoding human Rab7 from a placenta cDNA library. The open reading frame for human Rab7 encodes a protein of 207 amino acids which exhibits high homology with the mouse, rat, and dog counterparts. Northern blot analysis of total RNAs isolated from different cell lines with a cDNA probe containing the entire open reading frame revealed two mRNA transcripts of 2.5 and 1.8 kilobases. The isolation of human Rab7 cDNA will allow further characterization of its function in normal and pathological states. q 1996 Academic Press
The Rab family of Ras-related GTP binding proteins regulates intracellular membrane traffic in eukaryotic cells. Rab proteins are localized to the cytoplasmic surface of specific intracellular membrane compartments and their involvement in the regulation of membrane traffic has been studied in yeast and higher eukaryotes (1, 2). These proteins are anchored to the membrane by a geranyl-geranyl group that is added to the C-terminal cysteines and that is important for their function (3). cDNAs for Rab7 have been previously isolated from rat, dog, mouse, yeast and plants (410). Functional data on yeast and plant Rab7 suggested an important role of this protein in the late endocytic pathway (7, 8). Recently the fundamental role of Rab7 in the late endocytic pathway has been demonstrated also in mammalian cells, using mutants that interfered with the ability of the protein to bind or hydrolyze GTP (11-13). The late endosomal compartment, where Rab7 resides, appears to be involved in the site of some human and mammal disease such as the Chediak-Higashi syndrome (14) or the vacuolar degeneration of epithelial cells induced by the VacA toxin of Helicobacter pylori (15). In both cases the formation of giant late endocytic organelles that bear Rab7 on them was observed (14, 15). The involvement of Rab7 in the cellular vacuolation induced by the Helicobacter pylori VacA cytotoxin has recently been demonstrated (16). Dominant negative mutants of Rab7 prevented vacuolisation, while the wild type or costitutively active mutants slightly increased the extent of vacuolisation. These data suggest that the vacuoles formed by the VacA toxin are probably formed by fusion of late endocytic structures in a process that requires a functional Rab7 (16). In order to be able to study the function of rab7 in normal and pathological states and to understand whether this protein could be involved in the etiology and/or in the pathogenesis 1 2
GenBank Accession No. is X93499. Corresponding author. Fax:/39-81-7703285. 887 0006-291X/96 $18.00 Copyright q 1996 by Academic Press All rights of reproduction in any form reserved.
AID
BBRC 5857
/
6914$$$621
12-06-96 07:12:33
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Vol. 229, No. 3, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. Comparison of the amino acid sequences of human, rat, mouse, dog and yeast Rab7. Potential geranylgeranylation sites are indicated by asterisks. Conserved residues in all the species are shaded.
of diseases that involve the late endosomal compartment, we have cloned and sequenced the human Rab7 cDNA from a placenta cDNA library. MATERIALS AND METHODS cDNA cloning and sequencing. A lgt 11 human placenta cDNA library (17) was screened. In detail 51105 phage plaques were plated and lifted onto nitrocellulose filters. Duplicate filters were screened using the rat Rab7 coding sequence (Bucci, 88). Hybridization was carried out using 7% SDS and 0.5M NaPi pH7.2 at 507C (18). Filters were washed with 1% SDS and 40mM NaPi pH7.2 at 507C as described (18). Restriction maps and sequence analysis of seven positive clones showed that they were all derived from the same RNA and that the longest one contained the full length open reading frame. This cDNA clone was studied in more detail. The cDNA clone is 2264 bp long. The cDNA insert was restricted with EcoRI and three EcoRI fragments of approximately 1.1, 0.7 and 0.4 kb were subcloned in pGEM4z and sequenced by dideoxynucleotide chain termination method on both strands (19). To ensure that the three fragments were colinear we have amplified and sequenced by PCR (polymerase chain reaction) on the phage DNA the two regions between the three fragments excluding the presence of small additional EcoRI fragments. The human Rab7 cDNA and deduced amino acid sequence were analyzed and compared with those of mouse, rat, dog and yeast using the GeneWorks program. Northern blot analysis. Total RNAs from different cell lines were prepared and denatured as described (6). The RNAs were then electrophoresed in a denatured 1.2% agarose gel and tranferred to a nylon membrane. The membrane was then hybridized with a 32P-labeled 1150bp EcoRI fragment spanning the coding region of human Rab7 or with a 32P-labeled 1400bp BamHI fragment containing the coding region of the human GAPDH, in 7% SDS and 0.5M NaPi pH7.2 at 607C (18). After 16 hrs the filters were washed at 557C as described (18) and were autoradiographed using Kodak XAR5 film for 16 hrs at 0707C.RNA.
RESULTS AND DISCUSSION
In order to investigate the structure and function of human Rab7, we cloned and sequenced the human cDNA encoding this protein. A human cDNA clone encoding rab7 was isolated from a placenta cDNA library in lgt 11 (17) by screening with the rat Rab7 coding sequence. The cDNA clone is 2264 bp long and it shows an open reading frame (ORF) encoding a protein of 207 amino acids. The complete sequence of 2264 bp is available from the EMBL/ Gene Bank Data Libraries under the accession number X93499. The nucleotide sequence of 888
AID
BBRC 5857
/
6914$$$621
12-06-96 07:12:33
bbrca
Vol. 229, No. 3, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 2. Northern blot analysis of human Rab7 mRNAs. Total RNAs (10 mg) were extracted from a meningioma and from HepG2, A1251, Hela, Aro and Fro cells. The RNAs were then electrophoresed in a denatured 1.2% agarose gel and tranferred to a nylon membrane. The membrane was then hybridized with a 32P-labeled 1150bp EcoRI fragment spanning the coding region of human Rab7 (A) or with a 32P-labeled 1400bp BamHI fragment containing the coding region of the human GAPDH (B) as described (18). After 16hrs the filters were washed at 507C as described (18) and were autoradiographed using Kodak XAR5 film for 16 hrs at 0707C. The relative migration of the ribosomal RNAs (28S and 18S) is indicated on the left of A.
the coding region of the human cDNA clone shares 91%, 92%, 95% homology with mouse, rat and dog respectively (data not shown). Figure 1 shows the deduced amino acid sequences of human Rab7 compared with that of mouse, rat, dog and yeast peptides. At the amino acid level the identity is 99% between human and mouse, rat, and dog and 61% between human and yeast. The conserved GTP-binding motifs GXXXXGKT/S, GIT, DXXG and NKXD (20) are all present in the G-domain of human Rab7. The two C-terminal cysteine required for the prenylation and conserved in the human cDNA clones are indicated by an asterisk (Fig. 1) To analyze the expression of human rab7 we performed a Northern blot analysis on total RNA extracted from a meningioma and from different human cells lines: HepG2 (liver carcinoma), A1251 (kidney carcinoma) , Hela (uterous carcinoma), ARO (human anaplastic thyroid carcinoma) and FRO (human thyroid follicular carcinoma) (21-24) (Fig. 2A). The filter was hybridized first with the EcoRI fragment of 1150 bp containing the entire ORF of the human rab7 cDNA and then with a probe of human GAPDH (Fig. 2B). The analysis showed two messenger RNA for Rab7 of about 2.5 and 1.7 kb similarly to what was been previously found in mouse and rat (4, 6). The two mRNAs were present in all the cell lines examined. However there was a strong difference in the amount of mRNAs among the different cell lines. The two mRNAs for Rab7 were very abundant in A1251 cells while they were barely visible in HepG2. The amount of the GAPDH mRNA, instead was quite similar in the different cell lines. The fact that the expression of the Rab7 mRNAs is altered in A1251 and HepG2 cells 889
AID
BBRC 5857
/
6914$$$621
12-06-96 07:12:33
bbrca
Vol. 229, No. 3, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
that come from two different kind of tumours, could suggest an important role of this protein in these pathologies. The high degree of conservation between the human and the yeast Rab7 protein (61%) indicates a critical structure-function relationship in the Rab7 protein conserved throughout the evolution similarly to what happens for other GTP-binding proteins of the same family (25). For instance the mammalian Rab1 protein non only shows a very high homology with the yeast counterpart YPT1 but is also able to complement its function (26). The isolation of the human Rab7 cDNA will allow us to study in detail the role of this protein in the endocytic pathway both in human cell lines and in normal or pathological states. This could be very significant especially considering that the late endosomal compartment appears to be involved in the pathogenesis of several important human diseases. ACKNOWLEDGMENTS Rosalba Vitelli is supported by a fellowship from the Associazione Italiana per la Ricerca sul Cancro (AIRC). This work was partially supported by a grant from the European Economic Community (to C.B.)
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.
Pfeffer, S. R. (1994) Curr. Opin. Cell Biol. 6, 522–526. Simons, K., and Zerial, M. (1993) Neuron 11, 789–799. Magee, T., and Newman, C. (1992) Trends in Cell Biol. 2, 318–323. Bucci, C., Frunzio, R., Chiariotti, L., Brown, A. L., Rechler, M. M., and Bruni, C. B. (1988) Nucleic Acids Res. 16, 9979–9993. Chavrier, P., Parton, R. G., Hauri, H. P., Simons, K., and Zerial, M. (1990) Cell 62, 317–329. Vitelli, R., Chiariello, M., Bruni, C. B., and Bucci, C. (1995) Biochim. Biophys. Acta 1264, 268–270. Wichmann, H., Hengst, L., and Gallwitz, D. (1992) Cell 71, 1131–1142. Cheon, C.-I., Lee, N.-G., Siddique, A.-B. M., Bal, A. K., and Verma, D. P. S. (1993) EMBO J. 12, 4125–4135. Drew, J. E., Brown, D., and Gatehouse, J. A. (1993) Plant Mol. Biol. 21, 1195–1199. Haizel, T., Merkle, T., Turk, F., and Nagy, F. (1995) Plant Physiol. 108, 59–67. Feng, Y., Press, B., and Wandinger-Ness, A. (1995) J. Cell Biol. 131, 1435–1452. Me´resse, S., Gorvel, J.-P., and Chavrier, P. (1995) J. Cell Sci. 108, 3349–3358. Vitelli, R., Santillo, M., Lattero, D., Chiariello, M., Bifulco, M., Bruni, C. B., and Bucci, C. (1996) J. Biol. Chem., in press. Burkhardt, J., Wiebel, F. A., Hester, S., and Argon, Y. (1993) J. Exp. Med 178, 1845–1856. Papini, E., de Bernard, M., Milia, E., Bugnoli, M., Zerial, M., Rappuoli, R., and Montecucco, C. (1994) Proc. Natl. Acad. Sci. USA 91, 9720–9724. Papini, E., Satin, B., Bucci, C., de Bernard, M., Telford, J., Rappuoli, R., Zerial, M., and Montecucco, C. (1996) EMBO J., in press. Young, R. A., and Davis, R. W. (1983) Proc. Natl. Acad. Sci. 80, 1194–1200. Church, G. M., and Gilbert, W. (1984) Proc. Natl. Acad. Sci. USA 81, 1991–1995. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463–5467. Bourne, H., Sanders, D., and McCormick, F. (1991) Nature 349, 117–127. Fagin, J. A., Matsuo, K., Karmakar, A., Chen, D. L., Tang, S. H., and Koeffler, H. P. (1987) J. Clin. Invest. 91, 179–184. Knowles, B. B., Howen, C. C., and Aden, D. P. (1980) Science 209, 497. Martin, G. G., and Shapiro, D. J. (1985) Biochim. Biophys. Acta 837, 163–172. Rosen, S. W., Weintraub, B. D., and Aaronson, S. A. (1980) J. Clin. Endocrinol. Metab. 50, 834–841. Valencia, A., Chardin, P., Wittinghofer, A., and Sander, C. (1991) Biochemistry 30, 4637–4648. Haubruck, H., Prange, R., Vorgias, C., and Gallwitz, D. (1989) EMBO J. 8, 1427–1432.
890
AID
BBRC 5857
/
6914$$$621
12-06-96 07:12:33
bbrca