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A homologue of the ras-related CDC42 gene from Schizosaccharomyces pombe
Gene. 114 (1992) 153-154
© 1992 ElsevierSciencePublishers B.V. All rightsreserved. 0378-1119/92/$05.00
153
GENE 06478
A homologue of the ras-related CDC42 gene from
Schizosaccharomycespombe*
(GTP-binding protein; fission yeast; cell polarity; G25K; Saccharomyces)
Erica Fawell, Sally Bowden and John Armstrong Membrane Molecular Biology Laboratoo,, hnperial Cancer Research Fund, London WC2A 3PX (UK)
Received by R.W. Davies: 9 December 1991 Accepted: 5 January 1992 Received at publishers: 2 March 1992
SUMMARY A cDNA was isolated from the fission yeast, Schizosaccharomycespombe, using mixed oligodeoxyribonucleotides encoding part of the GTP-binding site of the ras superfamily. The encoded protein is the homologue of the budding yeast CDC42 gene product and the human proteins, CDC42Hs and G25K.
The ras superfamily comprises a large group of small GTP-binding proteins, members of which have been implicated in the regulation of a variety of processes including signal transduction, membrane traffic and organisation of the cytoskeleton (Bourne et al., 1990). Examples of these proteins are found in all eukaryotes including the budding yeast S. cerevisiae, in which the functions of several have been investigated by genetic methods. One of these is the CDC42 gene product. Temperature-sensitive mutants in this gene do not form buds; instead material is deposited in a delocalised fashion on the cell surface, suggesting that CDC42 is involved in controlling polarity in the yeast cell (Adams et al., 1990). The gene product falls within the
Correspondenceto: Dr. J. Armstrong,MembraneMolecularBiologyLab-
oratory, Imperial Cancer Research Fund, P.O. Box 123, Lincoln's Inn Fields, London WC2A 3PX (UK) Tel. (44-71)269-3393; Fax (44-71)430-2666. * On request, the authors will supply detailed experimentalevidencefor the conclusionsreached in this BriefNote. Abbreviations: oligo, oligodeoxyribonucleotide;S., Saccharmnyces; Sc., Schi=osaccharomyces; SDS, sodium dodecyi sulfate; Sp (also Sp), S. pombe.
subgroup of ras-related proteins which include the Rho and Rac proteins (Johnson and Pringle, 1990). Surprisingly, close homologues of this protein are found in non-budding eukaryotic cells: two human proteins, CDC42Hs and G25K, are 86% identical to the yeast protein (Shinjo et al., 1990; Munemitsu et al., 1990). G25K has attracted further interest as a good substrate in vitro for the tyrosine kinase activity of the epidermal growth factor receptor (Hart et al., 1990) and for nucleotide exchange by the dbl oncogene product (Hart et al., 1991). The yeast Sc. pombe reproduces by elongation and fission rather than budding. It is very different from S. cerevisiae and in several respects appears to be a closer model of higher eukaryotic cells (Nasim et ai., 1989). Using mixed oligos encoding part of the GTP-binding site conserved throughout the ras family (Touchot et al., 1987), we have isolated several ras-related genes from this organism, and have previously described three members of the ypt/rab group (Fawell et al., 1989; 1990). Here we report a further sequence, isolated from a eDNA library (Fig. 1). The encoded protein, CDC42Sp, is 87%, 82% and 81% identical to CDC42Sc, CDC42Hs and G25K, respectively. Since Sc. pombe, like S. cerevisiae, is amenable to genetic analysis, it may be a useful organism in which to resolve the apparent discrepancy between the proposed function of CDC42 protein in polarised bud formation and its presence in non-budding cells.
154 1 55
GAA ATG 1 M 109 CTG 19L 163 TTC 37 F 217 T T A 55 L 271 CCT 73 P 325 G A A 91 E 379 CCG 109 P 433 CAG 127 Q 487 TTA 145 L 5,11 AAA 163 K 595 GTT 181 V 649 CTT 703 CTT 757 TTT 811 GGC 865 AAC 919 GCA
TTC CCC P CTT L GAT D TTC F CAA Q AAT N TGC C AAA K GCT A GGG G CCT P TCC AGC CAT TTT GAC GGA
AAT ACC T ATT I AAT N GAT D ACA T GTG V TTA L CTA L CGT
R TTA L CAC H TTT TTT CCT ACA GAT ATT
AAA ATT I TCC S TAT Y ACC T GAC D AAA K ATT I GCT A GAG E AAG K AAG K TCC CTT AGC TAT TTT C
GTG AAG K TAT Y GCT A GCT A GTC V GAA E GTT V CGC R TTG L AAT N AAA K GAA TAA TTT CTG GCT
AAG TGT C ACT T GTC V GGT G TTT F AAG K GGT G CAG Q GGT G GTT V AAG K ATT CGA TGT TGT ATA
CAA GTC V ACA T ACT T CAG Q TTG L TGG W ACC T CAT H GCT A TTT F TCA S TCC TCC GCT TTT CTT
AGC GTA V AAC N GTC V GAG E GTT V TTT F CAA Q CAG Q GTC V GAT D AAG K TTA CTC TCT CTG TTG
TTT GTA V AAG K ATG M GAT D TGT C CCC P ATT I CAT H AAG K GAA E TGT C ATA ATA TCA GCT CTA
ACG GGA G TTT F ATC I TAT Y TTT F GAA E GAT D CCC P TAT Y GCT A TTG L CAG TTT TTC AGG CAT
ATT GAC D CCT P GGT G GAT D AGT S GTT V TTA L CTT L GTT V ATT I GTA V CTA GCA ATT CAT TAT
AAT GGT G AGT S GAT D CGC R GTA V CAT H CGT
TAT GCT A GAC D GAA E TTG L ACT T CAT H GAT
TTT GTA V TAT Y CCA P CGT R TCT S CAT H GAC
TTG GGA G GTG V TAC Y CCT P CCT P TGT C CCT CAA Q GCT A CTT L AAC
TGA AAG K CCA P ACT T TTA L GCC A CCG P TCT S GGT G TTG L GAT D CCT
AAT ACC T ACT T CTT L TCC S AGT S GGC G GTG V GAA E ACC T CCT P GGT
AGT TGT C GTA V GGT G TAT Y TTT F GTC V CAA Q CGA R CAA Q CCT P TTT
R
D
D
P
ACA T GAG E GTA V CTG L GTT AAG CTG ATT TGT
CAT H TGT C GCC A TAA
GAG E TCC S GCT A TTT
TAC ATA TCA TCC ATT
AAC TTT ATC TTT CTT
AGT GCT ACA TTC CTG
AAA ATT CAC CGT CCA
CAA TGT TTC TAA TTA
TGT TAT TTG ATA CTC
Fig. 1. The sequence of the CDC42Sp cDNA and encoded protein. The cDNA was isolated from a eDNA library of Sc. pombe in 2gtl I (provided by V. Simanis and P. Nurse, University of Oxford) by hybridisation to an equimolar mixture of the oligos 5'-GATACKGCKGGKCAGGAGCG and 5'-CGTrCTrGKCCKGCKGTGTC (K = G or T) (Touchot et al., 1987). Hybridisation was in 0.5 M Na.phosphate, pH 7.2/8% SDS/I mM EDTA/ 1% bovine serum albumin (Church and Gilbert, 1984) and filters were washed in 3 M tetramethyl ammonium chloride/50 mM Tris.HCI, pH 7.6/2 mM EDTA/0.1% SDS (Wood et al., 1985) at 59 ° C. After plaque purification, the eDNA was excised with EcoRl, subcloned into plasmids and the sequence determined using modified T7 polymerase (Tabor and Richardson, 1987). The sequence will appear in the GenBank/EMBL Nucleotide Sequence Databases with the accession No. M83650. ACKNOWLEDGEMENTS
We thank Viesturs Simanis and Paul Nurse for providing the eDNA library, and Aiister Craig for advice concerning oligo hybridisation.
REFERENCES Adams, A.E,M., Johnson, D,I., Longnecker, R.M., Sloat, B.F. and Pringle, J.R.: CDC42 and CDC43, two additional genes involved in budding and the establishment of cell polarity in the yeast Soccharomyces cerevisiae. J. Cell Biol. 111 ( ! 990) ! 3 ! - 142. Bourne, H.R., Sanders, D.A. and McCormick, F.: The GTPase superfamily: a conserved switch for diverse cell functions. Nature 348 (1990) 125-132. Church, G,M. and Gilbert, W.: Genomic sequencing. Proc. Natl. Acad. Sci. USA 81 (1984) 1991-1995. Fawell, E., Hook, S. and Armstrong, J.: Nucleotide sequence of a gene encoding a YPTl-related protein from Schizosaccharo,o,ces pombe. Nucleic Acids Res. 17 (1989)4373. Fawell, E., Hook, S., Sweet, D. and Armstrong, J.: Novel YPTl-related genes from Schizosaccharo,o,ces pombe. Nucleic Acids Res. 18 (1990) 4264. Hart, MJ., Polakis, P.G., Evans, T. and Cerione, R.A.: The identification and characterization of an epidermal growth factor-stimulated phosphorylation of a specific low molecular weight GTP-binding protein in a reconstituted phospholipid vesicle system. J. Biol. Chem. 265 (1990) 5990-6001.
Hart, M.J., Eva, A., Evans, T., Aaronson, S.A. and Cerione, R.A.: Catalysis of guanine nucleotide exchange on the CDC42Hs protein by the dbl oncogene product. Nature 354 ( 1991) 311-314. Johnson, D.I. and Pringle, J.R.: Molecular characterization of CDC42, a Saccham,O,ces cerevisiae gene involved in the development of cell polarity. J. Cell Biol. 1 ! ! (1990) 143-152. Munemitsu, S., Innis, M.A., Clark, R., McCormick, F., Ullrich, A. and Polakis, P.: Molecular cloning and expression of a G25K eDNA, th~ human homolog of the yeast cell cycle gene CDC42. Mol. Cell. Biol. 10 (1990) 5977-5982. Nasim, A., Young, P. and Johnson, B.F. (Eds.), Molecular Biology of the Fission Yeast. Academic Press, San Diego, CA, 1989. Shinjo, K., Koland, J.G., Hart, M.J., Narasimhan, V., Johnson, D.I., Evans, T. and Cerione, R.A.: Molecular cloning of the gene for the human placental GTP-binding protein Gp (G25K): identification of this GTP-binding protein as the human homolog of the yeast celldivision.cycle protein CDC42. Proc. Natl. Acad. Sci. L ,~A 87 (1990) 9853-9857. Tabor, S. and Richardson, C.C.: DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc. Natl. Acad. Sci. USA 84 (! 987) 4767-4771. Touchot, N., Chardin, P. and Tavitian, A.: Four additional members of the ras gene superfamily isolated by an oligonucleotide strategy: molecular cloning of YPT-related cDNAs from a rat brain library. Proc. Natl. Acad. Sci. USA 84 (1987) 8210-8214. Wood, W.I., Gitschier, J., Lasky, L.A. and Lawn, R.M.: Base composition-independent hybridization in tetramethylammonium chloride: a method for oligonucleotide screening of highly complex gene libraries. Proe. Natl. Acad. Sci. USA 82 (1985) 1585-1588.
© 1992 ElsevierSciencePublishers B.V. All rightsreserved. 0378-1119/92/$05.00
153
GENE 06478
A homologue of the ras-related CDC42 gene from
Schizosaccharomycespombe*
(GTP-binding protein; fission yeast; cell polarity; G25K; Saccharomyces)
Erica Fawell, Sally Bowden and John Armstrong Membrane Molecular Biology Laboratoo,, hnperial Cancer Research Fund, London WC2A 3PX (UK)
Received by R.W. Davies: 9 December 1991 Accepted: 5 January 1992 Received at publishers: 2 March 1992
SUMMARY A cDNA was isolated from the fission yeast, Schizosaccharomycespombe, using mixed oligodeoxyribonucleotides encoding part of the GTP-binding site of the ras superfamily. The encoded protein is the homologue of the budding yeast CDC42 gene product and the human proteins, CDC42Hs and G25K.
The ras superfamily comprises a large group of small GTP-binding proteins, members of which have been implicated in the regulation of a variety of processes including signal transduction, membrane traffic and organisation of the cytoskeleton (Bourne et al., 1990). Examples of these proteins are found in all eukaryotes including the budding yeast S. cerevisiae, in which the functions of several have been investigated by genetic methods. One of these is the CDC42 gene product. Temperature-sensitive mutants in this gene do not form buds; instead material is deposited in a delocalised fashion on the cell surface, suggesting that CDC42 is involved in controlling polarity in the yeast cell (Adams et al., 1990). The gene product falls within the
Correspondenceto: Dr. J. Armstrong,MembraneMolecularBiologyLab-
oratory, Imperial Cancer Research Fund, P.O. Box 123, Lincoln's Inn Fields, London WC2A 3PX (UK) Tel. (44-71)269-3393; Fax (44-71)430-2666. * On request, the authors will supply detailed experimentalevidencefor the conclusionsreached in this BriefNote. Abbreviations: oligo, oligodeoxyribonucleotide;S., Saccharmnyces; Sc., Schi=osaccharomyces; SDS, sodium dodecyi sulfate; Sp (also Sp), S. pombe.
subgroup of ras-related proteins which include the Rho and Rac proteins (Johnson and Pringle, 1990). Surprisingly, close homologues of this protein are found in non-budding eukaryotic cells: two human proteins, CDC42Hs and G25K, are 86% identical to the yeast protein (Shinjo et al., 1990; Munemitsu et al., 1990). G25K has attracted further interest as a good substrate in vitro for the tyrosine kinase activity of the epidermal growth factor receptor (Hart et al., 1990) and for nucleotide exchange by the dbl oncogene product (Hart et al., 1991). The yeast Sc. pombe reproduces by elongation and fission rather than budding. It is very different from S. cerevisiae and in several respects appears to be a closer model of higher eukaryotic cells (Nasim et ai., 1989). Using mixed oligos encoding part of the GTP-binding site conserved throughout the ras family (Touchot et al., 1987), we have isolated several ras-related genes from this organism, and have previously described three members of the ypt/rab group (Fawell et al., 1989; 1990). Here we report a further sequence, isolated from a eDNA library (Fig. 1). The encoded protein, CDC42Sp, is 87%, 82% and 81% identical to CDC42Sc, CDC42Hs and G25K, respectively. Since Sc. pombe, like S. cerevisiae, is amenable to genetic analysis, it may be a useful organism in which to resolve the apparent discrepancy between the proposed function of CDC42 protein in polarised bud formation and its presence in non-budding cells.
154 1 55
GAA ATG 1 M 109 CTG 19L 163 TTC 37 F 217 T T A 55 L 271 CCT 73 P 325 G A A 91 E 379 CCG 109 P 433 CAG 127 Q 487 TTA 145 L 5,11 AAA 163 K 595 GTT 181 V 649 CTT 703 CTT 757 TTT 811 GGC 865 AAC 919 GCA
TTC CCC P CTT L GAT D TTC F CAA Q AAT N TGC C AAA K GCT A GGG G CCT P TCC AGC CAT TTT GAC GGA
AAT ACC T ATT I AAT N GAT D ACA T GTG V TTA L CTA L CGT
R TTA L CAC H TTT TTT CCT ACA GAT ATT
AAA ATT I TCC S TAT Y ACC T GAC D AAA K ATT I GCT A GAG E AAG K AAG K TCC CTT AGC TAT TTT C
GTG AAG K TAT Y GCT A GCT A GTC V GAA E GTT V CGC R TTG L AAT N AAA K GAA TAA TTT CTG GCT
AAG TGT C ACT T GTC V GGT G TTT F AAG K GGT G CAG Q GGT G GTT V AAG K ATT CGA TGT TGT ATA
CAA GTC V ACA T ACT T CAG Q TTG L TGG W ACC T CAT H GCT A TTT F TCA S TCC TCC GCT TTT CTT
AGC GTA V AAC N GTC V GAG E GTT V TTT F CAA Q CAG Q GTC V GAT D AAG K TTA CTC TCT CTG TTG
TTT GTA V AAG K ATG M GAT D TGT C CCC P ATT I CAT H AAG K GAA E TGT C ATA ATA TCA GCT CTA
ACG GGA G TTT F ATC I TAT Y TTT F GAA E GAT D CCC P TAT Y GCT A TTG L CAG TTT TTC AGG CAT
ATT GAC D CCT P GGT G GAT D AGT S GTT V TTA L CTT L GTT V ATT I GTA V CTA GCA ATT CAT TAT
AAT GGT G AGT S GAT D CGC R GTA V CAT H CGT
TAT GCT A GAC D GAA E TTG L ACT T CAT H GAT
TTT GTA V TAT Y CCA P CGT R TCT S CAT H GAC
TTG GGA G GTG V TAC Y CCT P CCT P TGT C CCT CAA Q GCT A CTT L AAC
TGA AAG K CCA P ACT T TTA L GCC A CCG P TCT S GGT G TTG L GAT D CCT
AAT ACC T ACT T CTT L TCC S AGT S GGC G GTG V GAA E ACC T CCT P GGT
AGT TGT C GTA V GGT G TAT Y TTT F GTC V CAA Q CGA R CAA Q CCT P TTT
R
D
D
P
ACA T GAG E GTA V CTG L GTT AAG CTG ATT TGT
CAT H TGT C GCC A TAA
GAG E TCC S GCT A TTT
TAC ATA TCA TCC ATT
AAC TTT ATC TTT CTT
AGT GCT ACA TTC CTG
AAA ATT CAC CGT CCA
CAA TGT TTC TAA TTA
TGT TAT TTG ATA CTC
Fig. 1. The sequence of the CDC42Sp cDNA and encoded protein. The cDNA was isolated from a eDNA library of Sc. pombe in 2gtl I (provided by V. Simanis and P. Nurse, University of Oxford) by hybridisation to an equimolar mixture of the oligos 5'-GATACKGCKGGKCAGGAGCG and 5'-CGTrCTrGKCCKGCKGTGTC (K = G or T) (Touchot et al., 1987). Hybridisation was in 0.5 M Na.phosphate, pH 7.2/8% SDS/I mM EDTA/ 1% bovine serum albumin (Church and Gilbert, 1984) and filters were washed in 3 M tetramethyl ammonium chloride/50 mM Tris.HCI, pH 7.6/2 mM EDTA/0.1% SDS (Wood et al., 1985) at 59 ° C. After plaque purification, the eDNA was excised with EcoRl, subcloned into plasmids and the sequence determined using modified T7 polymerase (Tabor and Richardson, 1987). The sequence will appear in the GenBank/EMBL Nucleotide Sequence Databases with the accession No. M83650. ACKNOWLEDGEMENTS
We thank Viesturs Simanis and Paul Nurse for providing the eDNA library, and Aiister Craig for advice concerning oligo hybridisation.
REFERENCES Adams, A.E,M., Johnson, D,I., Longnecker, R.M., Sloat, B.F. and Pringle, J.R.: CDC42 and CDC43, two additional genes involved in budding and the establishment of cell polarity in the yeast Soccharomyces cerevisiae. J. Cell Biol. 111 ( ! 990) ! 3 ! - 142. Bourne, H.R., Sanders, D.A. and McCormick, F.: The GTPase superfamily: a conserved switch for diverse cell functions. Nature 348 (1990) 125-132. Church, G,M. and Gilbert, W.: Genomic sequencing. Proc. Natl. Acad. Sci. USA 81 (1984) 1991-1995. Fawell, E., Hook, S. and Armstrong, J.: Nucleotide sequence of a gene encoding a YPTl-related protein from Schizosaccharo,o,ces pombe. Nucleic Acids Res. 17 (1989)4373. Fawell, E., Hook, S., Sweet, D. and Armstrong, J.: Novel YPTl-related genes from Schizosaccharo,o,ces pombe. Nucleic Acids Res. 18 (1990) 4264. Hart, MJ., Polakis, P.G., Evans, T. and Cerione, R.A.: The identification and characterization of an epidermal growth factor-stimulated phosphorylation of a specific low molecular weight GTP-binding protein in a reconstituted phospholipid vesicle system. J. Biol. Chem. 265 (1990) 5990-6001.
Hart, M.J., Eva, A., Evans, T., Aaronson, S.A. and Cerione, R.A.: Catalysis of guanine nucleotide exchange on the CDC42Hs protein by the dbl oncogene product. Nature 354 ( 1991) 311-314. Johnson, D.I. and Pringle, J.R.: Molecular characterization of CDC42, a Saccham,O,ces cerevisiae gene involved in the development of cell polarity. J. Cell Biol. 1 ! ! (1990) 143-152. Munemitsu, S., Innis, M.A., Clark, R., McCormick, F., Ullrich, A. and Polakis, P.: Molecular cloning and expression of a G25K eDNA, th~ human homolog of the yeast cell cycle gene CDC42. Mol. Cell. Biol. 10 (1990) 5977-5982. Nasim, A., Young, P. and Johnson, B.F. (Eds.), Molecular Biology of the Fission Yeast. Academic Press, San Diego, CA, 1989. Shinjo, K., Koland, J.G., Hart, M.J., Narasimhan, V., Johnson, D.I., Evans, T. and Cerione, R.A.: Molecular cloning of the gene for the human placental GTP-binding protein Gp (G25K): identification of this GTP-binding protein as the human homolog of the yeast celldivision.cycle protein CDC42. Proc. Natl. Acad. Sci. L ,~A 87 (1990) 9853-9857. Tabor, S. and Richardson, C.C.: DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc. Natl. Acad. Sci. USA 84 (! 987) 4767-4771. Touchot, N., Chardin, P. and Tavitian, A.: Four additional members of the ras gene superfamily isolated by an oligonucleotide strategy: molecular cloning of YPT-related cDNAs from a rat brain library. Proc. Natl. Acad. Sci. USA 84 (1987) 8210-8214. Wood, W.I., Gitschier, J., Lasky, L.A. and Lawn, R.M.: Base composition-independent hybridization in tetramethylammonium chloride: a method for oligonucleotide screening of highly complex gene libraries. Proe. Natl. Acad. Sci. USA 82 (1985) 1585-1588.