Bni1p, a yeast formin linking Cdc42p and the actin cytoskeleton during polarized morphogenesis

Science

Volumn 276, Issue 5309, 1997, Pages 118-122

  Evangelista, Marie ^a   Blundell, Kelly ^a   Longtine, Mark S ^b   Chow, Clinton J ^a   Adames, Neil ^a   Pringle, John R ^b   Peter, Matthias ^c   Boone, Charles ^a  

^a SIMON FRASER UNIVERSITY   (Canada)

^b UNIVERSITY OF NORTH CAROLINA   (United States)

^c SWISS INST FOR EXP CANCER RESEARCH   (Switzerland)

Author keywords

[No Author keywords available]

Indexed keywords

ACTIN;

ARTICLE; CELL POLARITY; CYTOKINESIS; CYTOSKELETON; GENE ISOLATION; NONHUMAN; PRIORITY JOURNAL; SACCHAROMYCES CEREVISIAE;

ACTINS; CDC42 GTP-BINDING PROTEIN, SACCHAROMYCES CEREVISIAE; CELL CYCLE PROTEINS; CONTRACTILE PROTEINS; CYTOSKELETON; FUNGAL PROTEINS; GTP-BINDING PROTEINS; MICROFILAMENT PROTEINS; MORPHOGENESIS; MUTAGENESIS; PROFILINS; RECOMBINANT FUSION PROTEINS; SACCHAROMYCES CEREVISIAE; SACCHAROMYCES CEREVISIAE PROTEINS; SIGNAL TRANSDUCTION;

SACCHAROMYCES CEREVISIAE; VERTEBRATA;

EID: 0030932405     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.276.5309.118     Document Type: Article

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57. 5 potential transformants, 11 positive plasmids were isolated, all of which contained full-length ACT1 cDNAs.
58. D. C. Amberg, J. E. Zahner, J. W. Mulholland, J. R. Pringle, D. Botstein, Mol. Biol. Cell, in press.
59. Y773 is an SY2625 (11) derivative carrying a bud6Δ::URA3 mutation, created with Hind III-Sst I-cut p1055 (26). Upon exposure to pheromone in a projection assay (12), 99% of the Y773 cells were unbudded, and 17% of these cells also formed a polarized mating projection. Under similar conditions, 68% of wild-type cells and 2% of bni1Δ::URA3 cells formed polarized projections (12).
60. Like bni1 and bud6 mutant cells (9, 10, 23), act1-120 cells are defective for bipolar budding and develop a round cell shape with wide bud necks [D. G. Drubin, H. D. Jones, K. F. Wertman, Mol. Biol. Cell. 4, 1277 (1993); S. Yang, K. Ayscough, D. G. Drubin, J. Cell Biol. 137, 111 (1997)]. DBY5551 (MATa/MATα act1-120::HIS3/ACT1 tub2/TUB2 his3-200/his3-200 leu2/leu2 ura3/ura3 ade2/ ADE2 can1/CAN1 cry1/CRY1 ade4/ADE4) [K. F. Wertman, D. G. Drubin, D. Botstein, Genetics 132, 337 (1992)] was transformed with Hind III-Xho I-cut p321 (26) to create strain Y821, heterozygous for a bni1Δ::URA3 mutation. Y821 was sporulated, and tetrads were dissected on rich medium at 23°C. From 86 tetrads, 79 of the viable spore progeny were bni1Δ::URA3 ACT1, 79 were BNI1 act1-120::HIS3, and 93 were BNI1 ACT1. The 93 presumed bni1Δ::URA3 act1-120::HIS3 double mutants either failed to germinate or formed microcolonies too small to characterize further.
61. Like bni1 and bud6 mutant cells (9, 10, 23), act1-120 cells are defective for bipolar budding and develop a round cell shape with wide bud necks [D. G. Drubin, H. D. Jones, K. F. Wertman, Mol. Biol. Cell. 4, 1277 (1993); S. Yang, K. Ayscough, D. G. Drubin, J. Cell Biol. 137, 111 (1997)]. DBY5551 (MATa/MATα act1-120::HIS3/ACT1 tub2/TUB2 his3-200/his3-200 leu2/leu2 ura3/ura3 ade2/ ADE2 can1/CAN1 cry1/CRY1 ade4/ADE4) [K. F. Wertman, D. G. Drubin, D. Botstein, Genetics 132, 337 (1992)] was transformed with Hind III-Xho I-cut p321 (26) to create strain Y821, heterozygous for a bni1Δ::URA3 mutation. Y821 was sporulated, and tetrads were dissected on rich medium at 23°C. From 86 tetrads, 79 of the viable spore progeny were bni1Δ::URA3 ACT1, 79 were BNI1 act1-120::HIS3, and 93 were BNI1 ACT1. The 93 presumed bni1Δ::URA3 act1-120::HIS3 double mutants either failed to germinate or formed microcolonies too small to characterize further.
62. Like bni1 and bud6 mutant cells (9, 10, 23), act1-120 cells are defective for bipolar budding and develop a round cell shape with wide bud necks [D. G. Drubin, H. D. Jones, K. F. Wertman, Mol. Biol. Cell. 4, 1277 (1993); S. Yang, K. Ayscough, D. G. Drubin, J. Cell Biol. 137, 111 (1997)]. DBY5551 (MATa/MATα act1-120::HIS3/ACT1 tub2/TUB2 his3-200/his3-200 leu2/leu2 ura3/ura3 ade2/ ADE2 can1/CAN1 cry1/CRY1 ade4/ADE4) [K. F. Wertman, D. G. Drubin, D. Botstein, Genetics 132, 337 (1992)] was transformed with Hind III-Xho I-cut p321 (26) to create strain Y821, heterozygous for a bni1Δ::URA3 mutation. Y821 was sporulated, and tetrads were dissected on rich medium at 23°C. From 86 tetrads, 79 of the viable spore progeny were bni1Δ::URA3 ACT1, 79 were BNI1 act1-120::HIS3, and 93 were BNI1 ACT1. The 93 presumed bni1Δ::URA3 act1-120::HIS3 double mutants either failed to germinate or formed microcolonies too small to characterize further.
63. Plasmid p182 carries an 8.5-kb fragment that contains BNI1 isolated from a yeast genomic library [C. Boone, K. L. Clark, G. F. Sprague Jr., Nucleic Acids Res. 20, 4661 (1992)] in vector pRS316 [R. S. Sikorski and P. Hieter, Genetics 122, 19 (1989)] by complementation of the bni1-11 mating defect. In p182, the BNI1 open reading frame is directed toward the Not I site of the pRS316 polylinker, and the entire BNI1 genomic insert can be removed from the vector as an 8.5-kb Bam HI to Not I fragment. To create plasmid p321, containing a bni1Δ::URA3 allele, we cloned a 2.4-kb Xho I-Hind III fragment from p182 into KS+ (Stratagene), and inserted a URA3 fragment into the BgI II sites within BNI1; this deleted the BNI1 sequence encoding amino acids 1229 to 1415 and left the downstream BNI1 sequence out of frame. pY39tet1 is a multicopy plasmid that contains full-length Bni1p with a COOH-terminal tag composed of four tandem copies of the HA epitope (9). To create plasmid p532, carrying BNI1 with a Bam HI site just upstream of its ATG codon, we amplified a 1.3-kb fragment of BNI1 by the polymerase chain reaction with primers (5′-TGGATCCGCGAAATGTTGAAGAATCTAGGCTCC-3′ and 5′-AGCGGCCGCTTAAGTGGCCATTTTCCTTGTAGCCAGTTTCGTAGAAAGTAAACC-3′) that incorporated Bam HI and Msc I sites (underlined), and the product was ligated into p182 cut with Bam HI and Msc I. p527 is a pRS316-based vector that carries the GAL1 promoter regulating a sequence encoding three copies of the HA epitope [P. A. Kolodziej and R. A. Young, Methods Enzymol. 194, 508 (1991)] followed by Bam HI and Not I cloning sites. p827 contains the 3.6-kb Bam HI to Eco 47III fragment of p532, encoding Bni1p(1-1214), ligated in frame with the HA epitope sequences of p527. p828 is an expression vector similar to p827 except that it lacks the HA epitope sequence. p1025 contains the 3.9-kb Eco 47III to Not I fragment of p182 [encoding Bni1p(1215-1953)], ligated in frame with the HA epitope sequence of p527. p907 contains a Bam HI to Not I fragment, containing a full-length profilin cDNA (16), ligated in frame with the GST sequences of pGEX-3X (Pharmacia). To create p1055, carrying a bud6::URA3 allele, we ligated a 2.2-kb Hind III-Sst I fragment of BUD6 (AIP3) (23) into KS+ and inserted a URA3 fragment at the Sna BI site, thus disrupting BUD6 after its ninth codon. For integration of GAL1-BNI1 and GAL1-BNI1ΔN at the ADE8 locus, the Bam HI to Not I fragment from p532 (encoding full-length Bni1p) and the Msc I to Not I fragment from p182 [encoding Bni1p(452-1953)] were ligated downstream of the GAL1 promoter in p407, creating p849 and p414, respectively. p407 is a Ylplac211-based vector [R. D. Gietz and A. Sugino, Gene 74, 527 (1988)] that contains the GAL1 promoter inserted within an ADE8 fragment. Digestion of p407-derived plasmids with Nru I (or Msc I) targeted integration to ADE8. For integration of GAL1-lexABNI1ΔN at ADE8, a fragment encoding the LexA DBD was introduced upstream of BNI1ΔN in p414, creating p529. p850 contains the BNI1Δ2 Eco47 III-Not I fragment from p182 [encoding Bni1p(1215-1953)] ligated downstream of the GAL1 promoter in p407. For integration of GAL1-lexABNI1ΔN2 at ADE8, a fragment encoding the LexA DBD was introduced upstream of BNI1ΔN2 in p850, creating p997. pSH18-34Δspe is an integrating plasmid that carries URA3 and a lexAop-lacZ reporter. pSH18-34Δspe was targeted for integration at URA3 by digestion with Apa I.
64. Plasmid p182 carries an 8.5-kb fragment that contains BNI1 isolated from a yeast genomic library [C. Boone, K. L. Clark, G. F. Sprague Jr., Nucleic Acids Res. 20, 4661 (1992)] in vector pRS316 [R. S. Sikorski and P. Hieter, Genetics 122, 19 (1989)] by complementation of the bni1-11 mating defect. In p182, the BNI1 open reading frame is directed toward the Not I site of the pRS316 polylinker, and the entire BNI1 genomic insert can be removed from the vector as an 8.5-kb Bam HI to Not I fragment. To create plasmid p321, containing a bni1Δ::URA3 allele, we cloned a 2.4-kb Xho I-Hind III fragment from p182 into KS+ (Stratagene), and inserted a URA3 fragment into the BgI II sites within BNI1; this deleted the BNI1 sequence encoding amino acids 1229 to 1415 and left the downstream BNI1 sequence out of frame. pY39tet1 is a multicopy plasmid that contains full-length Bni1p with a COOH-terminal tag composed of four tandem copies of the HA epitope (9). To create plasmid p532, carrying BNI1 with a Bam HI site just upstream of its ATG codon, we amplified a 1.3-kb fragment of BNI1 by the polymerase chain reaction with primers (5′-TGGATCCGCGAAATGTTGAAGAATCTAGGCTCC-3′ and 5′-AGCGGCCGCTTAAGTGGCCATTTTCCTTGTAGCCAGTTTCGTAGAAAGTAAACC-3′) that incorporated Bam HI and Msc I sites (underlined), and the product was ligated into p182 cut with Bam HI and Msc I. p527 is a pRS316-based vector that carries the GAL1 promoter regulating a sequence encoding three copies of the HA epitope [P. A. Kolodziej and R. A. Young, Methods Enzymol. 194, 508 (1991)] followed by Bam HI and Not I cloning sites. p827 contains the 3.6-kb Bam HI to Eco 47III fragment of p532, encoding Bni1p(1-1214), ligated in frame with the HA epitope sequences of p527. p828 is an expression vector similar to p827 except that it lacks the HA epitope sequence. p1025 contains the 3.9-kb Eco 47III to Not I fragment of p182 [encoding Bni1p(1215-1953)], ligated in frame with the HA epitope sequence of p527. p907 contains a Bam HI to Not I fragment, containing a full-length profilin cDNA (16), ligated in frame with the GST sequences of pGEX-3X (Pharmacia). To create p1055, carrying a bud6::URA3 allele, we ligated a 2.2-kb Hind III-Sst I fragment of BUD6 (AIP3) (23) into KS+ and inserted a URA3 fragment at the Sna BI site, thus disrupting BUD6 after its ninth codon. For integration of GAL1-BNI1 and GAL1-BNI1ΔN at the ADE8 locus, the Bam HI to Not I fragment from p532 (encoding full-length Bni1p) and the Msc I to Not I fragment from p182 [encoding Bni1p(452-1953)] were ligated downstream of the GAL1 promoter in p407, creating p849 and p414, respectively. p407 is a Ylplac211-based vector [R. D. Gietz and A. Sugino, Gene 74, 527 (1988)] that contains the GAL1 promoter inserted within an ADE8 fragment. Digestion of p407-derived plasmids with Nru I (or Msc I) targeted integration to ADE8. For integration of GAL1-lexABNI1ΔN at ADE8, a fragment encoding the LexA DBD was introduced upstream of BNI1ΔN in p414, creating p529. p850 contains the BNI1Δ2 Eco47 III-Not I fragment from p182 [encoding Bni1p(1215-1953)] ligated downstream of the GAL1 promoter in p407. For integration of GAL1-lexABNI1ΔN2 at ADE8, a fragment encoding the LexA DBD was introduced upstream of BNI1ΔN2 in p850, creating p997. pSH18-34Δspe is an integrating plasmid that carries URA3 and a lexAop-lacZ reporter. pSH18-34Δspe was targeted for integration at URA3 by digestion with Apa I.
65. Plasmid p182 carries an 8.5-kb fragment that contains BNI1 isolated from a yeast genomic library [C. Boone, K. L. Clark, G. F. Sprague Jr., Nucleic Acids Res. 20, 4661 (1992)] in vector pRS316 [R. S. Sikorski and P. Hieter, Genetics 122, 19 (1989)] by complementation of the bni1-11 mating defect. In p182, the BNI1 open reading frame is directed toward the Not I site of the pRS316 polylinker, and the entire BNI1 genomic insert can be removed from the vector as an 8.5-kb Bam HI to Not I fragment. To create plasmid p321, containing a bni1Δ::URA3 allele, we cloned a 2.4-kb Xho I-Hind III fragment from p182 into KS+ (Stratagene), and inserted a URA3 fragment into the BgI II sites within BNI1; this deleted the BNI1 sequence encoding amino acids 1229 to 1415 and left the downstream BNI1 sequence out of frame. pY39tet1 is a multicopy plasmid that contains full-length Bni1p with a COOH-terminal tag composed of four tandem copies of the HA epitope (9). To create plasmid p532, carrying BNI1 with a Bam HI site just upstream of its ATG codon, we amplified a 1.3-kb fragment of BNI1 by the polymerase chain reaction with primers (5′-TGGATCCGCGAAATGTTGAAGAATCTAGGCTCC-3′ and 5′-AGCGGCCGCTTAAGTGGCCATTTTCCTTGTAGCCAGTTTCGTAGAAAGTAAACC-3′) that incorporated Bam HI and Msc I sites (underlined), and the product was ligated into p182 cut with Bam HI and Msc I. p527 is a pRS316-based vector that carries the GAL1 promoter regulating a sequence encoding three copies of the HA epitope [P. A. Kolodziej and R. A. Young, Methods Enzymol. 194, 508 (1991)] followed by Bam HI and Not I cloning sites. p827 contains the 3.6-kb Bam HI to Eco 47III fragment of p532, encoding Bni1p(1-1214), ligated in frame with the HA epitope sequences of p527. p828 is an expression vector similar to p827 except that it lacks the HA epitope sequence. p1025 contains the 3.9-kb Eco 47III to Not I fragment of p182 [encoding Bni1p(1215-1953)], ligated in frame with the HA epitope sequence of p527. p907 contains a Bam HI to Not I fragment, containing a full-length profilin cDNA (16), ligated in frame with the GST sequences of pGEX-3X (Pharmacia). To create p1055, carrying a bud6::URA3 allele, we ligated a 2.2-kb Hind III-Sst I fragment of BUD6 (AIP3) (23) into KS+ and inserted a URA3 fragment at the Sna BI site, thus disrupting BUD6 after its ninth codon. For integration of GAL1-BNI1 and GAL1-BNI1ΔN at the ADE8 locus, the Bam HI to Not I fragment from p532 (encoding full-length Bni1p) and the Msc I to Not I fragment from p182 [encoding Bni1p(452-1953)] were ligated downstream of the GAL1 promoter in p407, creating p849 and p414, respectively. p407 is a Ylplac211-based vector [R. D. Gietz and A. Sugino, Gene 74, 527 (1988)] that contains the GAL1 promoter inserted within an ADE8 fragment. Digestion of p407-derived plasmids with Nru I (or Msc I) targeted integration to ADE8. For integration of GAL1-lexABNI1ΔN at ADE8, a fragment encoding the LexA DBD was introduced upstream of BNI1ΔN in p414, creating p529. p850 contains the BNI1Δ2 Eco47 III-Not I fragment from p182 [encoding Bni1p(1215-1953)] ligated downstream of the GAL1 promoter in p407. For integration of GAL1-lexABNI1ΔN2 at ADE8, a fragment encoding the LexA DBD was introduced upstream of BNI1ΔN2 in p850, creating p997. pSH18-34Δspe is an integrating plasmid that carries URA3 and a lexAop-lacZ reporter. pSH18-34Δspe was targeted for integration at URA3 by digestion with Apa I.
66. Plasmid p182 carries an 8.5-kb fragment that contains BNI1 isolated from a yeast genomic library [C. Boone, K. L. Clark, G. F. Sprague Jr., Nucleic Acids Res. 20, 4661 (1992)] in vector pRS316 [R. S. Sikorski and P. Hieter, Genetics 122, 19 (1989)] by complementation of the bni1-11 mating defect. In p182, the BNI1 open reading frame is directed toward the Not I site of the pRS316 polylinker, and the entire BNI1 genomic insert can be removed from the vector as an 8.5-kb Bam HI to Not I fragment. To create plasmid p321, containing a bni1Δ::URA3 allele, we cloned a 2.4-kb Xho I-Hind III fragment from p182 into KS+ (Stratagene), and inserted a URA3 fragment into the BgI II sites within BNI1; this deleted the BNI1 sequence encoding amino acids 1229 to 1415 and left the downstream BNI1 sequence out of frame. pY39tet1 is a multicopy plasmid that contains full-length Bni1p with a COOH-terminal tag composed of four tandem copies of the HA epitope (9). To create plasmid p532, carrying BNI1 with a Bam HI site just upstream of its ATG codon, we amplified a 1.3-kb fragment of BNI1 by the polymerase chain reaction with primers (5′-TGGATCCGCGAAATGTTGAAGAATCTAGGCTCC-3′ and 5′-AGCGGCCGCTTAAGTGGCCATTTTCCTTGTAGCCAGTTTCGTAGAAAGTAAACC-3′) that incorporated Bam HI and Msc I sites (underlined), and the product was ligated into p182 cut with Bam HI and Msc I. p527 is a pRS316-based vector that carries the GAL1 promoter regulating a sequence encoding three copies of the HA epitope [P. A. Kolodziej and R. A. Young, Methods Enzymol. 194, 508 (1991)] followed by Bam HI and Not I cloning sites. p827 contains the 3.6-kb Bam HI to Eco 47III fragment of p532, encoding Bni1p(1-1214), ligated in frame with the HA epitope sequences of p527. p828 is an expression vector similar to p827 except that it lacks the HA epitope sequence. p1025 contains the 3.9-kb Eco 47III to Not I fragment of p182 [encoding Bni1p(1215-1953)], ligated in frame with the HA epitope sequence of p527. p907 contains a Bam HI to Not I fragment, containing a full-length profilin cDNA (16), ligated in frame with the GST sequences of pGEX-3X (Pharmacia). To create p1055, carrying a bud6::URA3 allele, we ligated a 2.2-kb Hind III-Sst I fragment of BUD6 (AIP3) (23) into KS+ and inserted a URA3 fragment at the Sna BI site, thus disrupting BUD6 after its ninth codon. For integration of GAL1-BNI1 and GAL1-BNI1ΔN at the ADE8 locus, the Bam HI to Not I fragment from p532 (encoding full-length Bni1p) and the Msc I to Not I fragment from p182 [encoding Bni1p(452-1953)] were ligated downstream of the GAL1 promoter in p407, creating p849 and p414, respectively. p407 is a Ylplac211-based vector [R. D. Gietz and A. Sugino, Gene 74, 527 (1988)] that contains the GAL1 promoter inserted within an ADE8 fragment. Digestion of p407-derived plasmids with Nru I (or Msc I) targeted integration to ADE8. For integration of GAL1-lexABNI1ΔN at ADE8, a fragment encoding the LexA DBD was introduced upstream of BNI1ΔN in p414, creating p529. p850 contains the BNI1Δ2 Eco47 III-Not I fragment from p182 [encoding Bni1p(1215-1953)] ligated downstream of the GAL1 promoter in p407. For integration of GAL1-lexABNI1ΔN2 at ADE8, a fragment encoding the LexA DBD was introduced upstream of BNI1ΔN2 in p850, creating p997. pSH18-34Δspe is an integrating plasmid that carries URA3 and a lexAop-lacZ reporter. pSH18-34Δspe was targeted for integration at URA3 by digestion with Apa I.
67. 2 1 mM dithiothreitol, 0.1% Triton X-100], the bound proteins were eluted by boiling in SDS-polyacrylamide gel electrophoresis sample buffer and subjected to immunoblot analysis with antibodies to Cdc42p [M. Peter et al., EMBO J. 15, 7046 (1996)] or HA.11 as described [M. Peter, A. Gartner, J. Horecka, G. Ammerer, I. Herskowitz, Cell 73, 747 (1993)].
68. 2 1 mM dithiothreitol, 0.1% Triton X-100], the bound proteins were eluted by boiling in SDS-polyacrylamide gel electrophoresis sample buffer and subjected to immunoblot analysis with antibodies to Cdc42p [M. Peter et al., EMBO J. 15, 7046 (1996)] or HA.11 as described [M. Peter, A. Gartner, J. Horecka, G. Ammerer, I. Herskowitz, Cell 73, 747 (1993)].
69. 2 1 mM dithiothreitol, 0.1% Triton X-100], the bound proteins were eluted by boiling in SDS-polyacrylamide gel electrophoresis sample buffer and subjected to immunoblot analysis with antibodies to Cdc42p [M. Peter et al., EMBO J. 15, 7046 (1996)] or HA.11 as described [M. Peter, A. Gartner, J. Horecka, G. Ammerer, I. Herskowitz, Cell 73, 747 (1993)].
70. 2 1 mM dithiothreitol, 0.1% Triton X-100], the bound proteins were eluted by boiling in SDS-polyacrylamide gel electrophoresis sample buffer and subjected to immunoblot analysis with antibodies to Cdc42p [M. Peter et al., EMBO J. 15, 7046 (1996)] or HA.11 as described [M. Peter, A. Gartner, J. Horecka, G. Ammerer, I. Herskowitz, Cell 73, 747 (1993)].
71. Two-hybrid experiments [E. M. Phizicky and S. Fields, Microbiol Rev. 59, 94 (1995)] were performed with strain Y704 (MATa lexAop-LEU2 lexAop-lacZ sst1Δ his3 trp1 ura3-52 leu2) or Y523 (MATa lexAop-lacZ leu2 his3 trp1 ade2 lys2 gal80 GAL4). These strains were transformed with both a LexA DNA-binding domain (DBD) plasmid and a transcriptional-activation domain (AD) plasmid, then assayed for expression of lexAop-lacZ. Cells were grown to mid-log phase in medium containing 2% raffinose, galactose was added to a concentration of 2%, and growth was continued for 4 hours before assaying for β-galactosidase activity (72). Each value represents the average and standard deviation for three independent cultures. The DBD plasmids were pEG202 [J. Gyuris, E. Golemis, H. Chertkov, R. Brent, Cell 75, 791 (1993)] derivatives expressing DBD fusion proteins. The AD plasmids were pJG4-5 (J. Gyuris et al., ibid., p. 791), pGAD-C [P. James, J. Halladay, E. A. Craig, Genetics 144, 1425 (1996)]. pACT [T. Durfee et al., Genes Dev. 7, 555 (1993)], and derivatives expressing AD fusion proteins. DBD-Cdc42p fusions (5) all incorporate the C188S substitution, which prevents prenylation. Other DBD plasmids used were p888, which contains a Bam HI to Not I fragment encoding a full-length profilin cDNA (76); p989, which encodes a mutant form of profilin, Pfy1p-3, lacking the last three amino acids (18); p890, which contains the BgI II to Stu I fragment from p182 (26), encoding Bni1p(1227-1397); p813, which contains the BgI II to Not I fragment from p182, encoding Bni1p(1414-1953); and p951, which contains the Hpa I to Not I fragment from p182, encoding Bni1p(1647-1953). The pJG4-5-derived plasmids were p561, which contains the Bam HI to Not I fragment from p532 (26), encoding Bni1p(1-1953); p717, which contains the Bam HI to Eco47 III fragment from p532, encoding Bni1p(1-1214); p558, which contains the Eco 47III to Not I fragment from p182, encoding Bni1p(1215-1953); p913, which contains the BgI II to Stu I fragment from p182, encoding Bni1p(1227-1397); p929, which contains the BgI II to Not I fragment from p182, encoding Bni1p(1414-1953); p952, which contains the Hpa I to Not I fragment from p182, encoding Bni1p(1647-1953); and p887, which contains the Bam HI to Not I fragment encoding a full-length profilin cDNA (76). The pACT-derived plasmid was p1124, encoding full-length Act1p as isolated in a catch and release screen (22). The pGAD-C-derived plasmid was p688, encoding the COOH-terminal 311 amino acids (478-788) of Bud6p, as isolated in a catch and release screen (22).
72. Two-hybrid experiments [E. M. Phizicky and S. Fields, Microbiol Rev. 59, 94 (1995)] were performed with strain Y704 (MATa lexAop-LEU2 lexAop-lacZ sst1Δ his3 trp1 ura3-52 leu2) or Y523 (MATa lexAop-lacZ leu2 his3 trp1 ade2 lys2 gal80 GAL4). These strains were transformed with both a LexA DNA-binding domain (DBD) plasmid and a transcriptional-activation domain (AD) plasmid, then assayed for expression of lexAop-lacZ. Cells were grown to mid-log phase in medium containing 2% raffinose, galactose was added to a concentration of 2%, and growth was continued for 4 hours before assaying for β-galactosidase activity (72). Each value represents the average and standard deviation for three independent cultures. The DBD plasmids were pEG202 [J. Gyuris, E. Golemis, H. Chertkov, R. Brent, Cell 75, 791 (1993)] derivatives expressing DBD fusion proteins. The AD plasmids were pJG4-5 (J. Gyuris et al., ibid., p. 791), pGAD-C [P. James, J. Halladay, E. A. Craig, Genetics 144, 1425 (1996)]. pACT [T. Durfee et al., Genes Dev. 7, 555 (1993)], and derivatives expressing AD fusion proteins. DBD-Cdc42p fusions (5) all incorporate the C188S substitution, which prevents prenylation. Other DBD plasmids used were p888, which contains a Bam HI to Not I fragment encoding a full-length profilin cDNA (76); p989, which encodes a mutant form of profilin, Pfy1p-3, lacking the last three amino acids (18); p890, which contains the BgI II to Stu I fragment from p182 (26), encoding Bni1p(1227-1397); p813, which contains the BgI II to Not I fragment from p182, encoding Bni1p(1414-1953); and p951, which contains the Hpa I to Not I fragment from p182, encoding Bni1p(1647-1953). The pJG4-5-derived plasmids were p561, which contains the Bam HI to Not I fragment from p532 (26), encoding Bni1p(1-1953); p717, which contains the Bam HI to Eco47 III fragment from p532, encoding Bni1p(1-1214); p558, which contains the Eco 47III to Not I fragment from p182, encoding Bni1p(1215-1953); p913, which contains the BgI II to Stu I fragment from p182, encoding Bni1p(1227-1397); p929, which contains the BgI II to Not I fragment from p182, encoding Bni1p(1414-1953); p952, which contains the Hpa I to Not I fragment from p182, encoding Bni1p(1647-1953); and p887, which contains the Bam HI to Not I fragment encoding a full-length profilin cDNA (76). The pACT-derived plasmid was p1124, encoding full-length Act1p as isolated in a catch and release screen (22). The pGAD-C-derived plasmid was p688, encoding the COOH-terminal 311 amino acids (478-788) of Bud6p, as isolated in a catch and release screen (22).
73. Two-hybrid experiments [E. M. Phizicky and S. Fields, Microbiol Rev. 59, 94 (1995)] were performed with strain Y704 (MATa lexAop-LEU2 lexAop-lacZ sst1Δ his3 trp1 ura3-52 leu2) or Y523 (MATa lexAop-lacZ leu2 his3 trp1 ade2 lys2 gal80 GAL4). These strains were transformed with both a LexA DNA-binding domain (DBD) plasmid and a transcriptional-activation domain (AD) plasmid, then assayed for expression of lexAop-lacZ. Cells were grown to mid-log phase in medium containing 2% raffinose, galactose was added to a concentration of 2%, and growth was continued for 4 hours before assaying for β-galactosidase activity (72). Each value represents the average and standard deviation for three independent cultures. The DBD plasmids were pEG202 [J. Gyuris, E. Golemis, H. Chertkov, R. Brent, Cell 75, 791 (1993)] derivatives expressing DBD fusion proteins. The AD plasmids were pJG4-5 (J. Gyuris et al., ibid., p. 791), pGAD-C [P. James, J. Halladay, E. A. Craig, Genetics 144, 1425 (1996)]. pACT [T. Durfee et al., Genes Dev. 7, 555 (1993)], and derivatives expressing AD fusion proteins. DBD-Cdc42p fusions (5) all incorporate the C188S substitution, which prevents prenylation. Other DBD plasmids used were p888, which contains a Bam HI to Not I fragment encoding a full-length profilin cDNA (76); p989, which encodes a mutant form of profilin, Pfy1p-3, lacking the last three amino acids (18); p890, which contains the BgI II to Stu I fragment from p182 (26), encoding Bni1p(1227-1397); p813, which contains the BgI II to Not I fragment from p182, encoding Bni1p(1414-1953); and p951, which contains the Hpa I to Not I fragment from p182, encoding Bni1p(1647-1953). The pJG4-5-derived plasmids were p561, which contains the Bam HI to Not I fragment from p532 (26), encoding Bni1p(1-1953); p717, which contains the Bam HI to Eco47 III fragment from p532, encoding Bni1p(1-1214); p558, which contains the Eco 47III to Not I fragment from p182, encoding Bni1p(1215-1953); p913, which contains the BgI II to Stu I fragment from p182, encoding Bni1p(1227-1397); p929, which contains the BgI II to Not I fragment from p182, encoding Bni1p(1414-1953); p952, which contains the Hpa I to Not I fragment from p182, encoding Bni1p(1647-1953); and p887, which contains the Bam HI to Not I fragment encoding a full-length profilin cDNA (76). The pACT-derived plasmid was p1124, encoding full-length Act1p as isolated in a catch and release screen (22). The pGAD-C-derived plasmid was p688, encoding the COOH-terminal 311 amino acids (478-788) of Bud6p, as isolated in a catch and release screen (22).
74. Two-hybrid experiments [E. M. Phizicky and S. Fields, Microbiol Rev. 59, 94 (1995)] were performed with strain Y704 (MATa lexAop-LEU2 lexAop-lacZ sst1Δ his3 trp1 ura3-52 leu2) or Y523 (MATa lexAop-lacZ leu2 his3 trp1 ade2 lys2 gal80 GAL4). These strains were transformed with both a LexA DNA-binding domain (DBD) plasmid and a transcriptional-activation domain (AD) plasmid, then assayed for expression of lexAop-lacZ. Cells were grown to mid-log phase in medium containing 2% raffinose, galactose was added to a concentration of 2%, and growth was continued for 4 hours before assaying for β-galactosidase activity (72). Each value represents the average and standard deviation for three independent cultures. The DBD plasmids were pEG202 [J. Gyuris, E. Golemis, H. Chertkov, R. Brent, Cell 75, 791 (1993)] derivatives expressing DBD fusion proteins. The AD plasmids were pJG4-5 (J. Gyuris et al., ibid., p. 791), pGAD-C [P. James, J. Halladay, E. A. Craig, Genetics 144, 1425 (1996)]. pACT [T. Durfee et al., Genes Dev. 7, 555 (1993)], and derivatives expressing AD fusion proteins. DBD-Cdc42p fusions (5) all incorporate the C188S substitution, which prevents prenylation. Other DBD plasmids used were p888, which contains a Bam HI to Not I fragment encoding a full-length profilin cDNA (76); p989, which encodes a mutant form of profilin, Pfy1p-3, lacking the last three amino acids (18); p890, which contains the BgI II to Stu I fragment from p182 (26), encoding Bni1p(1227-1397); p813, which contains the BgI II to Not I fragment from p182, encoding Bni1p(1414-1953); and p951, which contains the Hpa I to Not I fragment from p182, encoding Bni1p(1647-1953). The pJG4-5-derived plasmids were p561, which contains the Bam HI to Not I fragment from p532 (26), encoding Bni1p(1-1953); p717, which contains the Bam HI to Eco47 III fragment from p532, encoding Bni1p(1-1214); p558, which contains the Eco 47III to Not I fragment from p182, encoding Bni1p(1215-1953); p913, which contains the BgI II to Stu I fragment from p182, encoding Bni1p(1227-1397); p929, which contains the BgI II to Not I fragment from p182, encoding Bni1p(1414-1953); p952, which contains the Hpa I to Not I fragment from p182, encoding Bni1p(1647-1953); and p887, which contains the Bam HI to Not I fragment encoding a full-length profilin cDNA (76). The pACT-derived plasmid was p1124, encoding full-length Act1p as isolated in a catch and release screen (22). The pGAD-C-derived plasmid was p688, encoding the COOH-terminal 311 amino acids (478-788) of Bud6p, as isolated in a catch and release screen (22).
75. Two-hybrid experiments [E. M. Phizicky and S. Fields, Microbiol Rev. 59, 94 (1995)] were performed with strain Y704 (MATa lexAop-LEU2 lexAop-lacZ sst1Δ his3 trp1 ura3-52 leu2) or Y523 (MATa lexAop-lacZ leu2 his3 trp1 ade2 lys2 gal80 GAL4). These strains were transformed with both a LexA DNA-binding domain (DBD) plasmid and a transcriptional-activation domain (AD) plasmid, then assayed for expression of lexAop-lacZ. Cells were grown to mid-log phase in medium containing 2% raffinose, galactose was added to a concentration of 2%, and growth was continued for 4 hours before assaying for β-galactosidase activity (72). Each value represents the average and standard deviation for three independent cultures. The DBD plasmids were pEG202 [J. Gyuris, E. Golemis, H. Chertkov, R. Brent, Cell 75, 791 (1993)] derivatives expressing DBD fusion proteins. The AD plasmids were pJG4-5 (J. Gyuris et al., ibid., p. 791), pGAD-C [P. James, J. Halladay, E. A. Craig, Genetics 144, 1425 (1996)]. pACT [T. Durfee et al., Genes Dev. 7, 555 (1993)], and derivatives expressing AD fusion proteins. DBD-Cdc42p fusions (5) all incorporate the C188S substitution, which prevents prenylation. Other DBD plasmids used were p888, which contains a Bam HI to Not I fragment encoding a full-length profilin cDNA (76); p989, which encodes a mutant form of profilin, Pfy1p-3, lacking the last three amino acids (18); p890, which contains the BgI II to Stu I fragment from p182 (26), encoding Bni1p(1227-1397); p813, which contains the BgI II to Not I fragment from p182, encoding Bni1p(1414-1953); and p951, which contains the Hpa I to Not I fragment from p182, encoding Bni1p(1647-1953). The pJG4-5-derived plasmids were p561, which contains the Bam HI to Not I fragment from p532 (26), encoding Bni1p(1-1953); p717, which contains the Bam HI to Eco47 III fragment from p532, encoding Bni1p(1-1214); p558, which contains the Eco 47III to Not I fragment from p182, encoding Bni1p(1215-1953); p913, which contains the BgI II to Stu I fragment from p182, encoding Bni1p(1227-1397); p929, which contains the BgI II to Not I fragment from p182, encoding Bni1p(1414-1953); p952, which contains the Hpa I to Not I fragment from p182, encoding Bni1p(1647-1953); and p887, which contains the Bam HI to Not I fragment encoding a full-length profilin cDNA (76). The pACT-derived plasmid was p1124, encoding full-length Act1p as isolated in a catch and release screen (22). The pGAD-C-derived plasmid was p688, encoding the COOH-terminal 311 amino acids (478-788) of Bud6p, as isolated in a catch and release screen (22).
76. For localization of Bni1p, SY2625 (11) cells carrying a multicopy plasmid encoding either HA-tagged Bni1p [pY39tet1 (9)] or nontagged Bni1p were induced to form mating projections (72). HA-Bni1p was localized by immunofluorescence with monoclonal antibody HA.11 (Berkeley Antibody Company) as described [J. R. Pringle, A. E. M. Adams, D. G. Drubin, B. K. Haarer, Methods Enzymol. 194, 565 (1991)]. For localization of Bud6p, SY2625 cells expressing GFP-Bud6p (23) or containing the control plasmid pRS316 (26) were induced to form mating projections (72), then observed by fluorescence microscopy with the use of a fluorescein isothiocyanate filter set.
77. 4 (pH 7.3)]. Glutathione-Sepharose beads with GST or GST-profilin bound were then added to the yeast extract containing HA-Bni1p(1215-1953) and incubated on ice. After 45 min, the beads were collected and washed twice with PBS. The GST proteins and associated proteins were eluted with glutathione [10 mM glutathione, 50 mM tris-HCl (pH 8.0)] and subjected to immunoblot analysis with antibodies to GST (Pharmacia) or the HA epitope (29) as described (27).
78. We thank D. Amberg, B. Andrews, R. Brent, R. Dorer, S. J. Elledge, S. Givan, B. K. Haarer, J. Horecka, P. James, I. Sadowski, M. Tyers, and J. Zahner for plasmids and yeast strains; B. Brandhorst, J. Brown, N. Davis, S. Kim, B. Nelson, and I. Pot for comments on the manuscript; and G. Poje and I. Pot for assistance with experiments. Supported by grants to C.B. from the Natural Sciences and Engineering Research Council of Canada and the National Cancer Institute of Canada; by a grant from the Swiss National Science Foundation to M.P.; and by NIH grant GM31006 to J.R.P.