Abstract
The Rho-type GTPase Cdc42p is required for cell polarization and bud emergence in Saccharomyces cerevisiae. To identify genes whose functions are linked to CDC42, we screened for (i) multicopy suppressors of a Ts- cdc42 mutant, (ii) mutants that require multiple copies of CDC42 for survival, and (iii) mutations that display synthetic lethality with a partial-loss-of-function allele of CDC24, which encodes a guanine nucleotide exchange factor for Cdc42p. In all three screens, we identified a new gene, BEM4. Cells from which BEM4 was deleted were inviable at 37 degrees C. These cells became unbudded, large, and round, consistent with a model in which Bem4p acts together with Cdc42p in polarity establishment and bud emergence. In some strains, the ability of CDC42 to serve as a multicopy suppressor of the Ts- growth defect of deltabem4 cells required co-overexpression of Rho1p, which is an essential Rho-type GTPase necessary for cell wall integrity. This finding suggests that Bem4p also affects Rho1p function. Bem4p displayed two-hybrid interactions with Cdc42p, Rho1p, and two of the three other known yeast Rho-type GTPases, suggesting that Bem4p can interact with multiple Rho-type GTPases. Models for the role of Bem4p include that it serves as a chaperone or modulates the interaction of these GTPases with one or more of their targets or regulators.
Full Text
The Full Text of this article is available as a PDF (468.1 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Adams A. E., Johnson D. I., Longnecker R. M., Sloat B. F., Pringle J. R. CDC42 and CDC43, two additional genes involved in budding and the establishment of cell polarity in the yeast Saccharomyces cerevisiae. J Cell Biol. 1990 Jul;111(1):131–142. doi: 10.1083/jcb.111.1.131. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bender A., Pringle J. R. Multicopy suppression of the cdc24 budding defect in yeast by CDC42 and three newly identified genes including the ras-related gene RSR1. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9976–9980. doi: 10.1073/pnas.86.24.9976. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bender A., Pringle J. R. Use of a screen for synthetic lethal and multicopy suppressee mutants to identify two new genes involved in morphogenesis in Saccharomyces cerevisiae. Mol Cell Biol. 1991 Mar;11(3):1295–1305. doi: 10.1128/mcb.11.3.1295. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bender A., Sprague G. F., Jr Pheromones and pheromone receptors are the primary determinants of mating specificity in the yeast Saccharomyces cerevisiae. Genetics. 1989 Mar;121(3):463–476. doi: 10.1093/genetics/121.3.463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Boguski M. S., McCormick F. Proteins regulating Ras and its relatives. Nature. 1993 Dec 16;366(6456):643–654. doi: 10.1038/366643a0. [DOI] [PubMed] [Google Scholar]
- Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
- Botstein D., Falco S. C., Stewart S. E., Brennan M., Scherer S., Stinchcomb D. T., Struhl K., Davis R. W. Sterile host yeasts (SHY): a eukaryotic system of biological containment for recombinant DNA experiments. Gene. 1979 Dec;8(1):17–24. doi: 10.1016/0378-1119(79)90004-0. [DOI] [PubMed] [Google Scholar]
- Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: conserved structure and molecular mechanism. Nature. 1991 Jan 10;349(6305):117–127. doi: 10.1038/349117a0. [DOI] [PubMed] [Google Scholar]
- Broach J. R., Strathern J. N., Hicks J. B. Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene. Gene. 1979 Dec;8(1):121–133. doi: 10.1016/0378-1119(79)90012-x. [DOI] [PubMed] [Google Scholar]
- Carlson M., Botstein D. Two differentially regulated mRNAs with different 5' ends encode secreted with intracellular forms of yeast invertase. Cell. 1982 Jan;28(1):145–154. doi: 10.1016/0092-8674(82)90384-1. [DOI] [PubMed] [Google Scholar]
- Chant J., Corrado K., Pringle J. R., Herskowitz I. Yeast BUD5, encoding a putative GDP-GTP exchange factor, is necessary for bud site selection and interacts with bud formation gene BEM1. Cell. 1991 Jun 28;65(7):1213–1224. doi: 10.1016/0092-8674(91)90016-r. [DOI] [PubMed] [Google Scholar]
- Chattoo B. B., Sherman F., Azubalis D. A., Fjellstedt T. A., Mehnert D., Ogur M. Selection of lys2 Mutants of the Yeast SACCHAROMYCES CEREVISIAE by the Utilization of alpha-AMINOADIPATE. Genetics. 1979 Sep;93(1):51–65. doi: 10.1093/genetics/93.1.51. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chenevert J., Corrado K., Bender A., Pringle J., Herskowitz I. A yeast gene (BEM1) necessary for cell polarization whose product contains two SH3 domains. Nature. 1992 Mar 5;356(6364):77–79. doi: 10.1038/356077a0. [DOI] [PubMed] [Google Scholar]
- Coleman K. G., Steensma H. Y., Kaback D. B., Pringle J. R. Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and characterization of the CDC24 gene and adjacent regions of the chromosome. Mol Cell Biol. 1986 Dec;6(12):4516–4525. doi: 10.1128/mcb.6.12.4516. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cvrcková F., De Virgilio C., Manser E., Pringle J. R., Nasmyth K. Ste20-like protein kinases are required for normal localization of cell growth and for cytokinesis in budding yeast. Genes Dev. 1995 Aug 1;9(15):1817–1830. doi: 10.1101/gad.9.15.1817. [DOI] [PubMed] [Google Scholar]
- Drubin D. G. Development of cell polarity in budding yeast. Cell. 1991 Jun 28;65(7):1093–1096. doi: 10.1016/0092-8674(91)90001-f. [DOI] [PubMed] [Google Scholar]
- Gietz R. D., Sugino A. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene. 1988 Dec 30;74(2):527–534. doi: 10.1016/0378-1119(88)90185-0. [DOI] [PubMed] [Google Scholar]
- Harper J. W., Adami G. R., Wei N., Keyomarsi K., Elledge S. J. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell. 1993 Nov 19;75(4):805–816. doi: 10.1016/0092-8674(93)90499-g. [DOI] [PubMed] [Google Scholar]
- Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
- Herskowitz I., Jensen R. E. Putting the HO gene to work: practical uses for mating-type switching. Methods Enzymol. 1991;194:132–146. doi: 10.1016/0076-6879(91)94011-z. [DOI] [PubMed] [Google Scholar]
- Hirano H., Tanaka K., Ozaki K., Imamura H., Kohno H., Hihara T., Kameyama T., Hotta K., Arisawa M., Watanabe T. ROM7/BEM4 encodes a novel protein that interacts with the Rho1p small GTP-binding protein in Saccharomyces cerevisiae. Mol Cell Biol. 1996 Aug;16(8):4396–4403. doi: 10.1128/mcb.16.8.4396. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Imai J., Toh-e A., Matsui Y. Genetic analysis of the Saccharomyces cerevisiae RHO3 gene, encoding a rho-type small GTPase, provides evidence for a role in bud formation. Genetics. 1996 Feb;142(2):359–369. doi: 10.1093/genetics/142.2.359. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Irie K., Nomoto S., Miyajima I., Matsumoto K. SGV1 encodes a CDC28/cdc2-related kinase required for a G alpha subunit-mediated adaptive response to pheromone in S. cerevisiae. Cell. 1991 May 31;65(5):785–795. doi: 10.1016/0092-8674(91)90386-d. [DOI] [PubMed] [Google Scholar]
- Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Johnson D. I., Pringle J. R. Molecular characterization of CDC42, a Saccharomyces cerevisiae gene involved in the development of cell polarity. J Cell Biol. 1990 Jul;111(1):143–152. doi: 10.1083/jcb.111.1.143. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jones J. S., Prakash L. Yeast Saccharomyces cerevisiae selectable markers in pUC18 polylinkers. Yeast. 1990 Sep-Oct;6(5):363–366. doi: 10.1002/yea.320060502. [DOI] [PubMed] [Google Scholar]
- Kao L. R., Peterson J., Ji R., Bender L., Bender A. Interactions between the ankyrin repeat-containing protein Akr1p and the pheromone response pathway in Saccharomyces cerevisiae. Mol Cell Biol. 1996 Jan;16(1):168–178. doi: 10.1128/mcb.16.1.168. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Keszenman-Pereyra D., Hieda K. A colony procedure for transformation of Saccharomyces cerevisiae. Curr Genet. 1988;13(1):21–23. doi: 10.1007/BF00365751. [DOI] [PubMed] [Google Scholar]
- Koshland D., Kent J. C., Hartwell L. H. Genetic analysis of the mitotic transmission of minichromosomes. Cell. 1985 Feb;40(2):393–403. doi: 10.1016/0092-8674(85)90153-9. [DOI] [PubMed] [Google Scholar]
- Levin D. E., Bartlett-Heubusch E. Mutants in the S. cerevisiae PKC1 gene display a cell cycle-specific osmotic stability defect. J Cell Biol. 1992 Mar;116(5):1221–1229. doi: 10.1083/jcb.116.5.1221. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Levin D. E., Fields F. O., Kunisawa R., Bishop J. M., Thorner J. A candidate protein kinase C gene, PKC1, is required for the S. cerevisiae cell cycle. Cell. 1990 Jul 27;62(2):213–224. doi: 10.1016/0092-8674(90)90360-q. [DOI] [PubMed] [Google Scholar]
- Madaule P., Axel R., Myers A. M. Characterization of two members of the rho gene family from the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1987 Feb;84(3):779–783. doi: 10.1073/pnas.84.3.779. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Masuda T., Tanaka K., Nonaka H., Yamochi W., Maeda A., Takai Y. Molecular cloning and characterization of yeast rho GDP dissociation inhibitor. J Biol Chem. 1994 Aug 5;269(31):19713–19718. [PubMed] [Google Scholar]
- Matsui Y., Toh-E A. Yeast RHO3 and RHO4 ras superfamily genes are necessary for bud growth, and their defect is suppressed by a high dose of bud formation genes CDC42 and BEM1. Mol Cell Biol. 1992 Dec;12(12):5690–5699. doi: 10.1128/mcb.12.12.5690. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Matsui Y., Toh-e A. Isolation and characterization of two novel ras superfamily genes in Saccharomyces cerevisiae. Gene. 1992 May 1;114(1):43–49. doi: 10.1016/0378-1119(92)90705-t. [DOI] [PubMed] [Google Scholar]
- Nonaka H., Tanaka K., Hirano H., Fujiwara T., Kohno H., Umikawa M., Mino A., Takai Y. A downstream target of RHO1 small GTP-binding protein is PKC1, a homolog of protein kinase C, which leads to activation of the MAP kinase cascade in Saccharomyces cerevisiae. EMBO J. 1995 Dec 1;14(23):5931–5938. doi: 10.1002/j.1460-2075.1995.tb00281.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Peterson J., Zheng Y., Bender L., Myers A., Cerione R., Bender A. Interactions between the bud emergence proteins Bem1p and Bem2p and Rho-type GTPases in yeast. J Cell Biol. 1994 Dec;127(5):1395–1406. doi: 10.1083/jcb.127.5.1395. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pringle J. R., Preston R. A., Adams A. E., Stearns T., Drubin D. G., Haarer B. K., Jones E. W. Fluorescence microscopy methods for yeast. Methods Cell Biol. 1989;31:357–435. doi: 10.1016/s0091-679x(08)61620-9. [DOI] [PubMed] [Google Scholar]
- Qadota H., Anraku Y., Botstein D., Ohya Y. Conditional lethality of a yeast strain expressing human RHOA in place of RHO1. Proc Natl Acad Sci U S A. 1994 Sep 27;91(20):9317–9321. doi: 10.1073/pnas.91.20.9317. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rose M. D., Novick P., Thomas J. H., Botstein D., Fink G. R. A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene. 1987;60(2-3):237–243. doi: 10.1016/0378-1119(87)90232-0. [DOI] [PubMed] [Google Scholar]
- Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
- Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Simon M. N., De Virgilio C., Souza B., Pringle J. R., Abo A., Reed S. I. Role for the Rho-family GTPase Cdc42 in yeast mating-pheromone signal pathway. Nature. 1995 Aug 24;376(6542):702–705. doi: 10.1038/376702a0. [DOI] [PubMed] [Google Scholar]
- Sloat B. F., Adams A., Pringle J. R. Roles of the CDC24 gene product in cellular morphogenesis during the Saccharomyces cerevisiae cell cycle. J Cell Biol. 1981 Jun;89(3):395–405. doi: 10.1083/jcb.89.3.395. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sloat B. F., Pringle J. R. A mutant of yeast defective in cellular morphogenesis. Science. 1978 Jun 9;200(4346):1171–1173. doi: 10.1126/science.349694. [DOI] [PubMed] [Google Scholar]
- Stevenson B. J., Ferguson B., De Virgilio C., Bi E., Pringle J. R., Ammerer G., Sprague G. F., Jr Mutation of RGA1, which encodes a putative GTPase-activating protein for the polarity-establishment protein Cdc42p, activates the pheromone-response pathway in the yeast Saccharomyces cerevisiae. Genes Dev. 1995 Dec 1;9(23):2949–2963. doi: 10.1101/gad.9.23.2949. [DOI] [PubMed] [Google Scholar]
- Sutton A., Immanuel D., Arndt K. T. The SIT4 protein phosphatase functions in late G1 for progression into S phase. Mol Cell Biol. 1991 Apr;11(4):2133–2148. doi: 10.1128/mcb.11.4.2133. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vojtek A. B., Hollenberg S. M., Cooper J. A. Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell. 1993 Jul 16;74(1):205–214. doi: 10.1016/0092-8674(93)90307-c. [DOI] [PubMed] [Google Scholar]
- Yamochi W., Tanaka K., Nonaka H., Maeda A., Musha T., Takai Y. Growth site localization of Rho1 small GTP-binding protein and its involvement in bud formation in Saccharomyces cerevisiae. J Cell Biol. 1994 Jun;125(5):1077–1093. doi: 10.1083/jcb.125.5.1077. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
- Zheng Y., Cerione R., Bender A. Control of the yeast bud-site assembly GTPase Cdc42. Catalysis of guanine nucleotide exchange by Cdc24 and stimulation of GTPase activity by Bem3. J Biol Chem. 1994 Jan 28;269(4):2369–2372. [PubMed] [Google Scholar]
- Ziman M., Johnson D. I. Genetic evidence for a functional interaction between Saccharomyces cerevisiae CDC24 and CDC42. Yeast. 1994 Apr;10(4):463–474. doi: 10.1002/yea.320100405. [DOI] [PubMed] [Google Scholar]
- Ziman M., O'Brien J. M., Ouellette L. A., Church W. R., Johnson D. I. Mutational analysis of CDC42Sc, a Saccharomyces cerevisiae gene that encodes a putative GTP-binding protein involved in the control of cell polarity. Mol Cell Biol. 1991 Jul;11(7):3537–3544. doi: 10.1128/mcb.11.7.3537. [DOI] [PMC free article] [PubMed] [Google Scholar]