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. 1992 Dec 1;119(5):1151–1162. doi: 10.1083/jcb.119.5.1151

Effects of null mutations and overexpression of capping protein on morphogenesis, actin distribution and polarized secretion in yeast

PMCID: PMC2289735  PMID: 1447293

Abstract

CAP1, the gene encoding the alpha subunit of Saccharomyces cerevisiae capping protein, was cloned using a probe prepared by PCR with primers based on the amino acid sequence of purified alpha subunit peptides. The sequence is similar to that of capping protein alpha subunits of other species but not to that of the S. cerevisiae capping protein beta subunit or any other protein. Null mutants of capping protein, prepared by deletion of the coding region of CAP1 and CAP2 separately or together, are viable and have a similar phenotype. Deletion of the gene for one subunit leads to a loss of protein for the other subunit. The null mutant has a severe deficit of actin cables and an increased number of actin spots in the mother. Cells are round and relatively large. These features are heterogeneous within a population of cells and vary with genetic background. Overexpression of CAP1 and CAP2 also causes loss of actin cables and cell enlargement, as well as the additional traits of aberrant morphogenesis and cell wall thickening. Capping protein null strains and overexpression strains exhibited normal polarized secretion during bud growth as demonstrated by labeling with fluoresceinated Con A. Projection formation and chitin deposition in response to mating pheromone, mating efficiency, and bud site selection were also normal in capping protein null strains. In addition, bulk secretion of invertase was unimpaired. These data indicate that actin cables are not required for polarized secretion in S. cerevisiae.

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Selected References

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  1. Adams A. E., Botstein D., Drubin D. G. A yeast actin-binding protein is encoded by SAC6, a gene found by suppression of an actin mutation. Science. 1989 Jan 13;243(4888):231–233. doi: 10.1126/science.2643162. [DOI] [PubMed] [Google Scholar]
  2. Adams A. E., Botstein D., Drubin D. G. Requirement of yeast fimbrin for actin organization and morphogenesis in vivo. Nature. 1991 Dec 5;354(6352):404–408. doi: 10.1038/354404a0. [DOI] [PubMed] [Google Scholar]
  3. Adams A. E., Pringle J. R. Relationship of actin and tubulin distribution to bud growth in wild-type and morphogenetic-mutant Saccharomyces cerevisiae. J Cell Biol. 1984 Mar;98(3):934–945. doi: 10.1083/jcb.98.3.934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  5. Amatruda J. F., Cannon J. F., Tatchell K., Hug C., Cooper J. A. Disruption of the actin cytoskeleton in yeast capping protein mutants. Nature. 1990 Mar 22;344(6264):352–354. doi: 10.1038/344352a0. [DOI] [PubMed] [Google Scholar]
  6. Amatruda J. F., Cooper J. A. Purification, characterization, and immunofluorescence localization of Saccharomyces cerevisiae capping protein. J Cell Biol. 1992 Jun;117(5):1067–1076. doi: 10.1083/jcb.117.5.1067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ankenbauer T., Kleinschmidt J. A., Walsh M. J., Weiner O. H., Franke W. W. Identification of a widespread nuclear actin binding protein. Nature. 1989 Dec 14;342(6251):822–825. doi: 10.1038/342822a0. [DOI] [PubMed] [Google Scholar]
  8. Baba M., Baba N., Ohsumi Y., Kanaya K., Osumi M. Three-dimensional analysis of morphogenesis induced by mating pheromone alpha factor in Saccharomyces cerevisiae. J Cell Sci. 1989 Oct;94(Pt 2):207–216. doi: 10.1242/jcs.94.2.207. [DOI] [PubMed] [Google Scholar]
  9. Bankaitis V. A., Malehorn D. E., Emr S. D., Greene R. The Saccharomyces cerevisiae SEC14 gene encodes a cytosolic factor that is required for transport of secretory proteins from the yeast Golgi complex. J Cell Biol. 1989 Apr;108(4):1271–1281. doi: 10.1083/jcb.108.4.1271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bowdish K., Tang Y., Hicks J. B., Hilvert D. Yeast expression of a catalytic antibody with chorismate mutase activity. J Biol Chem. 1991 Jun 25;266(18):11901–11908. [PubMed] [Google Scholar]
  11. 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]
  12. Carlson M., Taussig R., Kustu S., Botstein D. The secreted form of invertase in Saccharomyces cerevisiae is synthesized from mRNA encoding a signal sequence. Mol Cell Biol. 1983 Mar;3(3):439–447. doi: 10.1128/mcb.3.3.439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Casella J. F., Casella S. J., Hollands J. A., Caldwell J. E., Cooper J. A. Isolation and characterization of cDNA encoding the alpha subunit of Cap Z(36/32), an actin-capping protein from the Z line of skeletal muscle. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5800–5804. doi: 10.1073/pnas.86.15.5800. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Chant J., Herskowitz I. Genetic control of bud site selection in yeast by a set of gene products that constitute a morphogenetic pathway. Cell. 1991 Jun 28;65(7):1203–1212. doi: 10.1016/0092-8674(91)90015-q. [DOI] [PubMed] [Google Scholar]
  16. Chant J., Pringle J. R. Budding and cell polarity in Saccharomyces cerevisiae. Curr Opin Genet Dev. 1991 Oct;1(3):342–350. doi: 10.1016/s0959-437x(05)80298-9. [DOI] [PubMed] [Google Scholar]
  17. Cooper J. A., Caldwell J. E., Gattermeir D. J., Torres M. A., Amatruda J. F., Casella J. F. Variant cDNAs encoding proteins similar to the alpha subunit of chicken CapZ. Cell Motil Cytoskeleton. 1991;18(3):204–214. doi: 10.1002/cm.970180306. [DOI] [PubMed] [Google Scholar]
  18. Cross F., Hartwell L. H., Jackson C., Konopka J. B. Conjugation in Saccharomyces cerevisiae. Annu Rev Cell Biol. 1988;4:429–457. doi: 10.1146/annurev.cb.04.110188.002241. [DOI] [PubMed] [Google Scholar]
  19. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Drubin D. G. Actin and actin-binding proteins in yeast. Cell Motil Cytoskeleton. 1990;15(1):7–11. doi: 10.1002/cm.970150103. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Drubin D. G., Miller K. G., Botstein D. Yeast actin-binding proteins: evidence for a role in morphogenesis. J Cell Biol. 1988 Dec;107(6 Pt 2):2551–2561. doi: 10.1083/jcb.107.6.2551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Dykstra C. C., Kitada K., Clark A. B., Hamatake R. K., Sugino A. Cloning and characterization of DST2, the gene for DNA strand transfer protein beta from Saccharomyces cerevisiae. Mol Cell Biol. 1991 May;11(5):2583–2592. doi: 10.1128/mcb.11.5.2583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Farkas V., Kovarík J., Kosinová A., Bauer S. Autoradiographic study of mannan incorporation into the growing cell walls of Saccharomyces cerevisiae. J Bacteriol. 1974 Jan;117(1):265–269. doi: 10.1128/jb.117.1.265-269.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Field C., Schekman R. Localized secretion of acid phosphatase reflects the pattern of cell surface growth in Saccharomyces cerevisiae. J Cell Biol. 1980 Jul;86(1):123–128. doi: 10.1083/jcb.86.1.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Gribskov M., Burgess R. R. Sigma factors from E. coli, B. subtilis, phage SP01, and phage T4 are homologous proteins. Nucleic Acids Res. 1986 Aug 26;14(16):6745–6763. doi: 10.1093/nar/14.16.6745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hartmann H., Noegel A. A., Eckerskorn C., Rapp S., Schleicher M. Ca2+-independent F-actin capping proteins. Cap 32/34, a capping protein from Dictyostelium discoideum, does not share sequence homologies with known actin-binding proteins. J Biol Chem. 1989 Jul 25;264(21):12639–12647. [PubMed] [Google Scholar]
  28. Hartwell L. H., Unger M. W. Unequal division in Saccharomyces cerevisiae and its implications for the control of cell division. J Cell Biol. 1977 Nov;75(2 Pt 1):422–435. doi: 10.1083/jcb.75.2.422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Hasek J., Rupes I., Svobodová J., Streiblová E. Tubulin and actin topology during zygote formation of Saccharomyces cerevisiae. J Gen Microbiol. 1987 Dec;133(12):3355–3363. doi: 10.1099/00221287-133-12-3355. [DOI] [PubMed] [Google Scholar]
  30. Haus U., Hartmann H., Trommler P., Noegel A. A., Schleicher M. F-actin capping by cap32/34 requires heterodimeric conformation and can be inhibited with PIP2. Biochem Biophys Res Commun. 1991 Dec 16;181(2):833–839. doi: 10.1016/0006-291x(91)91265-e. [DOI] [PubMed] [Google Scholar]
  31. Helms C., Dutchik J. E., Olson M. V. A lambda DNA protocol based on purification of phage on DEAE-cellulose. Methods Enzymol. 1987;153:69–82. doi: 10.1016/0076-6879(87)53048-8. [DOI] [PubMed] [Google Scholar]
  32. Hug C., Miller T. M., Torres M. A., Casella J. F., Cooper J. A. Identification and characterization of an actin-binding site of CapZ. J Cell Biol. 1992 Feb;116(4):923–931. doi: 10.1083/jcb.116.4.923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Jackson C. L., Hartwell L. H. Courtship in S. cerevisiae: both cell types choose mating partners by responding to the strongest pheromone signal. Cell. 1990 Nov 30;63(5):1039–1051. doi: 10.1016/0092-8674(90)90507-b. [DOI] [PubMed] [Google Scholar]
  34. Jackson C. L., Konopka J. B., Hartwell L. H. S. cerevisiae alpha pheromone receptors activate a novel signal transduction pathway for mating partner discrimination. Cell. 1991 Oct 18;67(2):389–402. doi: 10.1016/0092-8674(91)90190-a. [DOI] [PubMed] [Google Scholar]
  35. Johnston G. C., Prendergast J. A., Singer R. A. The Saccharomyces cerevisiae MYO2 gene encodes an essential myosin for vectorial transport of vesicles. J Cell Biol. 1991 May;113(3):539–551. doi: 10.1083/jcb.113.3.539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Kilmartin J. V., Adams A. E. Structural rearrangements of tubulin and actin during the cell cycle of the yeast Saccharomyces. J Cell Biol. 1984 Mar;98(3):922–933. doi: 10.1083/jcb.98.3.922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  38. Liu H., Bretscher A. Characterization of TPM1 disrupted yeast cells indicates an involvement of tropomyosin in directed vesicular transport. J Cell Biol. 1992 Jul;118(2):285–299. doi: 10.1083/jcb.118.2.285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Marczak J. E., Brandriss M. C. Isolation of constitutive mutations affecting the proline utilization pathway in Saccharomyces cerevisiae and molecular analysis of the PUT3 transcriptional activator. Mol Cell Biol. 1989 Nov;9(11):4696–4705. doi: 10.1128/mcb.9.11.4696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Novick P., Botstein D. Phenotypic analysis of temperature-sensitive yeast actin mutants. Cell. 1985 Feb;40(2):405–416. doi: 10.1016/0092-8674(85)90154-0. [DOI] [PubMed] [Google Scholar]
  41. Novick P., Schekman R. Secretion and cell-surface growth are blocked in a temperature-sensitive mutant of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1858–1862. doi: 10.1073/pnas.76.4.1858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. Pringle J. R. Staining of bud scars and other cell wall chitin with calcofluor. Methods Enzymol. 1991;194:732–735. doi: 10.1016/0076-6879(91)94055-h. [DOI] [PubMed] [Google Scholar]
  45. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Schekman R., Brawley V. Localized deposition of chitin on the yeast cell surface in response to mating pheromone. Proc Natl Acad Sci U S A. 1979 Feb;76(2):645–649. doi: 10.1073/pnas.76.2.645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Schekman R. Protein localization and membrane traffic in yeast. Annu Rev Cell Biol. 1985;1:115–143. doi: 10.1146/annurev.cb.01.110185.000555. [DOI] [PubMed] [Google Scholar]
  48. Seeger M., Payne G. S. A role for clathrin in the sorting of vacuolar proteins in the Golgi complex of yeast. EMBO J. 1992 Aug;11(8):2811–2818. doi: 10.1002/j.1460-2075.1992.tb05348.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Shaw J. A., Mol P. C., Bowers B., Silverman S. J., Valdivieso M. H., Durán A., Cabib E. The function of chitin synthases 2 and 3 in the Saccharomyces cerevisiae cell cycle. J Cell Biol. 1991 Jul;114(1):111–123. doi: 10.1083/jcb.114.1.111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. 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]
  51. Tkacz J. S., Lampen J. O. Surface distributon of invertase on growing Saccharomyces cells. J Bacteriol. 1973 Feb;113(2):1073–1075. doi: 10.1128/jb.113.2.1073-1075.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Tkacz J. S., Lampen J. O. Wall replication in saccharomyces species: use of fluorescein-conjugated concanavalin A to reveal the site of mannan insertion. J Gen Microbiol. 1972 Sep;72(2):243–247. doi: 10.1099/00221287-72-2-243. [DOI] [PubMed] [Google Scholar]
  53. Wright R., Rine J. Transmission electron microscopy and immunocytochemical studies of yeast: analysis of HMG-CoA reductase overproduction by electron microscopy. Methods Cell Biol. 1989;31:473–512. doi: 10.1016/s0091-679x(08)61624-6. [DOI] [PubMed] [Google Scholar]
  54. van Tuinen E., Riezman H. Immunolocalization of glyceraldehyde-3-phosphate dehydrogenase, hexokinase, and carboxypeptidase Y in yeast cells at the ultrastructural level. J Histochem Cytochem. 1987 Mar;35(3):327–333. doi: 10.1177/35.3.3546482. [DOI] [PubMed] [Google Scholar]

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