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Abstract. Budding cells of the yeast Saccharomyces cerevisiae possess a ring of 10-nm-diameter filaments, of unknown biochemical nature, that lies just inside the plasma membrane in the neck connecting the mother cell to its bud. Electron microscopic observations suggest that these filaments assemble at the budding site coincident with bud emergence and disassemble shortly before cytokinesis (Byers, B. and L. Goetsch. 1976. J. Cell Biol. 69:717-721). Mutants defective in any of four genes (CDC3, CDCIO, CDC11, or CDC12) lack these filaments and display a pleiotropic phenotype that involves abnormal bud growth and an inability to complete cytokinesis. We showed previously by immunofluorescence that the CDC12 gene product is probably a constituent of the ring of 10-nm filaments (Haarer, B. and J. Pringle. 1987. Mol. Cell. BioL 7:3678-3687). We now report the use of fusion proteins to generate polyclonal antibodies specific for the CDC3 gene product. In immunofluorescence experiments, these antibodies decorated the neck regions of wild-type and mutant cells in patterns suggesting that the CDC3 gene product is also a constituent of the ring of 10-nm filaments. We also used the CDC3-specific and CDC12-specific antibodies to investigate the timing of localization of these proteins to the budding site. The results suggest that the CDC3 protein is organized into a ring at the budding site well before bud emergence and remains so organized for some time after cytokinesis. The CDC12 product appears to behave similarly, but may arrive at the budding site closer to the time of bud emergence, and disappear from that site more quickly after cytokinesis, than does the CDC3 product. Examination of mating cells and cells responding to purified mating pheromone revealed novel arrangements of the CDC3 and CDC12 products in the regions of cell wall reorganization. Both proteins were present in normallooking ring structures at the bases of the first zygotic buds.

Budding cells of the yeast Saccharomyces cerevisiae possess a ring of 10-nm-diameter filaments, of unknown biochemical nature, that lies just inside the plasma membrane in the neck connecting the mother cell to its bud (B. Byers and L. Goetsch, J. Cell Biol. 69:717-721, 1976). Mutants defective in any of four genes (CDC3, CDCIO, CDCII, and CDC12) lack these filaments and display a pleiotropic phenotype that involves abnormal bud growth and cell-wail deposition and an inability to complete cytokinesis. We fused the cloned CDC12 gene to the Escherichia coli lacZ and trpE genes and used the resulting fusion proteins to raise polyclonal antibodies specific for the CDC12 gene product. In immunofluorescence experiments with affinity-purified antibodies, the neck regions of wild-type and mutant cells stained in patterns consistent with the hypothesis that the CDC12 gene product is a constituent of the ring of 10-nm filaments. Without careful affinity purification of the CDC12-specific antibodies, these staining patterns were completely obscured by the staining of residual cell wall components in the neck region by antibodies present even in the "preimmune" sera of all rabbits tested.One of the fundamental problems of cell biology is to understand the morphogenetic mechanisms by which cellular shape and three-dimensional organization are generated. In the yeast Saccharomyces cerevisiae, the mitotic cell division cycle involves a variety of morphogenetic events (8,25,26) including (i) selection of a nonrandom budding site; (ii) formation of a chitin ring in the largely nonchitinous cell wall at that site; (iii) localization of new cell wall growth to the region bounded by the chitin ring, resulting in the appearance and selective growth of a bud; (iv) localization of new cell wall growth to the tip of the growing bud; (v) nuclear migration to the region of the mother-bud neck; and (vi) cytokinesis and the localized formation of septal cell wall.One promising approach to the understanding of cellular morphogenesis in yeasts is the investigation of mutants with morphogenetic abnormalities. In this regard, the temperature-sensitive cdc3, cdc10, cdcll, and cdc12 mutants (11,25,30) are of interest. At restrictive temperature, these mutants are defective in cytokinesis, yet continue budding, DNA synthesis, and nuclear division; thus, multibudded, multinucleate cells are produced. Moreover, mutants in all four genes fail to form normal chitin rings at the bases of buds formed at restrictive temperature (A. E. M. Adams, Ph.D. thesis, The University of Michigan, Ann Arbor, 1984), and these buds are grossly elongated in comparison with normal buds (1, 26). This hyperpolarization of bud growth is apparent from the time the buds are first visible and is accompanied by an apparent hyperpolarization of the cellular actin network (1, 26), which is believed to be involved in the localized deposition of new cell surface material (1,15,23,26).A clue to the possible molecular defects in the cdc3, cdcJO, cdcll, and cdcl2 mutants was provide...

Abstract. We have isolated profilin from yeast (Saccharomyces cerevisiae) and have microsequenced a portion of the protein to confirm its identity; the region microsequenced agrees with the predicted amino acid sequence from a profilin gene recently isolated from S. cerevisiae (Magdolen, V., U. Oechsner, G. Miiller, and W. Bandlow. 1988. Mol. Cell. Biol. 8:5108-5115). Yeast profilin resembles profilins from other organisms in molecular mass and in the ability to bind to polyproline, retard the rate of actin polymerization, and inhibit hydrolysis of ATP by monomeric actin.Using strains that carry disruptions or deletions of the profilin gene, we have found that, under appropriate conditions, cells can survive without detectable profilin. Such cells grow slowly, are temperature sensitive, lose the normal ellipsoidal shape of yeast cells, often become multinucleate, and generally grow much larger than wild-type cells. In addition, these cells exhibit delocalized deposition of cell wall chitin and have dramatically altered actin distributions.