Abstract. Myo2 protein (Myo2p), an unconventional myosin in the budding yeast Saccharomyces cerevisiae, has been implicated in polarized growth and secretion by studies of the temperature-sensitive myo2-66 mutant. Overexpression of Smylp, which by sequence is a kinesin-related protein, can partially compensate for defects in the myo2 mutant (Lillie, S. H., and S. S. Brown. 1992. Nature (Lond.). 356:358-361). We have now immunolocalized Smylp and Myo2p. Both are concentrated in regions of active growth, as caps at incipient bud sites and on small buds, at the mother-bud neck just before cell separation, and in mating cells as caps on shmoo tips and at the fusion bridge of zygotes. Double labeling of cells with either Myo2p or Smylp antibody plus phalloidin was used to compare the localization of Smylp and Myo2p to actin, and by extrapolation, to each other. These studies confirmed that Myo2p and Smylp colocalize, and are concentrated in the same general regions of the cell as actin spots. However, neither colocalizes with actin. We noted a correlation in the behavior of Myo2p, Smylp, and actin, but not microtubules, under a number of circumstances. In cdc4 and cdcll mutants, which produce multiple buds, Myo2p and Smylp caps were found only in the subset of buds that had accumulations of actin. Mutations in actin or secretory genes perturb actin, Smylp and Myo2p localization. The rearrangements of Myo2p and Smylp correlate temporally with those of actin spots during the cell cycle, and upon temperature and osmotic shift. In contrast, microtubules are not grossly affected by these perturbations. Although wild-type Myo2p localization does not require Smylp, Myo2p staining is brighter when SMY1 is overexpressed. The myo2 mutant, when shifted to restrictive temperature, shows a permanent loss in Myo2p localization and actin polarization, both of which can be restored by SMY1 overexpression. However, the lethality of MY02 deletion is not overcome by SMY1 overexpression. We noted that the myo2 mutant can recover from osmotic shift (unlike actin mutants; Novick, P., and D. Botstein. 1985. Cell. 40:405--416). We have also determined that the myo2-66 allele encodes a Lys instead of a Glu at position 511, which lies at an actin-binding face in the motor domain. M vo2 protein (Myo2p) is an unconventional myosin in yeast (Saccharomyces cerevisiae), which hasbeen classified as a class V myosin on the basis of the sequence of the putative motor domain (Cheney et al., 1993). Although Myo2p has not yet been tested for actinbased motor activity, such activity has been demonstrated for a chicken class V myosin (Espindola et al., 1992).Studies of the temperature-sensitive myo2-66 mutant have implicated Myo2p in polarized growth (Johnston et al., 1991). Yeast grow by budding, a process in which virtually all growth is directed to the bud. This process is disturbed in the myo2 mutant; cells at restrictive temperature continue Address all correspondence to Dr. S. S. Brown, Department of Anatomy and Cell Biology, University of Michiga...

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.

Treatment of chick embryo fibroblasts with 0.5% Triton X-100 extracts most of the cell protein, leaving an organized part of the cell structure attached to the tissue culture dish. This "Triton cytoskeleton" consists largely of intermediate-sized filaments and bundles of microfilaments. SDS polyacrylamide gel electrophoresis reveals that this cytoskeleton is made up of three main proteins. One protein component is 42,000 daltons and co-migrates with muscle actin. The other two components are 52,000 and 230,000 daltons and remain quantitatively associated with the cytoskeleton during the detergent extraction. The possible identity of these three protein components and their organization into a supramolecular structure is discussed.

Motor proteins in cells include myosin, which is actin-based, and kinesin, dynein and dynamin, which are microtubule-based. Several proteins have recently been identified that have amino-acid sequences with similarity to the motor domains of either myosin or kinesin, but are otherwise dissimilar. This has led to the suggestion that these may all be motor proteins, but that they are specialized for moving different cargos. Genetic analysis can address the question of the different functions of these new proteins. Studies of a temperature-sensitive mutation (myo2-66) in a gene of the myosin superfamily (MYO2) have implicated the Myo2 protein (Myo2p) in the process of polarized secretion in yeast (Saccharomyces cerevisiae). To understand more about the role of Myo2p, we have looked for 'multicopy suppressors' (heterologous genes that, when overexpressed, can correct the temperature sensitivity of the myo2-66 mutant). Here we report the identification of such a suppressor (SMY1) that (surprisingly) encodes a predicted polypeptide sharing sequence similarity with the motor portion of proteins in the kinesin superfamily.

CAP is a component of the S. cerevisiae adenylyl cyclase complex. The N-terminal domain is required for cellular RAS responsiveness. Loss of the C-terminal domain is associated with morphological and nutritional defects. Here we report that cap- cells bud randomly and are defective in actin distribution. The morphological and nutritional defects associated with loss of the CAP C-terminal domain are suppressed by over-expression of PFY, the gene encoding profilin, an actin- and polyphosphoinositide-binding protein. The phenotype of cells lacking PFY resembles that of cells lacking the CAP C-terminal domain. Study of mutated yeast profilins and profilins from Acanthamoeba suggests that the ability of profilin to suppress cap- cells is dependent upon a property other than, or in addition to, its ability to bind actin. This property may be its ability to bind polyphosphoinositides. We propose that CAP and profilin provide a link between growth signals and remodeling of the cellular cytoskeleton.