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.

SummaryRio1p was identified as a protein serine kinase founding a novel subfamily. It is highly conserved from Archaea to man and only distantly related to previously established protein kinase families. Nevertheless, analysis of multiple protein sequence alignments shows that those amino acid residues that are important for either structure or catalytic activity in conventional protein kinases are also conserved in members of the Rio1p family at the respective positions (corresponding to domains I-XI of protein kinases). Recombinant Rio1p from Escherichia coli and tagged Rio1p from yeast has kinase activity in vitro, and mutation of amino acid residues that are conserved and indispensable for catalytic activity (i.e. ATP-binding motif, catalytic centre) abrogates activity. RIO1 is essential in yeast and plays a role in cell cycle progression. After sporulation of RIO1/rio1 diploids, RIO1-disrupted progeny cease growth after one to three cell divisions and arrest as either large unbudded or large-budded cells. Cells deprived of Rio1p are enlarged and arrest either in G1 or in mitosis mainly with the DNA at the bud neck and short spindles (a phenotype also seen in cells carrying a weak allele), suggesting that Rio1p activity is required for at least at two steps during the cell division cycle: for entrance into S phase and for exit from mitosis. The weak RIO1 allele leads to increased plasmid loss. IntroductionProtein kinases are important regulators of a plethora of processes in eukaryotes. The cell division cycle is a Novel class of protein kinases 311The RIO1 gene encodes a protein of 484 amino acids (deduced molecular mass 56.0 kDa). It is transcribed constitutively at a very low level (Angermayr and Bandlow, 1997). Little is known about the biological role of Rio1p, and reports on its function are conflicting. In Emericella nidulans, a mutant orthologue of RIO1, sudD, has been identified as a suppressor of a mutant blocked in mitosis (bimD; Anaya et al., 1998). BimD is the orthologue of yeast Pds5p, a component of a macromolecular complex that holds sister chromatids together after replication until the onset of anaphase (Hartman et al., 2000), suggesting that Rio1p might be involved in the control of chromatid segregation during mitosis. Recently, Rio1p was isolated in a screen for mutants synthetically lethal with a mutant allele of GAR1, an essential gene involved in 18S rRNA maturation (Vanrobays et al., 2001). Depletion of Rio1p led to the accumulation of 20S prerRNA.We report here that Rio1p is a protein serine kinase. Although only distantly related to previously established protein kinase families, it displays the subdomain structure characteristic of protein kinases and has protein kinase activity in vitro. Some of the conserved residues are shown to be essential for viability and enzymatic activity. Thus, Rio1p constitutes the founding member of a novel subfamily of protein kinases. The protein is mainly located in the cytoplasm. It plays an important role in G1 to S transition and in the control ...

The gene coding for profilin (PFY), an actin-binding protein, occurs as a single copy in the haploid genome of Saccharomyces cerevisiae and is required for spore germination and cell viability. Displacement of one gene copy in a diploid cell by a nonfunctional allele is recessively lethal: tetrad analysis yields only two viable spores per ascus. The PFY gene maps on chromosome XV and is linked to the ADE2 marker. The primary transcript of about 1,000 bases contains an intron of 209 bases and is spliced into a messenger of about 750 bases. The intron was identified by comparison with a cDNA clone, which also revealed the 3' end of the transcript. The 5' end of the mRNA was mapped by primer elongation. The gene is transcribed constitutively and has a coding capacity for a protein of 126 amino acids. The deduced molecular weight of

Overproduction of actin is lethal to yeast cells. In contrast, overexpression of the profilin gene, PFYI, encoding an actin‐binding protein, leads to no very obvious phenotype. Interestingly, profilin over‐production can compensate for the deleterious effects of too much actin in a profilin concentration‐dependent manner. Our results, thus, document that actin and profilin interact in vivo. Immunofluorescence studies suggest that suppression works by reducing actin assembly. We observed, however, that even massive overproduction of profilin fails to fully restore the wild‐type phenotype (e.g. the wild‐type appearance of the actin microfilament system). This may indicate that actin monomer sequestration is not the only mechanism by which the balance of actin polymerization is controlled.

Saccharomyces cerevisiaeGuanine nucleotide-binding proteins (G proteins) are important regulators of a wide spectrum of signal-transducing systems. G proteins consist of ␤ and ␥ subunits and the GTPbinding ␣ subunit. The activity of these regulatory complexes is controlled by GDP-GTP exchange, which is accomplished by a transmembrane receptor and followed by dissociation of the ␣ subunit from the ␤␥ subcomplex. Then, either the free ␣ subunit or the ␤␥ dimer, or occasionally both, regulates downstream effectors. The signaling system is shut off by hydrolysis of GTP, followed by reassociation of the inactive ␣␤␥ complex. In higher eucaryotes, trimeric GTP-binding proteins are involved in the regulation of a large number of effectors, including adenylyl cyclase, phospholipase C␤, phospholipase A2, phosphoinositide 3-kinase, and ion channels (for reviews, see references 8 and 25