Protein farnesyltransferase (FTase), a heterodimer enzyme consisting of alpha and beta subunits, catalyzes the addition of farnesyl groups to the C termini of proteins such as Ras. In this paper, we report that the protein substrate specificity of yeast FTase can be switched to that of a closely related enzyme, geranylgeranyltransferase type I (GGTase I) by a single amino acid change at one of the three residues: Ser-159, Tyr-362, or Tyr-366 of its beta-subunit, Dpr1. All three Dpr1 mutants can function as either FTase or GGTase I beta subunit in vivo, although some differences in efficiency were observed. These results point to the importance of two distinct regions (one at 159 and the other at 362 and 366) of Dpr1 for the recognition of the protein substrate. Analysis of the protein, after site directed mutagenesis was used to change Ser-159 to all possible amino acids, showed that either asparagine or aspartic acid at this position allowed FTase beta to function as GGTase I beta. A similar site-directed mutagenesis study on Tyr-362 showed that leucine, methionine, or isoleucine at this position also resulted in the ability of mutant FTase beta to function as GGTase I beta. Interestingly, in both position 159 and 362 substitutions, amino acids that could change the protein substrate specificity had similar van der Waals volumes. Biochemical characterization of the S159N and Y362L mutant proteins showed that their kcat/Km values for GGTase I substrate are increased about 20-fold compared with that of the wild type protein. These results demonstrate that the conversion of the protein substrate specificity of FTase to that of GGTase I can be accomplished by introducing a distinct size amino acid at either of the two residues, 159 and 362.

Staurosporine is an antibiotic that specifically inhibits protein kinase C. Fourteen staurosporine- and temperature-sensitive (stt) mutants of Saccharomyces cerevisiae were isolated and characterized. These mutants were divided into ten complementation groups, and characterized for their cross-sensitivity to K-252a, neomycin, or CaCl2. The STT1 gene was cloned and sequenced. The nucleotide sequence of the STT1 gene revealed that STT1 is the same gene as PKC1. The STT1 gene conferred resistance to staurosporine on wild-type cells, when present on a high copy number plasmid. STT1/stt1::HIS3 diploid cells were more sensitive to staurosporine than STT1/STT1 diploid cells. Analysis of temperature-sensitive stt1 mutants showed that the STT1 gene product functioned in S or G2/M phase. These results suggest that a protein kinase (the STT1 gene product) is one of the essential targets of staurosporine in yeast cells.

Both the gene and the cDNA encoding the Rpb4 subunit of RNA polymerase II were cloned from the fission yeast Schizosaccharomyces pombe. The cDNA sequence indicates that Rpb4 consists of 135 amino acid residues with a molecular weight of 15,362. As in the case of the corresponding subunits from higher eukaryotes such as humans and the plant Arabidopsis thaliana, Rpb4 is smaller than RPB4 from the budding yeast Saccharomyces cerevisiae and lacks several segments, which are present in the S. cerevisiae RPB4 subunit, including the highly charged sequence in the central portion. The RPB4 subunit of S. cerevisiae is not essential for normal cell growth but is required for cell viability under stress conditions. In contrast, S. pombe Rpb4 was found to be essential even under normal growth conditions. The fraction of RNA polymerase II containing RPB4 in exponentially growing cells of S. cerevisiae is about 20%, but S. pombe RNA polymerase II contains the stoichiometric amount of Rpb4 even at the exponential growth phase. In contrast to the RPB4 homologues from higher eukaryotes, however, S. pombe Rpb4 formed stable hybrid heterodimers with S. cerevisiae RPB7, suggesting that S. pombe Rpb4 is similar, in its structure and essential role in cell viability, to the corresponding subunits from higher eukaryotes. However, S. pombe Rpb4 is closer in certain molecular functions to S. cerevisiae RPB4 than the eukaryotic RPB4 homologues.RNA polymerase II in eukaryotes is composed of more than 10 different polypeptides (for example, see reference 29). The genes coding for all 12 putative subunits of RNA polymerase II have been isolated from the budding yeast Saccharomyces cerevisiae (reviewed in references 26 and 27) and humans (10). Sometime ago we reported that the purified RNA polymerase II from the fission yeast Schizosaccharomyces pombe contained at least 10 polypeptides, devoid of the components corresponding to RPB4 and RPB9 of S. cerevisiae (21, 24; for a recent review, see reference 8). Later we cloned the gene and the cDNA for Rpb9 by PCR using the sequence knowledge of subunit 9 from other organisms (23). By Western blot analysis with antibodies against the Rpb9 protein expressed in Escherichia coli, we found that the purified S. pombe RNA polymerase II does indeed contain Rpb9, which had not been detected in the gel pattern because of its comigration with Rpb8 and Rpb11 (23).Recently, the genes coding for subunit 4 were cloned from humans (10) and the plant Arabidopsis thaliana (15). Human cDNA for RPB4 was cloned by two-hybrid screening of cDNA coding for a protein which interacts with human RPB7 (hRPB7) (10), because S. cerevisiae RPB4 forms a binary complex with RPB7 (6, 11). On the other hand, the gene for the A. thaliana RPB15.9 (AtRPB15.9) subunit, which is a homologue of S. cerevisiae RPB4, was cloned by cross-hybridization using the homologous expressed sequence tag (EST) clone of oilseed rape (Brassica napus) as the probe (15). Both hRPB4 and AtRPB15.9 are smaller than S. cerevisiae RPB4, lacking a segment cor...

In the yeast Saccharomyces cerevisiae, Ras regulates adenylate cyclase, which is essential for progression through the G1 phase of the cell cycle. However, even when the adenosine 3',5'-monophosphate (cAMP) pathway was bypassed, the double disruption of RAS1 and RAS2 resulted in defects in growth at both low and high temperatures. Furthermore, the simultaneous disruption of RAS1, RAS2, and the RAS-related gene RSR1 was lethal at any temperature. The triple-disrupted cells were arrested late in the mitotic (M) phase, which was accompanied by an accumulation of cells with divided chromosomes and sustained histone H1 kinase activity. The lethality of the triple disruption was suppressed by the multicopies of CDC5, CDC15, DBF2, SPO12, and TEM1, all of which function in the completion of the M phase. Mammalian ras also suppressed the lethality, which suggests that a similar signaling pathway exists in higher eukaryotes. These results demonstrate that S. cerevisiae Ras functions in the completion of the M phase in a manner independent of the Ras-cAMP pathway.

Entry into meiosis in Saccharomyces cerevisiae cells is regulated by starvation through the adenylate cyclase/cAMP‐dependent protein kinase (AC/PK) pathway. The gene IME1 is also involved in starvation control of meiosis. Multicopy IME1 plasmids overcome the meiotic deficiency of bcy1 and of RASval19 diploids. Double mutants ime1 cdc25 and ime1 ras2 are sporulation deficient. These results suggest that IME1 comes after the AC/PK cascade. Furthermore, the level of IME1 transcripts is affected by mutations in the AC/PK genes CDC25, CYR1 and BCY1. Moreover, the addition of cAMP to a cyr1‐2 diploid suppresses IME1 transcription. The presence in a bcy1 diploid of IME1 multicopy plasmids does not cure the failure of bcy1 cells to arrest as unbudded cells following starvation and to enter the G0 state (thermotolerance, synthesis of unique G0 proteins). This indicates that the pathway downstream of the AC/PK cascade branches to control meiosis through IME1, and to control entry into G0 and cell cycle initiation, independently of IME1.