Ste5 is a scaffold for the mitogen-activated protein kinase (MAPK) cascade components in a yeast pheromone response pathway. Ste5 also associates with Ste4, the beta subunit of a heterotrimeric guanine nucleotide-binding protein, potentially linking receptor activation to stimulation of the MAPK cascade. A RING-H2 motif at the Ste5 amino terminus is apparently essential for function because Ste5(C177S) and Ste5(C177A C180A) mutants did not rescue the mating defect of a ste5Delta cell. In vitro Ste5(C177A C180A) bound each component of the MAPK cascade, but not Ste4. Unlike wild-type Ste5, the mutant did not appear to oligomerize; however, when fused to a heterologous dimerization domain (glutathione S-transferase), the chimeric protein restored mating in an ste5Delta cell and an ste4Delta ste5Delta double mutant. Thus, the RING-H2 domain mediates Ste4-Ste5 interaction, which is a prerequisite for Ste5-Ste5 self-association and signaling.
The gene cluster (ery) responsible for production of the macrolide antibiotic erythromycin by Saccharopolyspora erythraea is also known to contain ermE, the gene conferring resistance to the antibiotic. The nucleotide sequence has been determined of a 4.5 kb portion of the biosynthetic gene cluster, from a region lying between 3.7 kb and 8.2 kb 3' of ermE. This has revealed the presence of four complete open reading frames, including the previously known ery gene eryG, which catalyses the last step in the biosynthetic pathway. Comparison of the amino acid sequence of EryG with the sequence of other S-adenosylmethionine (SAM)-dependent methyltransferases has revealed that one of the sequence motifs previously suggested to be part of the SAM-binding site is present not only in EryG but also in many other recently sequenced SAM-dependent methyltransferases. Previous genetic studies have shown that this region also contains gene(s) involved in hydroxylation of the intermediate 6-deoxyerythronolide B. One of the three other open reading frames (eryF) in fact shows very high sequence similarity to known cytochrome P450 hydroxylases. An adjacent gene (ORF5) shows a strikingly high degree of similarity to prokaryotic and eukaryotic acyltransferases and thioesterases.
Silencing at HMR requires silencers, and one of the roles of the silencer is to recruit Sir proteins. This work focuses on the function of Sir1p once it is recruited to the silencer. We have generated mutants of Sir1p that are recruited to the silencer but are unable to silence, and we have utilized these mutants to identify four proteins, Sir3p, Sir4p, Esc2p, and Htz1p, that when overexpressed, restored silencing. The isolation of Sir3p and Sir4p validated this screen. Molecular analysis suggested that Esc2p contributed to silencing in a manner similar to Sir1p and probably helped recruit or stabilize the other Sir proteins, while Htz1p present at HMR assembled a specialized chromatin structure necessary for silencing.
The GCS1 gene of the budding yeast Saccharomyces cerevisiae mediate the resumption of cell proliferation from the starved, stationary-phase state. Here we identify yeast genes that, in increased dosages, overcome the growth defect of gcs1 delta mutant cells. Among these are YCK1 (CK12) and YCK2 (CKI1), encoding membrane-associated casein kinase I, and YCK3, encoding a novel casein kinase I isoform. Some Yck3p gene product was found associated with the plasma membrane, like Yck1p and Yck2p, but most confractionated with the nucleus, like another yeast casein kinase I isoform, Hrr25p. Genetic studies showed that YCK3 and HRR25 constitute an essential gene family and that Yck3p can weakly substitute for Yck1p-Yck2p. For gcs1 delta suppression, both a protein kinase domain and a C-terminal prenylation motif were shown to be necessary. An impairment in endocytosis was found for gcs1 delta mutant cells, which was alleviated by an increased YCK2 gene dosage. The ability of an increased casein kinase I gene dosage to suppress the effects caused by the absence of Gcs1p suggests that Gcs1p and Yck1p-Yck2p affect parallel pathways.
We have identified two novel genes designated hhp1+ and hhp2+ in the fission yeast Schizosaccharomyces pombe. The hhp1+ and hhp2+ genes encode two closely related protein kinases that share significant sequence identities with Hrr25p from Saccharomyces cerevisiae. Characterization of strains harboring single and double mutations in the hhp+ genes reveals DNA repair defects in these cells. Schizosaccharomyces pombe strains lacking either or both Hhp activities reveal differences in their ability to withstand DNA lesions caused by either methyl methanesulfonate (MMS) or gamma‐rays which correlate with their ability to repair DNA strand breaks caused by these agents. We suggest that Hhp1 and Hhp2 are involved in the regulation of distinct and overlapping DNA repair pathways in S. pombe.
The absolute requirement for the histone deacetylase activity of Sir2p in silencing coupled with the conservation of Sir2p‐like proteins in larger eukaryotes suggests that this molecule plays an important role in gene regulation in all organisms. Here we report the purification and characterization of two Sir2p‐containing protein complexes; one of which contains Sir4p and the other Net1p. The Sir4p‐containing complex has an NAD‐dependent histone deacetylase activity, while the Net1p‐containing complex possesses deacetylase activity but only weak NAD‐dependent histone deacetylase activity. Finally, we demonstrate that the Sir2p‐containing complexes bind nucleosomes efficiently and partially restrict accessibility of the linker DNA to enzymatic probes.
The cloning and molecular characterization of the GCS1 gene from the budding yeast Saccharomyces cerevisiae show that stationary phase is in fact a unique developmental state, with requirements to resume cell proliferation that can be distinct from those for maintenance of proliferation. Deletion of the GCS1 gene products a novel phenotype: stationary‐phase mutant cells do not resume proliferation at a restrictive temperature of 15 degrees C, but mutant cells lacking Gcs1p that are proliferating at the permissive temperature of 29 degrees C continue to proliferate after transfer to 15 degrees C as long as nutrients are available. The GCS1 gene sequence predicts a 39 kDa polypeptide with a novel ‘Zn‐finger’ motif. A point mutation within the finger motif produces a phenotype that mimics that of deletion of the GCS1 gene, showing that the finger motif is essential for full Gcs1p activity. Gcs1p and the products of two newly identified genes, SPS18 and GLO3, constitute a family of novel Zn‐finger proteins.
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