Rapid regulation of protein activity in fission yeast

Background: The fission yeast Schizosaccharomyces pombe is widely-used as a model organism for the study of a broad range of eukaryotic cellular processes such as cell cycle, genome stability and cell morphology. Despite the availability of extensive set of genetic, molecular biological, biochemical and cell biological tools for analysis of protein function in fission yeast, studies are often hampered by the lack of an effective method allowing for the rapid regulation of protein level or protein activity. Results: In order to be able to regulate protein function, we have made use of a previous finding that the hormone binding domain of steroid receptors can be used as a regulatory cassette to subject the activity of heterologous proteins to hormonal regulation. The approach is based on fusing the protein of interest to the hormone binding domain (HBD) of the estrogen receptor (ER). The HBD tag will attract the Hsp90 complex, which can render the fusion protein inactive. Upon addition of estradiol the protein is quickly released from the Hsp90 complex and thereby activated. We have tagged and characterised the induction of activity of four different HBD-tagged proteins. Here we show that the tag provided the means to effectively regulate the activity of two of these proteins. Conclusion: The estradiol-regulatable hormone binding domain provides a means to regulate the function of some, though not all, fission yeast proteins. This system may result in very quick and reversible activation of the protein of interest. Therefore it will be a powerful tool and it will open experimental approaches in fission yeast that have previously not been possible. Since fission yeast is a widely-used model organism, this will be valuable in many areas of research

Global transcriptional response after exposure of fission yeast cells to ultraviolet light

Background In many cell types, including the fission yeast Schizosaccharomyces pombe, a set of checkpoints are induced by perturbations of the cell cycle or by DNA damage. Many of the checkpoint responses include a substantial change of the transcriptional pattern. As part of characterising a novel G1/S checkpoint in fission yeast we have investigated whether a transcriptional response is induced after irradiation with ultraviolet light. Results Microarray analyses were used to measure the global transcription levels of all open reading frames of fission yeast after 254 nm ultraviolet irradiation, which is known to induce a G1/S checkpoint. We discovered a surprisingly weak transcriptional response, which is quite unlike the marked changes detected after some other types of treatment and in several other checkpoints. Interestingly, the alterations in gene expression after ultraviolet irradiation were not similar to those observed after ionising radiation or oxidative stress. Pathway analysis suggests that there is little systematic transcriptional response to the irradiation by ultraviolet light, but a marked, coordinated transcriptional response was noted on progression of the cells from G1 to S phase. Conclusion There is little response in fission yeast to ultraviolet light at the transcriptional level. Amongst the genes induced or repressed after ultraviolet irradiation we found none that are likely to be involved in the G1/S checkpoint mechanism, suggesting that the checkpoint is not dependent upon transcriptional regulation

Germinating fission yeast spores delay in G1 in response to UV irradiation

Background Checkpoint mechanisms prevent cell cycle transitions until previous events have been completed or damaged DNA has been repaired. In fission yeast, checkpoint mechanisms are known to regulate entry into mitosis, but so far no checkpoint inhibiting S phase entry has been identified. Results We have studied the response of germinating Schizosaccharomyces pombe spores to UV irradiation in G1. When germinating spores are irradiated in early G1 phase, entry into S phase is delayed. We argue that the observed delay is caused by two separate mechanisms. The first takes place before entry into S phase, does not depend on the checkpoint proteins Rad3, Cds1 and Chk1 and is independent of Cdc2 phosphorylation. Furthermore, it is not dependent upon inhibiting the Cdc10-dependent transcription required for S phase entry, unlike a G1/S checkpoint described in budding yeast. We show that expression of Cdt1, a protein essential for initiation of DNA replication, is delayed upon UV irradiation. The second part of the delay occurs after entry into S phase and depends on Rad3 and Cds1 and is probably due to the intra-S checkpoint. If the germinating spores are irradiated in late G1, they enter S phase without delay and arrest in S phase, suggesting that the delay we observe upon UV irradiation in early G1 is not caused by nonspecific effects of UV irradiation. Conclusions We have studied the response of germinating S. pombe spores to UV irradiation in G1 and shown that S phase entry is delayed by a mechanism that is different from classical checkpoint responses. Our results point to a mechanism delaying expression of proteins required for S phase entry

Activation of Gcn2 in response to different stresses

<div><p>All organisms have evolved pathways to respond to different forms of cellular stress. The Gcn2 kinase is best known as a regulator of translation initiation in response to starvation for amino acids. Work in budding yeast has showed that the molecular mechanism of GCN2 activation involves the binding of uncharged tRNAs, which results in a conformational change and GCN2 activation. This pathway requires GCN1, which ensures delivery of the uncharged tRNA onto GCN2. However, Gcn2 is activated by a number of other stresses which do not obviously involve accumulation of uncharged tRNAs, raising the question how Gcn2 is activated under these conditions. Here we investigate the requirement for ongoing translation and tRNA binding for Gcn2 activation after different stresses in fission yeast. We find that mutating the tRNA-binding site on Gcn2 or deleting Gcn1 abolishes Gcn2 activation under all the investigated conditions. These results suggest that tRNA binding to Gcn2 is required for Gcn2 activation not only in response to starvation but also after UV irradiation and oxidative stress.</p></div

Mutating the tRNA-binding sites of Gcn2 abolishes activation in response to UVC irradiation.

<p><b>A</b> Alignment of the HisRS-like domain of budding yeast and fission yeast Gcn2. <b>B</b> The indicated strains were irradiated with 1100 J/m² and samples were taken at the indicated times after irradiation. eIF2α phosphorylation was detected by immunoblotting, α-tubulin levels are shown to check even loading.</p

Gcn1 is required for Gcn2 activation after H₂O₂- treatment.

<p>eIF2α phosphorylation after H₂O₂ treatment in wild-type, <i>gcn2Δ</i> and <i>gcn1Δ</i> cells. The indicated strains were grown in EMM medium and treated with H₂O₂ at the concentrations shown, for 15 minutes. eIF2α phosphorylation was detected by immunoblotting, α-tubulin levels are shown to check even loading.</p

Inhibition of translation does not prevent Gcn2 activation after UV irradiation in fission yeast.

<p>Inhibition of translation does not prevent Gcn2 activation after UV irradiation in fission yeast.</p

Strains used in this study.

<p>Strains used in this study.</p