Satoshi Katayama

Covalent modifier NEDD8 is essential for SCF ubiquitin-ligase in fission yeast

A ubiquitin‐like modifier, NEDD8, is covalently attached to cullin‐family proteins, but its physiological role is poorly understood. Here we report that the NEDD8‐modifying pathway is essential for cell viability and function of Pcu1 (cullin‐1 orthologue) in fission yeast. Pcu1 assembled on SCF ubiquitin‐ligase was completely modified by NEDD8. Pcu1K713R defective for NEDD8 conjugation lost the ability to complement lethality due to pcu1 deletion. Forced expression of Pcu1K713R or depletion of NEDD8 in cells resulted in impaired cell proliferation and marked stabilization of the cyclin‐dependent kinase inhibitor Rum1, which is a substrate of the SCF complex. Based on these findings, we propose that covalent modification of cullin‐1 by the NEDD8 system plays an essential role in the function of SCF in fission yeast.

Phosphorylation of Mei2 and Ste11 by Pat1 Kinase Inhibits Sexual Differentiation via Ubiquitin Proteolysis and 14-3-3 Protein in Fission Yeast

Fission yeast Pat1 kinase inhibits sexual differentiation by phosphorylating the meiotic inducer Mei2 and the transcription factor Ste11. Here, we show how Pat1 downregulates these proteins. Mei2 is degraded via a ubiquitin-proteasome pathway in a phosphorylation-dependent fashion. The E2 Ubc2 and the E3 Ubr1 are required for this proteolysis. In addition, Pat1 negatively regulates Ste11 via Rad24/14-3-3, thereby repressing mei2+ transcription. The Pat1 phosphorylation sites of Ste11 match the consensus recognition sequence for 14-3-3. Rad24 binds preferentially to phosphorylated Ste11, and this binding results in inhibition of the transcriptional activation capacity of Ste11. Overall, therefore, these results show that Pat1 coordinates concerted molecular mechanisms that govern the sexual differentiation developmental decision.

Rho‐dependence of Schizosaccharomyces pombe Pck2

In metazoans, the HR1 domain, a motif found in a number of proteins including the protein kinase C‐related PRKs, is responsible for an interaction with Rho‐GTPases. The structural similarity between the Schizosaccaromyces pombe Pck proteins and the mammalian Rho‐dependent protein kinase C‐related family, has led us to investigate the relationship between the function of Rho and that of Pck1/2.

The spike of S phase cyclin Cig2 expression at the G1-S border in fission yeast requires both APC and SCF ubiquitin ligases

We describe a novel set of oscillation mechanisms for the fission yeast S phase cyclin Cig2, which contains an authentic destruction box and is destroyed at anaphase via the APC/cyclosome (APC/C). Unlike the mitotic cyclin Cdc13, however, Cig2 mRNA and protein peak at the G1/S boundary and decline to low levels in G2 and M phases. We show here that SCF(Pop1, Pop2) plays a role in transcriptional periodicity, as pop mutations result in constitutive cig2(+) transcripts. The instability of Cig2 during G2 and M is independent of either the APC/C or Pop1/Pop2, but requires Skp1, a core component of SCF. These data indicate that the APC/C and SCF control Cig2 levels differentially at different stages of the cell cycle.

Molecular interactions of fission yeast Skp1 and its role in the DNA damage checkpoint

Skp1 is a central component of the E3 ubiquitin ligase SCF (Skp1‐Cullin‐1‐F‐box). It forms an adapter bridge between Cullin‐1 and the substrate‐determining component, the F‐box protein. In order to establish the role of Skp1, a temperature sensitive (ts) screen was carried out using mutagenic PCR (polymerase chain reaction) and 9 independent ts mutants were isolated. Mapping the mutated residues on the 3‐D structure of human Skp1 suggested that the mutants would be compromised in binding to F‐box proteins but not Cullin‐1 (Pcu1). In order to assess the binding properties of ts Skp1, 12 F‐box proteins and Pcu1 were epitope‐tagged, and co‐immunoprecipitation performed. This systematic analysis showed that ts Skp1 retains binding to Pcu1. However, binding to three specific F‐box proteins, essential Pof1, Pof3 involved in maintaining genome integrity, and nonessential Pof10, was reduced. skp1ts cells exhibit a G2 cell cycle delay, which is attributable to activation of the DNA damage checkpoint. Intriguingly, contrary to pof3 mutants, in which this checkpoint is required for survival, checkpoint abrogation in skp1ts suppresses a G2 delay and furthermore almost rescues the ts phenotype. The activation mechanism of the DNA damage checkpoint therefore differs between pof3Δ and skp1ts, implicating a novel role for Skp1 in the checkpoint‐signalling cascade.

Nuclear Protein Quality Is Regulated by the Ubiquitin-Proteasome System through the Activity of Ubc4 and San1 in Fission Yeast

Eukaryotic cells monitor and maintain protein quality through a set of protein quality control (PQC) systems whose role is to minimize the harmful effects of the accumulation of aberrant proteins. Although these PQC systems have been extensively studied in the cytoplasm, nuclear PQC systems are not well understood. The present work shows the existence of a nuclear PQC system mediated by the ubiquitin-proteasome system in the fission yeast Schizosaccharomyces pombe. Asf1-30, a mutant form of the histone chaperone Asf1, was used as a model substrate for the study of the nuclear PQC. A temperature-sensitive Asf1-30 protein localized to the nucleus was selectively degraded by the ubiquitin-proteasome system. The Asf1-30 mutant protein was highly ubiquitinated at higher temperatures, and it remained stable in an mts2-1 mutant, which lacks proteasome activity. The E2 enzyme Ubc4 was identified among 11 candidate proteins as the ubiquitin-conjugating enzyme in this system, and San1 was selected among 100 candidates as the ubiquitin ligase (E3) targeting Asf1-30 for degradation. San1, but not other nuclear E3s, showed specificity for the mutant nuclear Asf1-30, but did not show activity against wild-type Asf1. These data clearly showed that the aberrant nuclear protein was degraded by a defined set of E1-E2-E3 enzymes through the ubiquitin-proteasome system. The data also show, for the first time, the presence of a nuclear PQC system in fission yeast.