The 26S proteasome of the fission yeast Schizosaccharomyces pombe

The 26S proteasome is the multiprotein complex that degrades proteins that have been marked for destruction by the ubiquitin pathway. It is made up of two multisubunit complexes, the 20S catalytic core and the 19S regulatory complex. We describe the isolation and characterization of conditional mutants in the regulatory complex and their use to investigate interactions between different subunits. In addition we have investigated the localization of the 26S proteasome in fission yeast, by immunofluorescence in fixed cells and live cells with the use of a GFP-tagged subunit. Surprisingly, we find that in mitotic cells the 26S proteasome occupies a discrete intracellular compartment, the nuclear periphery. Electron microscopic analysis demonstrates that the complex resides inside the nuclear envelope. During meiosis the localization showed a more dynamic distribution. In meiosis I the proteasome remained around the nuclear periphery. However, during meiosis II there was a dramatic relocalization: initially, the signal occupied the area between the dividing nuclei, but at the end of mitosis the signal dispersed, returning to the nuclear periphery on ascospore formation. This observation implies that the nuclear periphery is a major site of proteolysis in yeast during mitotic growth and raises important questions about the function of the 26S proteasome in protein degradation.     

Segregation of the nucleolus during mitosis in budding and fission yeast.

The segregation of the nucleolus during mitosis was examined in Saccharomyces cerevisiae and Schizosaccharomyces pombe by indirect immunofluorescence using antibodies directed to highly conserved anti-nucleolus antigens. In mitotic S. pombe cells, the nucleolus appears to trail the bulk of the DNA. In wild-type cells of S. cerevisiae, the nucleolus segregates alongside the bulk of the genomic DNA. Based on its distance from the centromere, we would expect the rDNA in both organisms to segregate behind the majority of the genomic DNA, if telomeric regions trail centromeric regions as in other eukaryotes. We therefore suggest that in S. cerevisiae the nucleolus is attached to other parts of the nucleus which enable it to segregate along with the bulk of the DNA. The segregation of the nucleolus in topoisomerase mutants and nuclear division mutants of S. cerevisiae was also investigated. In cdc14 mutants which arrest at late anaphase, the vast majority of the DNA is separated, but the nucleolar antigens remain extended between the mother and daughter cells. Thus, the CDC14 gene of S. cerevisiae appears to be important for the separation of the nucleolus at mitosis.     

A chromodomain protein, Swi6, performs imprinting functions in fission yeast during mitosis and meiosis

Inheritance of stable states of gene expression is essential for cellular differentiation. In fission yeast, an epigenetic imprint marking the mating-type (mat2/3) region contributes to inheritance of the silenced state, but the nature of the imprint is not known. We show that a chromodomain-containing Swi6 protein is a dosage-critical component involved in imprinting the mat locus. Transient overexpression of Swi6 alters the epigenetic imprint at the mat2/3 region and heritably converts the expressed state to the silenced state. The establishment and maintenance of the imprint are tightly coupled to the recruitment and the persistence of Swi6 at the mat2/3 region during mitosis as well as meiosis. Remarkably, Swi6 remains bound to the mat2/3 interval throughout the cell cycle and itself seems to be a component of the imprint. Our analyses suggest that the unit of inheritance at the mat2/3 locus comprises the DNA plus the associated Swi6 protein complex.     

Nuclear compartmentalization is abolished during fission yeast meiosis

In eukaryotic cells, the nuclear envelope partitions the nucleus from the cytoplasm. The fission yeast Schizosaccharomyces pombe undergoes closed mitosis in which the nuclear envelope persists rather than being broken down, as in higher eukaryotic cells. It is therefore assumed that nucleocytoplasmic transport continues during the cell cycle. Here we show that nuclear transport is, in fact, abolished specifically during anaphase of the second meiotic nuclear division. During that time, both nucleoplasmic and cytoplasmic proteins disperse throughout the cell, reminiscent of the open mitosis of higher eukaryotes, but the architecture of the S. pombe nuclear envelope itself persists. This functional alteration of the nucleocytoplasmic barrier is likely induced by spore wall formation, because ectopic induction of sporulation signaling leads to premature dispersion of nucleoplasmic proteins. A photobleaching assay demonstrated that nuclear envelope permeability increases abruptly at the onset of anaphase of the second meiotic division. The permeability was not altered when sporulation was inhibited by blocking the trafficking of forespore-membrane vesicles from the endoplasmic reticulum to the Golgi. The evidence indicates that yeast gametogenesis produces vesicle transport-mediated forespore membranes by inducing nuclear envelope permeabilization.     

Reconstruction of the kinetochore during meiosis in fission yeast Schizosaccharomyces pombe

During the transition from mitosis to meiosis, the kinetochore undergoes significant reorganization, switching from a bipolar to a monopolar orientation. To examine the centromere proteins that are involved in fundamental reorganization in meiosis, we observed the localization of 22 mitotic and 2 meiotic protein components of the kinetochore during meiosis in living cells of the fission yeast. We found that the 22 mitotic proteins can be classified into three groups: the Mis6-like group, the NMS (Ndc80-Mis12-Spc7) group, and the DASH group, based on their meiotic behavior. Mis6-like group proteins remain at the centromere throughout meiosis. NMS group proteins disappear from the centromere at the onset of meiosis and reappear at the centromere in two steps in late prophase. DASH group proteins appear shortly before metaphase of meiosis I. These observations suggest that Mis6-like group proteins constitute the structural basis of the centromere and that the NMS and DASH group proteins reassemble to establish the functional metaphase kinetochore. On the other hand, the meiosis-specific protein Moa1, which plays an important role in forming the meiotic monopolar kinetochore, is loaded onto the centromere significantly earlier than the NMS group, whereas another meiosis-specific protein, Sgo1, is loaded at times similar to the NMS group.     

Commitment to meiosis in fission yeast

Summary Mutants of Schizosaccharomyces pombe blocked during meiosis were analysed with respect to the induction of diploid mitotic division. Wild type zygotes of this yeast can form diploid colonies with a low probability (ca. 1%) when they are transferred to fresh growth medium. Mutants of three genes affecting meiosis responded to the shift by forming diploid colonies with high yield (ca. 80% of the zygotes). Hence, commitment to meiosis could not be reached by such zygotes. Zygotes of a fourth meiosis-I-deficient mutant were unable to yield diploid colonies more effectively than wild type zygotes. Morphological changes were prominent in the mutant zygotes not reaching commitment (as well as in mutant cells that could not complete conjugation), indicating that activities needed during early stages of conjugation were not turned off in these zygotes.This work was supported by NIH grant GM 13234 initially, and by DFG (SFB 46).     

Uch2/Uch37 is the major deubiquitinating enzyme associated with the 26S proteasome in fission yeast

Conjugation of proteins to ubiquitin plays a central role for a number of cellular processes including endocytosis, DNA repair and degradation by the 26S proteasome. However, ubiquitination is reversible as a number of deubiquitinating enzymes mediate the disassembly of ubiquitin-protein conjugates. Some deubiquitinating enzymes are associated with the 26S proteasome contributing to and regulating the particle's activity. Here, we characterise fission yeast Uch2 and Ubp6, two proteasome associated deubiquitinating enzymes. The human orthologues of these enzymes are known as Uch37 and Usp14, respectively. We report that the subunit Uch2/Uch37 is the major deubiquitinating enzyme associated with the fission yeast 26S proteasome. In contrast, the activity of Ubp6 appears to play a more regulatory and/or structural role involving the proteasome subunits Mts1/Rpn9, Mts2/Rpt2 and Mts3/Rpn12, as Ubp6 becomes essential when activity of these subunits is compromised by conditional mutations. Finally, when the genes encoding Uch2/Uch37 and Ubp6 are disrupted, the cells are viable without showing obvious signs of impaired ubiquitin-dependent proteolysis, indicating that other deubiquitinating enzymes may remedy for the redundancy of these enzymes.     

Identification of a 26S proteasome-associated UCH in fission yeast

We have identified a 26S proteasome-associated ubiquitin carboxyl-terminal hydrolase (UCH) in Schizosaccharomyces pombe. The gene (designated uch2+) encodes a protein containing a UCH catalytic domain at its N-terminus and a short extension at its C-terminus. uch2+ is nonessential as the uch2 null mutant strain showed no significant difference from the wild-type strain. The GFP-tagged Uch2p is localized predominantly to the nuclear periphery, which is similar to the 26S proteasome localization. Deletion of the C-terminal extension of Uch2p resulted in a drastic change of its subcellular localization: it showed a generally diffused distribution instead of a perinuclear pattern. Glycerol gradient centrifugation analysis and coimmunoprecipitation studies of fission yeast extracts using anti-Mts4p antiserum suggest that Uch2p is associated with the 26S proteasome and the association of Uch2p with the 26S proteasome is mediated by its C-terminal extension.     

Spindle pole body components are reorganized during fission yeast meiosis

During meiosis, the centrosome/spindle pole body (SPB) must be regulated in a manner distinct from that of mitosis to achieve a specialized cell division that will produce gametes. In this paper, we demonstrate that several SPB components are localized to SPBs in a meiosis-specific manner in the fission yeast Schizosaccharomyces pombe. SPB components, such as Cut12, Pcp1, and Spo15, which stay on the SPB during the mitotic cell cycle, disassociate from the SPB during meiotic prophase and then return to the SPB immediately before the onset of meiosis I. Interestingly, the polo kinase Plo1, which normally localizes to the SPB during mitosis, is excluded from them in meiotic prophase, when meiosis-specific, horse-tail nuclear movement occurs. We found that exclusion of Plo1 during this period was essential to properly remodel SPBs, because artificial targeting of Plo1 to SPBs resulted in an overduplication of SPBs. We also found that the centrin Cdc31 was required for meiotic SPB remodeling. Thus Plo1 and a centrin play central roles in the meiotic SPB remodeling, which is essential for generating the proper number of meiotic SPBs and, thereby provide unique characteristics to meiotic divisions.     

The assembly pathway of the 19S regulatory particle of the yeast 26S proteasome

The 26S proteasome consists of the 20S proteasome (core particle) and the 19S regulatory particle made of the base and lid substructures, and it is mainly localized in the nucleus in yeast. To examine how and where this huge enzyme complex is assembled, we performed biochemical and microscopic characterization of proteasomes produced in two lid mutants, rpn5-1 and rpn7-3, and a base mutant DeltaN rpn2, of the yeast Saccharomyces cerevisiae. We found that, although lid formation was abolished in rpn5-1 mutant cells at the restrictive temperature, an apparently intact base was produced and localized in the nucleus. In contrast, in DeltaN rpn2 cells, a free lid was formed and localized in the nucleus even at the restrictive temperature. These results indicate that the modules of the 26S proteasome, namely, the core particle, base, and lid, can be formed and imported into the nucleus independently of each other. Based on these observations, we propose a model for the assembly process of the yeast 26S proteasome.     

Distinct temporal requirements for autophagy and the proteasome in yeast meiosis

Meiosis is a special type of cellular renovation that involves 2 successive cell divisions and a single round of DNA replication. Two major degradation systems, the autophagy-lysosome and the ubiquitin-proteasome, are involved in meiosis, but their roles have yet to be elucidated. Here we show that autophagy mainly affects the initiation of meiosis but not the nuclear division. Autophagy works not only by serving as a dynamic recycling system but also by eliminating some negative meiotic regulators such as Ego4 (Ynr034w-a). In a quantitative proteomics study, the proteasome was found to be significantly upregulated during meiotic divisions. We found that proteasomal activity is essential to the 2 successive meiotic nuclear divisions but not for the initiation of meiosis. Our study defines the roles of autophagy and the proteasome in meiosis: Autophagy mainly affects the initiation of meiosis, whereas the proteasome mainly affects the 2 successive meiotic divisions.     

Interaction of the Doa4 deubiquitinating enzyme with the yeast 26S proteasome

e Saccharomyces cerevisiae Doa4 deubiquitinating enzyme is required for the rapid degradation of protein substrates of the ubiquitin-proteasome pathway. Previous work suggested that Doa4 functions late in the pathway, possibly by deubiquitinating (poly)-ubiquitin-substrate intermediates associated with the 26S proteasome. We now provide evidence for physical and functional interaction between Doa4 and the proteasome. Genetic interaction is indicated by the mutual enhancement of defects associated with a deletion of DOA4 or a proteasome mutation when the two mutations are combined. Physical association of Doa4 and the proteasome was investigated with a new yeast 26S proteasome purification procedure, by which we find that a sizeable fraction of Doa4 copurifies with the protease. Another yeast deubiquitinating enzyme, Ubp5, which is related in sequence to Doa4 but cannot substitute for it even when overproduced, does not associate with the proteasome. DOA4-UBP5 chimeras were made by a novel PCR/yeast recombination method and used to identify an N-terminal 310-residue domain of Doa4 that, when appended to the catalytic domain of Ubp5, conferred Doa4 function, consistent with Ubp enzymes having a modular architecture. Unlike Ubp5, a functional Doa4-Ubp5 chimera associates with the proteasome, suggesting that proteasome binding is important for Doa4 function. Together, these data support a model in which Doa4 promotes proteolysis through removal of ubiquitin from proteolytic intermediates on the proteasome before or after initiation of substrate breakdown.     

Virtual breakdown of the nuclear envelope in fission yeast meiosis

Asymmetric localization of Ran regulators (RanGAP1 and RanGEF/RCC1) produces a gradient of RanGTP across the nuclear envelope. In higher eukaryotes, the nuclear envelope breaks down as the cell enters mitosis (designated "open" mitosis). This nuclear envelope breakdown (NEBD) leads to collapse of the RanGTP gradient and the diffusion of nuclear and cytoplasmic macromolecules in the cell, resulting in irreversible progression of the cell cycle. On the other hand, in many fungi, chromosome segregation takes place without NEBD (designated "closed" mitosis). Here we report that in the fission yeast Schizosaccharomyces pombe, despite the nuclear envelope and the nuclear pore complex remaining intact throughout both the meiotic and mitotic cell cycles, nuclear proteins diffuse into the cytoplasm transiently for a few minutes at the onset of anaphase of meiosis II. We also found that nuclear protein diffusion into the cytoplasm occurred coincidently with nuclear localization of Rna1, an S. pombe RanGAP1 homolog that is usually localized in the cytoplasm. These results suggest that nuclear localization of RanGAP1 and depression of RanGTP activity in the nucleus may be mechanistically tied to meiosis-specific diffusion of nuclear proteins into the cytoplasm. This nucleocytoplasmic shuffling of RanGAP1 and nuclear proteins represents virtual breakdown of the nuclear envelope.     

Subcellular localization, stoichiometry, and protein levels of 26 S proteasome subunits in yeast.

The 26 S proteasome of eukaryotes is responsible for the degradation of proteins targeted for proteolysis by the ubiquitin system. Yeast has been an important model organism for understanding eukaryotic proteasome structure and function. Toward a quantitative characterization of the proteasome, we have determined the localization, cellular levels, and stoichiometry of proteasome subunits. The subcellular localization of two ATPase components of the regulatory complex of the proteasome, Sug2/Rpt4 and Sug1/Rpt6, and a subunit of the 20 S proteasome, Pre1, were determined by immunofluorescence. In contrast to findings in multicellular organisms, these proteins are localized almost exclusively to the nucleus throughout the cell cycle. We have also determined the cellular abundance and stoichiometry of these proteasome subunits. Sug1/Rpt6, Sug2/Rpt4, and Pre1 are present in roughly equal stoichiometry with an abundance of 15,000-30,000 molecules/cell, corresponding to a concentration of 13-26 microM in the nucleus. Also, in contrast to mammalian cells, we find no evidence of a p27-containing "modulator" of the proteasome in yeast. This information will be useful in comparing and contrasting the yeast and mammalian proteasomes and should contribute to a mechanistic understanding of how this complex functions.