In Xenopus oocytes, the spindle assembly checkpoint (SAC) kinase Bub1 is required for cytostatic factor (CSF)-induced metaphase arrest in meiosis II. To investigate whether matured mouse oocytes are kept in metaphase by a SAC-mediated inhibition of the anaphase-promoting complex/cyclosome (APC/C) complex, we injected a dominant-negative Bub1 mutant (Bub1dn) into mouse oocytes undergoing meiosis in vitro. Passage through meiosis I was accelerated, but even though the SAC was disrupted, injected oocytes still arrested at metaphase II. Bub1dn-injected oocytes released from CSF and treated with nocodazole to disrupt the second meiotic spindle proceeded into interphase, whereas noninjected control oocytes remained arrested at metaphase. Similar results were obtained using dominant-negative forms of Mad2 and BubR1, as well as checkpoint resistant dominant APC/C activating forms of Cdc20. Thus, SAC proteins are required for checkpoint functions in meiosis I and II, but, in contrast to frog eggs, the SAC is not required for establishing or maintaining the CSF arrest in mouse oocytes.

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Eukaryotic proteasomes are unusually large protein complexes with characteristic sets of subunits and have been classified into two isoforms with apparent sedimentation coefficients of 20S and 26S, respectively. The 20S proteasome (previously named the multicatalytic proteinase complex) is a cylindrical particle with a molecular weight (MW) of approximately 750 kD. It is a dimeric assembly of two symmetrical discs, each consisting of 7 α-type subunits and 7 ß-type subunits, having the molecular organization an[1-7)ßn[1-7)ßn[1-7)an[1-7), where 'n' indicates the number of heterogeneous 7 subunits with MWs of 21-32 kD. The α-type and ß-type subunits constitute a unique multi-gene family encoding previously unidentified, but homologous, polypeptides that have been conserved during evolution. Interestingly, some ß-type subunits with catalytic functions appear to be replaced by very homologous, but distinct, gene products that might be generated by gene duplication in response to extracellular signals, such as γ-interferon, suggesting that the 20S proteasome exists in cells as a heterogeneous population with functional diversity. The 26S proteasome is a eukaryotic ATP-dependent protease, selectively degrading various cellular proteins with specific degradation signals such as a multi-ubiquitin chain. It is a cylindrical caterpillar-shaped complex with a MW of about 2,000 kD. The 26S proteasome is a symmetrical assembly of a central 20S proteasome and a large terminal polypeptide complex with an apparent sedimentation coefficient of 22S. The terminal 22S subset consists of multiple components with MWs of 30-110 kD, which possibly have regulatory functions, and contains multiple ATPases, a de-ubiquitinating enzyme and the recognition molecule(s) for the target proteins. Thus the 26S proteasome is a multi-molecular assembly, consisting of the 20S proteasome and the 22S regulatory subunit complex.

The 26S proteasome is a large multisubunit protease complex, the largest regulatory subunit of which is a component named p112. Molecular cloning of cDNA encoding human p112 revealed a polypeptide predicted to have 953 amino acid residues and a molecular mass of 105,865. The human p112 gene was mapped to the q37.1-q37.2 region of chromosome 2. Computer analysis showed that p112 has strong similarity to the Saccharomyces cerevisiae Sen3p, which has been listed in a gene bank as a factor affecting tRNA splicing endonuclease. The SEN3 also was identified in a synthetic lethal screen with the nin1-1 mutant, a temperature-sensitive mutant of NIN1. NIN1 encodes p31, another regulatory subunit of the 26S proteasome, which is necessary for activation of Cdc28p kinase. Disruption of the SEN3 did not affect cell viability, but led to temperature-sensitive growth. The human p112 cDNA suppressed the growth defect at high temperature in a SEN3 disruptant, indicating that p112 is a functional homologue of the yeast Sen3p. Maintenance of SEN3 disruptant cells at the restrictive temperature resulted in a variety of cellular dysfunctions, including defects in proteolysis mediated by the ubiquitin pathway, in the N-end rule system, in the stress response upon cadmium exposure, and in nuclear protein transportation. The functional abnormality induced by SEN3 disruption differs considerably from various phenotypes shown by the nin1-1 mutation, suggesting that these two regulatory subunits of the 26S proteasome play distinct roles in the various processes mediated by the 26S proteasome.